Recovered ip-10 antibody, immunoconjugate and biospecific molecule thereof, compositions thereof, method of treating (versions), coding molecule of nucleic acid, relative expression vector, host cell and hybridoma

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented invention refers to immunology. There are presented versions of recovered monoclonal antibodies and an antigen-binding portion thereof specific to human IP-10. There are described: an immunoconjugate, bispecific molecule thereof, as well as versions of a composition of the antibody, immunoconjugate or bispecific molecule - for treating autoimmune and inflammatory diseases. There are also disclosed: a coding nucleic acid, an expression vector thereof and a host cell carrying this vector to produce the antibody. What is described is using the antibody or antigen-binding portion thereof for preparing a medicine for treating: either a viral or bacterial infection entailing undesired IP-10 activity, or autoimmune and inflammatory diseases caused by undesired IP-10 activity. What is presented is a hybridoma producing the antibody, derived from a transgenic mouse splenocyte cross-linked to an immortalised cell.

EFFECT: use of the invention provides the novel antibodies that can be find application in medicine to treat a variety of diseases associated with IP-10 activity.

22 cl, 30 dwg, 9 tbl, 8 ex

 

The technical field to which the invention relates.

The invention relates to the field of biotechnology, pharmakologie and medicine, specifically to a technology for selected antibodies, particularly human antibodies that bind to IP-10 with high affinity, on the basis of which the proposed pharmaceutical compositions and methods of treatment of various inflammatory and autoimmune diseases.

The level of technology

Induced by interferon-γ protein 10 (IP-10) (also known as SHSI) is a chemokine molecular weight of 10 KD, which is the source identified on the basis of gene expression of IP-10 in cells treated with interferon-γ (IFN-γ) (see Luster A.D. et al., (1985), Nature, 315:672-676). IP-10 shows homology with a protein with chemotactic activity, such as platelet factor 4 and β-thromboglobulin, and protein with mitogenic activity, such as peptide III activation of the connective tissue (see Luster A.D. et al., (1987), Proc. Natl. Acad. Sci. USA, 84:2868-2871). IP-10 is secreted by many cells, including endothelial cells, monocytes, fibroblasts and keratinocytes in response to IFN-γ (see A.D. Luster and Ravetch J.V., (1987), J. Exp. Med., 166:1084-1097). It is also shown that IP-10 is present in dermal macrophages and endothelial cells during reactions of delayed-type hypersensitivity (DTH) in human skin (the m article KaplanG. et al., (1987), J. Exp. Med., 166:1098-1108). Despite the fact that he is the source identified on the basis of what is induced by IFN-α, IP-10 can also to the induction of IFN-α, for example, dendritic cells (see Padovan E. et al., (2002), J. Leukoc. Biol., 71:669-676). The expression of IP-10 can be induced in cells of the Central nervous system, such as astrocytes and microglia, such incentives as IFN-γ, viruses, and lipopolysaccharides (see article Vanguri R. and Farber J.M., (1994), J. Immunol., 152:1411-1418; Ren, L.Q., et al., (1998), Brain Res. Mol. Brain Res., 59:256-263). A review of immunology IP-10 are presented in the article Neville L.F. et al., (1997), Cytokine Growth Factor Rev., 8:207-219.

The receptor for IP-10 identified as CXCR3, the seventh transmembrane receptor (see Loetscher, M. et al., (1996), J. Exp. Med., 184:963-969). It is shown that CXCR3 is expressed on activated T-lymphocytes, but not on resting T-lymphocytes or b-lymphocytes, monocytes or granulocytes (Loetscher, M. et al., see above). It is shown that increasing regulation of expression of CXCR3 is happening in NK (natural killer) cells upon stimulation with TGF-β 1 (growth factor T-cell β 1) (see Inngjerdingen, M. et al., (2001)Blood, 97:367-375).

Identified two other ligand for CXCR3: MIG (Loetscher, M. et al., see above) and GUS (see Cole KE et al., (1998), J. Exp. Med., 187:2009-2021).

It is shown that the binding of IP-10 to CXCR3 mediates the mobilization of calcium and chemotaxis in activated T-cells (Loetscher, M. et al., see above). Hemotek the IP and mobilization of intracellular calcium induced also by the binding of IP-10 to CXCR3 on activated NK-cells (see article A.A. Maghazachi et al., (1997), FASEB J., 11:765-774). In the thymus IP-10, as shown, is a chemoattractant for T cells TCRαβ+ CD8+T cells TCRγδ+ cells and NK-type (see Romagnani P. et al., (2001)Blood, 97:601-607).

IP-10 or its receptor CXCR3 identified in a number of various inflammatory and autoimmune conditions, including multiple sclerosis (see, for example, article t. l Sorensen et al., (1999), J. Clin. Invest., 103:807-815), rheumatoid arthritis (see, for example, article D.D. Patel et al., (2001), Clin. Immunol., 98:39-45), ulcerative colitis (see, for example, article Uguccioni M. et al., (1999), Am. J. Pathol., 155:331-336), hepatitis (see, for example, article Narumi S. et al., (1997), J. Immunol., 158:5536-5544), damage to the spinal cord (see, for example, articles McTigue D.M. et al., (1998), J. Neurosci. Res., 53:368-376; Gonzalez et al., 2003, Exp. Neurol., 184:456-463), systemic lupus erythematosus (see, for example, article Narumi S. et al., (2000), Cytokine 12:1561-1565), graft rejection (see, for example, Zhang Z. et al., (2002), J. Immunol., 168:3205-3212), Sjogren syndrome (see, for example, article N. Ogawa et al., (2002), Arthritis Rheum., 46:2730-2741). Therefore, the necessary therapeutic agents that inhibit this activity, in particular, the agents that are suitable for use in humans.

Disclosure of inventions

The present invention is selected monoclonal antibodies, particularly human monoclonal antibodies that bind to IP-10 and which exhibit many desirable properties. These properties include the high affinity binding to human IP-10, as well as cross-reactivity with IP-10 rhesus macaques, but in the absence of significant cross-reactivity or human MIG, human ITAC or murine IP-10. In addition, the antibodies inhibit the binding of IP-10 to its receptor, CXCR3, inhibit the calcium current induced IP-10 in cells expressing the receptor, and inhibit induced IP-10 cell migration (chemotaxis). Moreover, it was shown that antibodies corresponding to the invention, associated with IP-10 in the areas of the human brain, who has been diagnosed with multiple sclerosis.

In preferred variants of the invention, the human IP-10 includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:121 [Genbank Registration No. NP_001556];

CXCR3 includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:122 [Genbank Registration No. NP_001495]; IP-10 rhesus macaques includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:123 [Genbank Registration No. AAK95955]; murine IP-10 includes a polypeptide having an amino acid sequence as shown in SEQ ID NO:124 [Genbank Registration No. NPJ367249]; human MIG comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:125 [Genbank Registration No. NP_002407] and/or the ITAC person includes polypeptid is, having an amino acid sequence as shown in SEQ ID NO:126 [Genbank Registration No. NP_005400].

In one embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody specifically binds to IP-10, and includes a variable region of the heavy chain, which is a product of or derives from gene germline VHman, selected from the group consisting of gene VH3-33 human gene VH3-30 .3 human gene VH5-51 person and gene VH4-61 person.

In another embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody specifically binds to IP-10, and includes a variable region light chain, which is a product of or derives from gene germline Vkman, selected from the group consisting of gene VkA27 human gene VkL15 human gene VkL6 person and gene VkL18 person.

In yet another embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody specifically binds to IP-10, and includes:

(a) the variable region of the heavy chain, which is a product or get the it from the gene in the germ line V Hman, selected from the group consisting of gene VH3-33 human gene VH3-30 .3 human gene VH5-51 person and gene VH4-61 person and (b) variable region light chain, which is a product of or derives from gene Vkgermline human, selected from the group consisting of gene VkA27 human gene VkL15 human gene VkL6 person and gene VkL18 person.

In one embodiment, the invention is selected monoclonal antibody or its antigennegative part, the antibody includes:

(a) the variable region of the heavy chain, which is a product of or derives from gene VH3-33 man and

(b) variable region light chain, which is a product of or derives from gene Vkman, selected from the group consisting of gene VkA27 human gene VkL15 person and gene VkL6 person, and the antibody specifically binds to IP-10.

In another embodiment, the invention is selected monoclonal antibody or its antigennegative part, the antibody includes:

(a) the variable region of the heavy chain, which is a product of or derives from gene VH3-30 .3 man and

(b) variable region light the ETUI, which is a product of or isolated from gene VkL6 person;

moreover, the antibody specifically binds to IP-10.

In yet another embodiment, the invention is selected monoclonal antibody or its antigennegative part, the antibody includes:

(a) the variable region of the heavy chain, which is a product of or derives from gene VH5-51 man and

(b) variable region light chain, which is a product of or derives from gene VkL18 person;

moreover, the antibody specifically binds to IP-10.

In yet another embodiment, the invention is selected monoclonal antibody or its antigennegative part, the antibody includes:

(a) the variable region of the heavy chain, which is a product of or derives from gene Vk4-61 man and

(b) variable region light chain, which is a product of or derives from gene VkA27 person;

moreover, the antibody specifically binds to IP-10.

In another aspect of the invention is selected monoclonal antibody or its antigennegative portion comprising the variable region of the heavy chain containing a sequence of CDR 1, CDR2 and CDR3 and a variable region light chain containing after which outermost CDR1, CDR2 and CDR3, and:

(a) sequence of the variable region of the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:24-34 and conservative modifications

(b) sequence of the variable region of the light chain CDR3 comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:73-83 and conservative modifications

(C) the antibody specifically binds to IP-10 and

(d) the antibody exhibits at least one of the following properties:

(i) the antibody inhibits the binding of IP-10 to CXCR3;

(ii) the antibody inhibits induced IP-10 current calcium;

(iii) the antibody inhibits induced IP-10 migration of cells;

(iv) the antibody cross-reacts with IP-10 rhesus macaques;

(v) the antibody is not cross-reactive with mouse IP-10;

(vi) the antibody is not cross-reactive with human MIG;

(vii) the antibody is not cross-reactive with human ITAC.

In a preferred embodiment, the sequence of the variable region of the heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:13-23 and conservative modifications, and sequence of the variable region l is gcoi chain CDR2 comprises the amino acid sequence, selected from the group consisting of amino acid sequence SEQ ID NONO:62-72 and conservative modifications.

In another preferred embodiment, the sequence of the variable region of the heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:1-12 and conservative modifications, and sequence of the variable region of the light chain CDR1 comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:51-61 and their conservative modifications.

The antibody may represent, for example, a human antibody, humanitariannet antibody or chimeric antibody.

In another aspect of the invention is selected monoclonal antibody or its antigennegative portion comprising the variable region of the heavy chain and the variable region of light chain, and:

(a) the variable region of the heavy chain comprises an amino acid sequence that is at least 80% homologous amino acid sequence selected from the group consisting of SEQ ID NONO:35-46,

(b) the variable region of the light chain comprises an amino acid sequence that is at least 80% homologous amino acid sequence, selected from the group consisting the th of SEQ ID NONO:84-94,

(c) the antibody specifically binds to IP-10 and

(d) the antibody exhibits at least one of the following functional properties:

(i) the antibody inhibits the binding of IP-10 to CXCR3;

(ii) the antibody inhibits induced IP-10 current calcium;

(iii) the antibody inhibits induced IP-10 migration of cells;

(iv) the antibody cross-reacts with IP-10 rhesus macaques;

(v) the antibody is not cross-reactive with mouse IP-10;

(vi) the antibody is not cross-reactive with human MIG;

(vii) the antibody is not cross-reactive with human ITAC.

The antibody may represent, for example, a human antibody, humanitariannet antibody or chimeric antibody.

In preferred embodiments of the invention selected monoclonal antibody or its antigennegative part includes:

(a) CDR1 variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:1-12,

(b) CDR2 variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:13-23,

(c) CDR3 variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:24-34,

(d) CDR1 variable region light chain comprising the sequence of amino the slot, selected from the group consisting of SEQ ID NONO:51-61,

(e) a CDR2 variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:62-72, and

(f) a CDR3 variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:73-83, the antibody specifically binds to IP-10.

In other preferred embodiments, the implementation of the invention is selected monoclonal antibody, or antigennegative part, including:

(a) variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:35-46, and

(b) variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:84-94, the antibody specifically binds to IP-10.

In another aspect of the invention presents antibodies or their antigennegative part, which compete for binding to IP-10 with any of the above antibodies.

Antibodies corresponding to the invention, can be, for example, full length antibodies, for example isotype IgG1 or IgG4. Alternative antibodies may represent fragments of antibodies such as Fab fragments or Fab'2, or single-chain antibodies.

The invention also presents immunoconjugate, including ant the body, corresponding to the invention, or its antigennegative part associated with a therapeutic agent such as a cytotoxin or a radioactive isotope.

The invention also presents bespecifically molecule comprising the antibody or antigennegative part corresponding to the invention, associated with the second functional group having a different binding specificity compared with the indicated antibody or antigennegative part.

Presents the composition comprising the antibody or antigennegative part or immunoconjugate or bespecifically molecule corresponding to the invention, and a pharmaceutically acceptable carrier.

In addition, the invention encompasses molecules of nucleic acids encoding antibodies or their antigennegative part corresponding to the invention, as well as expression vectors comprising data nucleic acids, and cells of the host containing such expression vectors.

Moreover, the invention is a transgenic mouse that carries the transgene heavy and light chain of human immunoglobulin, and the mouse expresses an antibody corresponding to the invention, as well as hybridoma producing the antibody corresponding to the invention.

In another aspect the invention provides a method of inhibiting will pospolite is inoveryourhead.net,operatemaintain T-cells or NK-cells, which consists in contacting the T-cells or NK-cells with the antibody or antigennegative part corresponding to the invention, thus, what happens inhibition of inflammatory or autoimmune response.

In another aspect of the invention presents a method of treating inflammatory or autoimmune disease in need of treatment of a subject, which consists in the introduction to the subject antibodies or antigennegative part corresponding to the invention, so is the treatment of inflammatory or autoimmune disease in the subject.

The disease can be, for example, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease), systemic lupus erythematosus, type 1 diabetes, inflammatory skin disorders (e.g. psoriasis, lichen planus), autoimmune thyroid disease (e.g., graves ' disease, Hashimoto's thyroiditis, Sjogren syndrome, pulmonary inflammation (e.g. asthma, chronic obstructive pulmonary disease, sarcoidosis lungs, autoimmunologic), graft rejection, spinal cord injury, brain damage (e.g. stroke), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, gingivitis, inflammation, caused by gene therapy, disease, and is giganta, inflammatory disease of the kidneys (e.g., IgA nephropathy, membranoproliferative glomerulonephritis, rapidly progressive glomerulonephritis) and atherosclerosis.

In another aspect the invention is a method of treating a viral or bacterial infection, which entail undesirable activity of IP-10 in need of treatment of a subject, which consists in the introduction to the subject antibodies or antigennegative part corresponding to the invention, so is the treatment of viral or bacterial infection in the subject. For example, antibodies can be used to treat viral meningitis, viral encephalitis and bacterial meningitis.

Viral infection, which provide treated according to the invention, may be mediated, for example, human immunodeficiency virus (HIV), hepatitis C virus (HCV), herpes simplex virus type I (HSV-1) or virus severe acute respiratory syndrome (SARS).

The invention also provides methods for producing antibodies against IP-10 "second generation" based on the sequences of antibodies against IP-10, as proposed in this context.

For example, the invention is a method of obtaining antibodies against IP-10, providing;

(a) obtaining: (i) sequence variable regions of the heavy chain of the antibody, including the surrounding sequence CDR1, selected from the group consisting of SEQ ID NONO:1-12, a CDR2 sequence selected from the group consisting of SEQ ID NONO:13-23 and/or a CDR3 sequence selected from the group consisting of SEQ ID NONO:24-34, and (ii) the sequence of the variable region of the light chain of the antibody comprising the CDR1 sequence selected from the group consisting of SEQ ID NONO:51-61, a CDR2 sequence selected from the group consisting of SEQ ID NONO:62-72 and/or a CDR3 sequence selected from the group consisting of SEQ ID NONO:73-83;

(b) modifying at least one amino acid residue in the sequence of variable regions of heavy chain antibodies and/or sequence of the variable region of the light chain of the antibody to create at least one altered sequences of the antibodies, and

(c) the expression of the modified sequence of the antibody in the form of protein.

Other characteristics and advantages of this invention will be apparent from the subsequent detailed description and examples, which should not be construed as limiting. The contents of all references, descriptions Genbank, patents and published patent applications cited throughout the material of this application, is specifically included in this context by reference.

Brief description of drawings

The Figure 1 presents the nucleotide sequence (SEQ ID NO:99) and the sequence aminoxy the lot (SEQ ID NO:35) variable regions of the heavy chain of human monoclonal antibody 1D4. Schematically plots CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:13) and CDR3 (SEQ ID NO:24) and identifies deviation from the germline V, D and J.

The Figure 2 presents the nucleotide sequence (SEQ ID NO:110) and the amino acid sequence (SEQ ID NO:84) variable region of the light chain of human monoclonal antibody 1D4. Schematically plots CDR1 (SEQ ID NO:51), CDR2 (SEQ ID NO:62) and CDR3 (SEQ ID NO:73) and identifies deviations from the germ line V, and J.

The Figure 3 presents the nucleotide sequence (SEQ ID NO:100) and the amino acid sequence (SEQ ID NO:36) variable regions of the heavy chain of human monoclonal antibodies E. Schematically plots CDR1 (SEQ ID NO:2), CDR2 (SEQ ID NO:14) and CDR3 (SEQ ID NO:25) and identifies deviations from the germ line V, and J.

The Figure 4 presents the nucleotide sequence (SEQ ID NO:111) and the amino acid sequence (SEQ ID NO:85) variable region of the light chain of human monoclonal antibodies E. Schematically plots CDR1 (SEQ ID NO:52), CDR2 (SEQ ID NO:63) and CDR3 (SEQ ID NO:74) and identifies deviations from the germ line V, and J.

The Figure 5 presents the nucleotide sequence (SEQ ID NO:101) and the amino acid sequence (SEQ ID NO:37) variable regions of the heavy chain of human monoclonal antibody 2G1. Schematically plots CDR1 (SEQ ID NO:3), CDR2 (SEQ ID NO:15) and CDR3 (SEQ ID NO:26) and is listed on the Devi ations from germline V and J.

The Figure 6 presents the nucleotide sequence (SEQ ID NO:112) and the amino acid sequence (SEQ ID NO:86) variable region of the light chain of human monoclonal antibody 2G1. Schematically plots CDR1 (SEQ ID NO:53), CDR2 (SEQ ID NO:64), and CDR3 (SEQ ID NO:75) and identifies deviations from the germ line V, and J.

The Figure 7 presents the nucleotide sequence (SEQ ID NO:102) and the amino acid sequence (SEQ ID NO:38) variable regions of the heavy chain of human monoclonal antibodies S. Schematically plots CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:16) and CDR3 (SEQ ID NO:27) and identifies deviation from the germline V, D and J.

The Figure 8 presents the nucleotide sequence (SEQ ID NO:113) and the amino acid sequence (SEQ ID NO:87) variable region of the light chain of human monoclonal antibodies S. Schematically plots CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:65) and CDR3 (SEQ ID NO:76) and identifies deviations from the germ line V, and J.

The Figure 9 presents the nucleotide sequence (SEQ ID NO:103) and the amino acid sequence (SEQ ID NO:39) variable regions of the heavy chain of human monoclonal antibodies A. Schematically plots CDR1 (SEQ ID NO:5), CDR2 (SEQ ID NO:17), and CDR3 (SEQ ID NO:28) and identifies deviation from the germline V, D and J.

The Figure 10 presents the nucleotide sequence (SEQ ID NO:114) and PEFC is the sequence of amino acids (SEQ ID NO:88) variable region of the light chain of human monoclonal antibodies A. Schematically plots CDR1 (SEQ ID NO:55), CDR2 (SEQ ID NO:66) and CDR3 (SEQ ID NO:77) and identifies deviations from the germ line V, and J.

The Figure 11 presents the nucleotide sequence (SEQ ID NO:104) and the amino acid sequence (SEQ ID NO:40) variable regions of the heavy chain of human monoclonal antibodies A. Schematically plots CDR1 (SEQ ID NO:6), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO:29) and identifies deviations from the germ line V, and J.

The Figure 12 presents the nucleotide sequence (SEQ ID NO:115) and the amino acid sequence (SEQ ID NO:89) variable region of the light chain of human monoclonal antibodies A. Schematically plots CDR1 (SEQ ID NO:56), CDR2 (SEQ ID NO:67) and CDR3 (SEQ ID NO:78) and identifies deviations from the germ line V, and J.

In the Figure 13 presents the nucleotide sequence (SEQ ID NO:105) and the amino acid sequence (SEQ ID NO:41) variable regions of the heavy chain of human monoclonal antibodies B. Schematically plots CDR1 (SEQ ID NO:7), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO:30) and identifies deviation from the germline V, D and J.

In the Figure 14 presents the nucleotide sequence (SEQ ID NO:116) and the amino acid sequence (SEQ ID NO:90) variable region of the light chain of human monoclonal antibodies B. Schematically plots CDR1 (SEQ ID NO:57), CDR2 (SEQ ID NO:68) and CDR3 (SEQ ID NO:79) and the decree which are deviations from the germ line V, and J.

The Figure 15 shows the nucleotide sequence (SEQ ID NO:106) and the amino acid sequence (SEQ ID NO:42) variable regions of the heavy chain of human monoclonal antibodies S. Schematically plots CDR1 (SEQ ID NO:8), CDR2 (SEQ ID NO:20), and CDR3 (SEQ ID NO:31) and identifies deviation from the germline V, D and J.

In the Figure 16 presents the nucleotide sequence (SEQ ID NO:117) and the amino acid sequence (SEQ ID NO:91) variable region of the light chain of human monoclonal antibodies S. Schematically plots CDR1 (SEQ ID NO:58), CDR2 (SEQ ID NO:69) and CDR3 (SEQ ID NO:80) and identifies deviations from the germ line V, and J.

Figure 17 presents the nucleotide sequence (SEQ ID NO:107) and the amino acid sequence (SEQ ID NO:43) variable regions of the heavy chain of human monoclonal antibodies 8F6. Schematically plots CDR1 (SEQ ID NO:9), CDR2 (SEQ ID NO:21) and CDR3 (SEQ ID NO:32) and identifies deviation from the germline V, D and J.

Figure 18 presents the nucleotide sequence (SEQ ID NO:118) and the amino acid sequence (SEQ ID NO:92) variable region of the light chain of human monoclonal antibodies 8F6. Schematically plots CDR1 (SEQ ID NO:59), CDR2 (SEQ ID NO:70) and CDR3 (SEQ ID NO:81) and identifies deviations from the germ line V, and J.

The Figure 19 shows the nucleotide sequence (SEQ ID NO:108) and sledovatelnot amino acids (SEQ ID NO:44) variable regions of the heavy chain of human monoclonal antibodies A. Schematically plots CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:22) and CDR3 (SEQ ID NO:33) and identifies deviation from the germline V and J. the Alternative amino acid residue 32 in CDR1 may be mutated from cysteine in the series (SEQ ID NO:11), leading to the sequence VHSEQ ID NO:45.

The Figure 20 shows the nucleotide sequence (SEQ ID NO:119) and the amino acid sequence (SEQ ID NO:93) variable region of the light chain of human monoclonal antibodies A.

Schematically plots CDR1 (SEQ ID NO:60), CDR2 (SEQ ID NO:71) and CDR3 (SEQ ID NO:82) and identifies deviations from the germ line V, and J.

Figure 21 presents the nucleotide sequence (SEQ ID NO:109) and the amino acid sequence (SEQ ID NO:46) variable regions of the heavy chain of human monoclonal antibodies S. Schematically plots CDR1 (SEQ ID NO:12), CDR2 (SEQ ID NO:23), and CDR3 (SEQ ID NO:34) and identifies deviation from the germline V, D and J.

Figure 22 presents the nucleotide sequence (SEQ ID NO:120) and the amino acid sequence (SEQ ID NO:94) variable region of the light chain of human monoclonal antibodies S. Schematically plots CDR1 (SEQ ID NO:61), CDR2 (SEQ ID NO:72) and CDR3 (SEQ ID NO:83) and identifies deviations from the germ line V, and J.

Figure 23 presents the results of alignment amino acid sequence of the variable region of the heavy chain 1D4, 1 is 1, 2G1, A, A, S and A with the amino acid sequence of human germline VH3-33 (SEQ ID NO:47).

Figure 24 presents the results of alignment amino acid sequence of the variable region of the heavy chain V and 8F6 with the amino acid sequence of human germline VH3-30 .3 (SEQ ID NO:48).

Figure 25 presents the results of alignment amino acid sequence of the variable region of the heavy chain C with the amino acid sequence of human germline VH5-51 (SEQ ID NO:49).

Figure 26 presents the results of alignment amino acid sequence of the variable region of the heavy chain C with the amino acid sequence of human germline VH4-61 (SEQ ID NO:50).

Figure 27 presents the results of alignment amino acid sequence of the variable region of the light chain 1D4, 2G1, A, A, A and C with the amino acid sequence of human germline VkA27 (SEQ ID NO:95).

Figure 28 presents the results of alignment amino acid sequence of the variable region of the light chain E, V and 8F6 with the amino acid sequence of human germline VkL6 (SEQ ID NO:96).

Figure 29 presents the results of alignment amino acid sequence of the variable region of the light chain S posledovatelnostyu amino acid of the human germline V kL18 (SEQ ID NO:97).

Figure 30 presents the results of alignment amino acid sequence of the variable region of the light chain 7C 10 with the amino acid sequence of human germline VkL15 (SEQ ID NO:98). The implementation of the invention

The present invention relates to the selection of monoclonal antibodies, particularly human monoclonal antibodies that specifically bind to IP-10 and which inhibit the functional properties of IP-10.

In some embodiments of the antibodies corresponding to the invention, selected from specific sequences of the heavy and light chain germline and/or include certain structural features, such as areas CDR containing specific amino acid sequence.

The invention is selected antibodies, methods of obtaining data antibodies, immunoconjugates and bespecifically molecules comprising these antibodies, and pharmaceutical compositions containing the antibodies, immunoconjugates or bespecifically molecules corresponding to the invention.

The invention relates also to methods of using the antibodies to inhibit the inflammatory or autoimmune answers, for example, in the treatment of various inflammatory or autoimmune diseases, as well as to methods for treating viral infections, including the surrounding unwanted activity of IP-10.

To help understand the present invention, first define some terms. Additional definitions are provided throughout the text detailed description.

The terms "protein 10 induced by interferon-γ", "IP-10 and CXCL10" are used interchangeably, and include variants, isoforms and species homologs of human IP-10. Accordingly, human antibodies, corresponding to the invention, in some cases, there may be cross-reactive with IP-10 species other than human. In other cases, the antibodies may be completely specific in relation to human IP-10 and may not be species or other types of cross-reactivity.

The complete amino acid sequence of human IP-10 has a registration number Genbank NP_001556 (SEQ ID NO:121). The complete amino acid sequence of IP-10 rhesus macaques has a registration number Genbank AAK95955 (SEQ ID NO:123). The complete amino acid sequence of murine IP-10 has a registration number Genbank NP_067249 (SEQ ID NO:124).

The term "CXCR3" refers to the receptor for IP-10 (CXCL10). The complete amino acid sequence of human CXCR3 has a registration number Genbank NP_001495 (SEQ ID NO:122).

The term "MIG" refers to the CXCR3 ligand, also known as manokin induced by γ-interferon, which is different from the IP 10. The complete amino acid sequence of human MIG has the registration number Genbank NP_002407 (SEQ ID NO:125).

The term "ITAC" refers to the CXCR3 ligand, also known as induced by interferon a chemoattractant for T-cells α, which is different from the IP 10. The complete amino acid sequence of human ITAC has a registration number Genbank NP_005400 (SEQ ID NO:126).

The term "immune response" refers deystviy, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement)that results in selective damage to, destruction of, or elimination from the human body invasive pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

The term "path of signal transduction" refers to the biochemical relationships between many signal transduction molecules that play a role in signal transmission from one part of cells in another part of the cell. As used in this context, the expression "cell surface receptor" includes, for example, molecules and complexes of molecules capable of receiving the signal and transfer the signal across the plasma membrane of cells. An example of a "cell surface receptor"corresponding to this is obreteniyu, is the receptor CXCR3, which binds the molecule of IP-10.

The term "antibody"as referred to in this context, includes whole antibodies and any antigennegative fragment (i.e., "antigennegative portion") or single chains. The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains, linked together by disulfide bonds, or antigennegative part. Each heavy chain consists of a variable region heavy chain (abbreviated in this context as VH) and the constant region of the heavy chain. The constant region of the heavy chain consists of three domains, a CH1CH2and CH3. Each light chain consists of a variable region light chain (abbreviated in this context as VL) and the constant region of the light chain. The constant region of the light chain consists of a single domain CL. Region VHand VLcan be further subdivided into areas of hypervariability, called plots, complementarity determining (CDR), between which there are areas that are more conservative, called skeletal (spanning) regions (FR). Each of the VHand Vkconsists of three CDRs and four FR, built from aminobenzo to carboxilic in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 FR4. Variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant region of the antibodies may mediate the binding of immunoglobulin with tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical system of complement.

The term "antigennegative part of the antibody (or simply "antibody", as used in this context, refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen (for example, IP-10). It is shown that antigennegative function of antibodies can be performed by fragments of a full length antibodies. Examples of binding fragments encompassed by the term "antigennegative part of the antibody include (i) a Fab fragment, a monovalent fragment consisting of domains VLVHCLand CH1; (ii) the fragment F(ab')2, a bivalent fragment comprising two Fab fragment linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domains and SN; (iv) a Fv fragment consisting of domains Vkand VHone shoulder antibody, (v) a dAb fragment (see Ward et al., (1989), Nature, 341:544-546), which consists of domain VHand (vi) the selection complementarity determining (CDR). Also, although the two domains of the Fv fragment, VLand VHthat are encoded by separate genes, they can be connected by recombinant methods, by a synthetic linker that enables to receive them as a single protein chain in which the region Vkand VHcoupled with the formation of monovalent molecules (known as single-chain Fv (scFv); see, for example, article Bird et al., (1988), Science, 242:423-426; and Huston et al., (1988), Proc. Natl. Acad. Sci. USA, 85:5879-5883). It is also envisaged that the term "antigennegative part of the antibody comprises the data of single-chain antibodies. These fragments of antibodies obtained using accepted techniques known to experts in the field of engineering, and the fragments are screened for use in the same manner as are intact antibodies.

The term "isolated antibody", as used in this context is intended to refer to an antibody which is almost free of other antibodies having excellent antigenic specificnosti (for example, the selected antibody that specifically binds to IP-10, contains almost no antibodies that specifically bind antigens other than IP-10). The selected antibody that specifically binds to IP-10, however, may have cross-reactivity with other antigens, such as molecules of IP-10 from other species. Moreover, the dedicated and tetelo can almost not contain other cellular material and/or chemical agents.

The terms "monoclonal antibody" or "composition of monoclonal antibodies", as used in this context, refers to a preparation of antibody molecules of the same molecular composition. The composition of monoclonal antibodies reveals a single binding specificity and affinity for a particular epitope.

The term "human antibody", as used in this context is the incorporation of antibodies having variable regions in which both skeletal region and areas CDR selected from the sequences of immunoglobulin germline person. Furthermore, if the antibody comprises a constant region, the constant region also is derived from immunoglobulin sequences of the germline of the person. Human antibodies corresponding to the invention may include amino acid residues not encoded by the sequences of immunoglobulin germ line person (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used in this context, does not provide for the inclusion of antibodies in which CDR sequence selected from the germline of another form of mammal, such as a mouse, grafted on human skeletal sequence./p>

The term "human monoclonal antibody" refers to canttell, showing a single binding specificity which have variable regions, where the skeletal region and areas CDR selected from the sequences of immunoglobulin germline person. In one embodiment, human monoclonal antibodies are produced by hybridomas, which includes In-cell obtained from a transgenic animal other than human, for example, traganou mouse, having a genome that carries the heavy chain transgene and a light chain transgene person, merged with immortalizing cell.

The term "recombinant human antibody", as used in this context, includes all human antibodies that are obtained, expressed, created or selected recombinant means, such as (a) antibodies isolated from an animal (e.g. a mouse)that is transgenic or transpromotional for genes of human immunoglobulin, or derived from hybridoma (additionally described below), (b) antibodies isolated from a host cell transformed so that they are expressed human antibody, for example, introspector, (C) antibodies isolated from a recombinant, combinatorial libraries human antibodies, and (d) antibodies obtained, expressed, is created or selected by other means, that involve splicing of immunoglobulin gene sequences person with other DNA sequences. Data of recombinant human antibodies have variable regions in which skeletal areas and areas CDR selected from the gene sequences of human immunoglobulin. However, in some embodiments, implementation, data recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using an animal transgenic sequences 1D human somatic mutagenesis in vivo and, thus, the amino acid sequence of regions VHand VLrecombinant antibodies are sequences that, when highlighted and related to the sequences of the VHand VLgermline person, may not exist in vivo in the repertoire of germline human antibodies in vivo.

As used in this context, the term "isotype" refers to the class of antibodies (e.g., IgM or IgGI), which encodes the genes for the constant region of the heavy chain.

The expression "antibody that recognizes the antigen and antibody specific against the antigen used in this context is interchangeable with the term "antibody that specifically binds to the antigen".

As used in this pin is XTE, the expression of the antibody that "specifically binds to human IP-10" is intended to refer to an antibody which binds to human IP-10 with KD5×10-9M or less, more preferably 2×10-9M or less and even more preferably 1×10-10M or less. The expression of the antibody that "cross-reactive with IP-10 rhesus macaques", is intended to refer to an antibody which binds to IP-10 rhesus macaques with KD0,5×10-8M or less, more preferably 5×10-9M or less and even more preferably 2×10-9M or less. The expression of the antibody that "does not cross-reactive with mouse IP-10 or not cross-reactive with human MIG" or "not cross-reactive with human ITAC" is intended to refer to an antibody which binds to murine IP-10, human MIG or human ITAC with KDof 1.5×10-8M or higher, more preferably a KD of 5-10×10-8M or higher, and even more preferably 1×10-7M or higher. In some embodiments these antibodies are not cross-reactive with mouse IP-10, human MIG and/or human ITAC are essentially undetectable binding against these proteins in standard analyses of the binding.

As used in this context, the expression of the antibody cat is PoE "inhibits the binding of IP-10 to CXCR3" is intended to refer to antibodies, that inhibits the binding of IP-10 to CXCR3 with Ki1 nm or less, more preferably of 0.75 nm or less, even more preferably 0.5 nm or less and even more preferably of 0.25 nm or less.

As used in this context, the expression of the antibody that "inhibits induced IP-10 current calcium, is intended to refer to an antibody that inhibits induced IP-10 current calcium with IC5010 nm or less, more preferably of 7.5 nm or less, even more preferably 5 nm or less and even more preferably 2.5 nm or less.

As used in this context, the expression of the antibody that "inhibits induced IP-10 migration of cells"is intended to refer to an antibody that inhibits human induced IP-10 migration of cells with IC502 mcg/ml or less, more preferably 1 μg/ml or less, even more preferably 0.5 μg/ml or less and even more preferably 0.25 microgram/ml or less.

The term "Kassoc" or "Kand"as used in this context, is intended to denote the velocity of the Association of the interaction of a particular antibody-antigen, whereas the term "Kdis" or "Kd"as used in this context, is intended to denote the rate of dissociation of the interaction of a particular antibody-antigen. The term "KD "as used in this context is intended to refer to the dissociation constant, which is obtained from the ratio of KdKa(i.e. Kd/Ka) and expressed as a molar concentration (M). Values of KDfor antibodies can be determined using methods well established in the field of engineering. The preferred method for the determination of KDantibodies is the use of surface plasma resonance, preferably using a biosensor system such as the Biacore system®.

As used in this context, the term "high affinity" for an IgG antibody refers to an antibody having KD10-8M or less, more preferably 10-9M or less and even more preferably 10-10M or less in relation to the target antigen. However, "high-affinity" binding may vary in relation to other antibody isotypes. For example, "high-affinity" binding in respect of the IgM isotype refers to an antibody having KD10-7M or less, more preferably 10-8M or less.

As used in this context, the term "subject" includes any person or animal other than man. The term "animal other than man" includes all vertebrates, e.g., mammals and insects memleketim, such as primates, non-human,sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in the following subsections.

Antibodies against IP-10

Antibodies corresponding to the invention are characterized by certain functional characteristics and properties of antibodies. For example, antibodies specifically associated with human IP-10. In addition, antibodies can cross-react with IP-10, obtained from one or more of primates other than humans, such as macaque-rhesus. Preferably, when antibodies are not cross-reactive with mouse IP-10. Moreover, although the MIG, and ITAC are also ligands for the receptor CXCR3 antibodies corresponding to the invention, preferably are not cross-reactive with human MIG or human ITAC.

Preferably, when the antibody corresponding to the invention, binds to IP-10 high affinity, for example, with KD10-8M or less, or 10-9M or less, or even 10-10M or less.

In addition, antibodies corresponding to the invention capable of inhibiting one or more functional activities of IP-10. For example, in one embodiment, the antibodies inhibit the binding of IP-10 to CXCR3. In another embodiment, the antibodies inhibit induced IP-10 current calcium. In yet another variant is NTE implementation antibodies inhibit induced IP-10 cell migration (chemotaxis).

In the field of engineering known standard tests aimed at assessing the binding ability of the antibodies against IP-10 different types and/or MIG or ITAC, including, for example, ELISA (enzyme-linked immunosorbent assays), Western blot and RIA (radioimmunoassay analyses). Suitable assays are described in detail in the Examples. The binding kinetics (e.g., affinity of binding of the antibodies also can be assessed by standard methods of analysis, known in the technical field, such as analysis using Biacore. Analyses aimed at assessing the effects of antibodies on functional properties of IP-10 (e.g., receptor binding, calcium current, chemotaxis), described in more detail in the Examples.

Accordingly, whereas, the expression of the antibody that "inhibits" one or more data functional properties of IP-10 (e.g., biochemical, immunochemical, cellular, physiological or other biological activities, or the like)as determined according to methodologies known in the art and are described in this context, refers to a statistically significant decrease in specific activity relative to the level observed in the absence of antibodies (or, for example, when there is a control antibody nesootwetstwujut the th specificity). Preferably, when the antibody that inhibits the activity of IP-10, causes such a statistically significant decrease in the measured parameter, by at least 10%, more preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and in some preferred embodiments, the implementation of the antibody corresponding to the invention, may inhibit more than 92%, 94%, 95%, 97%, 98% or 99% of the functional activity of IP-10.

Monoclonal antibody 1D4, E, 2G1, S, A, A, V, S, 8F6, A and C

Preferred antibodies corresponding to the invention are the human monoclonal antibody 1D4, E, 2G1, S, A, A, V, S, 8F6, A and C, isolated and structurally characterized as described in Examples 1 and 2. Another preferred antibody is 10A12S, in which the remainder of the 32 amino acids of the heavy chain A (CDR1 VH) mutated from cysteine to serine. Amino acid sequence of the Vk1E1, 2G1, S, A, A, V, S, 8F6, A, 10A12S and IS presented in SEQ ID NONO:35-46, respectively. Amino acid sequence of the VkE, 2G1, S, A, A, V, S, 8F6, A and C presented in SEQ ID NONO:84-94, respectively.

Based on the fact that each of these antibodies can bind with IP-10, the sequence VHand VLcan be "mixed and matched" to create other binding molecules against IP-10, sootvetstvuyushchuyu. The binding of IP-10 data "mixed and matched" antibodies can be tested using the assays of the binding described above and in the Examples (e.g., ELISA). Preferably, when VHand VL-circuit mixed and selected, the sequence VHfrom a certain pair of VH/VLwas replaced with structurally similar sequences VH. Similarly, it is preferred when the sequence VLfrom a certain pair of VH/VLreplace structurally similar sequence VL. For example, the sequence VHand Vk1D4, 2G1, A, A, A or 10A12S particularly suitable for mixing and matching, because the antibodies used sequence VHand VLisolated from the same germline sequences (VH 3-33 and Vk A27) and, thus, they exhibit structural proximity. Similarly, the sequence VHand VLV and 8F6 are also particularly suitable for mixing and matching, because they also used the sequence VHand VLisolated from the same germline sequences (VH 3-30 .3 and Vk L6) and, thus, they exhibit structural proximity. Alternatively, for example, the sequence VH1D4, 2G1, A, A, A or 10A12S can be paired CV L13C4, since the sequence VH1D4, 2G1, A, A, A and 10A12S source coupled with a sequence of Vkgermline Vk A27 and a sequence Vk13C4 also comes from the germline Vk A27. Similarly, the sequence VL7C10 or E can be paired with VH1D4, 2G1, A, A, A or 10A12S, poskolkuutvarey VLS and A source coupled with a sequence of Vkgermline VH 3-33 and sequence VH1D4, 2G1, A, A, A and 10A12S also isolated from germline VH 3-33. Ordinary competent specialist will be easy to see the possibility of creating another mating VH/VLstructurally close sequences of sequences VHand VLdescribed in this context for monoclonal antibody 1D4, E, 2G1, S, A, A, V, S, 8F6, A and 13C4.

Accordingly in one aspect the invention is selected monoclonal antibody or its antigennegative part, including:

(a) the variable region of the heavy chain containing an amino acid sequence selected from the group consisting of SEQ ID NONO:35-46; and

(b) the variable region of the light chain containing the amino acid sequence selected from the group consisting of SEQ ID NONO:84-94;

moreover, the antibody specifically binds to IP-10.

The preferred combination is heavy and light chains include:

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:35; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:84; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:36; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:85; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:37; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:86; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:38; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:87; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:39; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:88; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:40; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:89; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:41; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO90; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:42; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:91; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:43; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:92; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:44 or 45; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:93; or

(a) the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:46; and (b) the variable region of the light chain containing the amino acid sequence of SEQ ID NO:94.

In another aspect, the invention provides antibodies that comprise CDR1, CDR2 and CDR3 of the heavy and light chains 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S and S or combinations thereof. Amino acid sequence CDR1 VH1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S and IS presented in SEQ ID NONO:1-12. Amino acid sequence CDR2 VH1D4, E, 2G1, S, A, A, V, S, 8F6, A and C presented in SEQ ID NONO:13-23. Amino acid sequence CDR3 VH1D4, E, 2G1, S, A, A, V, S, 8F6, A and S presents with SEQ ID NONO:24-34. Amino acid sequence CDR1 Vk1D4, E, 2G1, S,A, A, V, S, 8F6, A and C presented in SEQ ID NONO:51-61. Amino acid sequence CDR2 Vk1D4, E, 2G1, S, A, A, V, S, 8F6, A and C presented in SEQ ID NONO:62-72. Amino acid sequence CDR1 Vk1 D4,1 E1, 2G1, S, A, A, V, S, 8F6, A and C presented in SEQ ID NONO:73-83. Lots CDR schematically represented by the Kabat system (see Kabat, E. A. et al., (1991), Sequences of Proteins of Immunological Interest (Sequences of proteins representing immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

Based on the fact that these antibodies can be contacted with IP-10, and that antigennegative specificity is provided primarily through sites CDR1,2 and 3 sequences of CDR1,2 and 3 VHand the sequence of CDR1,2 and 3 VLyou can "mix and match" (i.e. you can mix and match CDRs from different antibodies, although each antibody must include the CDR1,2 and 3 VHand CDR1,2 and 3 VLto create other binding molecules against IP-10, corresponding to the invention. The binding of IP-10 data "mixed and matched" antibodies can be tested using the assays of the binding described above and in the Examples (e.g., ELISA). Preferably, when mixed and pick a sequence of CDR of the VHthe sequence of CDR1, CDR2 and/or CDR3 of sequence VHwas replaced structural Blimber is some sequence(s) CDR. Similarly, when mixed and pick a sequence of CDR of the VL, preferably, the sequence of CDR1, CDR2 and/or CDR3 of sequence VLwas replaced with structurally similar sequence(s) CDR. For example, CDR1 VH1D4, E, 2G1, A, A, V, S, 8F6, A and 10A12S have some structural proximity and, consequently, ideal for mixing and matching, whereas CDR1 VH3C4 and S are not structurally similar CDR1 VH1D4, E, 2G1, A, A, V, S, 8F6, A and 10A12S and, thus, they should not be mix and match with them. Ordinary competent specialist will be easy to see that the new sequence VHand VLcan be created by replacing one or more sequences of plots CDR VHand/or VLstructurally similar sequences from the CDR sequences described in this context for monoclonal antibody 1D4, E, 2G1, 3C4, A, A, V, S, 8F6, A and S.

Accordingly, in another aspect, the invention is selected monoclonal antibody or its antigennegative part, including:

(a) CDR1 variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:1-12;

(b) CDR2 variable region heavy chain comprising an amino acid sequence that is selected from the group consisting of SEQ ID NONO:13-23;

(c) CDR3 variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:24-34;

(d) CDR1 variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:51-61;

(e) a CDR2 variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:62-72; and

(f) a CDR3 variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:73-83;

where the antibody specifically binds to IP-10.

In a preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:1;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:13;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:24;

(d) CDR1 variable region light chain comprising SEQ ID NO:51;

(e) a CDR2 variable region light chain comprising SEQ ID NO:62; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:73.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:2;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:14;

(c) the CDR3 of the variable region of the heavy chain consisting of SEQID NO:25;

(d) CDR1 variable region light chain comprising SEQ ID NO:52;

(e) a CDR2 variable region light chain comprising SEQ ID NO:63; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:74.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:3;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:15;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:26;

(d) CDR1 variable region light chain comprising SEQ ID NO:53;

(e) a CDR2 variable region light chain comprising SEQ ID NO:64; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:75.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:4;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:16;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:27;

(d) CDR1 variable region light chain comprising SEQ ID NO:54;

(e) a CDR2 variable region light chain comprising SEQ ID NO:65; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:76.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:5;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:17;

(c) CR3 variable regions of the heavy chain, comprising SEQ ID NO:28;

(d) CDR1 variable region light chain comprising SEQ ID NO:55;

(e) a CDR2 variable region light chain comprising SEQ ID NO:66; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:77.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:6;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:18;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:29;

(d) CDR1 variable region light chain comprising SEQ ID NO:56;

(e) a CDR2 variable region light chain comprising SEQ ID NO:67; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:78.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:7;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:19;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:30;

(d) CDR1 variable region light chain comprising SEQ ID NO:57;

(e) a CDR2 variable region light chain comprising SEQ ID NO:68; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:79.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:8;

(b) variable regions CDR2 of the heavy chain, include the s SEQ ID NO:20;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:31;

(d) CDR1 variable region light chain comprising SEQ ID NO:58;

(e) a CDR2 variable region light chain comprising SEQ ID NO:69; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:80.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:9;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:21;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:32;

(d) CDR1 variable region light chain comprising SEQ ID NO:59;

(e) a CDR2 variable region light chain comprising SEQ ID NO:70; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:81.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:10 or 11;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:22;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:33;

(d) CDR1 variable region light chain comprising SEQ ID NO:60;

(e) a CDR2 variable region light chain comprising SEQ ID NO:71; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:82.

In another preferred embodiment, the antibody includes:

(a) CDR1 variable region heavy chain comprising SEQ ID NO:12;

(b) CDR2 variable region heavy chain comprising SEQ ID NO:23;

(c) CDR3 variable region heavy chain comprising SEQ ID NO:34;

(d) CDR1 variable region light chain comprising SEQ ID NO:61;

(e) a CDR2 variable region light chain comprising SEQ ID NO:72; and

(f) a CDR3 variable region light chain comprising SEQ ID NO:83.

Antibodies having the sequence of certain germ-lines

In some embodiments, the antibody corresponding to the invention, includes a variable region of the heavy chain of the gene heavy chain immunoglobulin specific germ line and/or the variable region of the light chain of the gene light chain immunoglobulin specific germ line.

As demonstrated in this context, derived human antibodies specific against IP-10, which include the variable region of the heavy chain, which is a product of or comes from a gene of the human germline VH 3-33, gene VH 3-30 .3, gene VH 5-51 gene VH 4-61. Accordingly, the invention is selected monoclonal antibody or its antigennegative part, the antibody comprises the variable region of the heavy chain, which is a product of or comes from a gene of the human germline VH selected from the group consisting of: VH 3-33, VH 3-30 .3, VH 5-51 and VH 4-61. Prefer the Ino, when antibody specific polypeptide IP-10 person (for example, including the sequence of Genbank Registration No. NP_001556).

In addition, as demonstrated in this context, derived human antibodies specific against IP-10, which include the variable region of light chain, which is a product of or comes from a gene of the human germline Vk A27 gene Vk L15, gen Vk L6 gene Vk L18. Accordingly, the invention is selected monoclonal antibody or its antigennegative part, the antibody comprises the variable region of light chain, which is a product of or comes from a gene of the human germline Vk selected from the group consisting of: VkA27, Vk L15, Vk and Vk L6 L18. Preferably, when the antibody specific polypeptide IP-10 person (for example, including the sequence of Genbank Registration No. NP_001556).

Preferred antibodies corresponding to the invention, represented by antibody comprising the variable region of the heavy chain, which is a product of or comes from one of the above genes in the human germline VH and comprising the variable region of light chain, which is a product of or derives from one of these human genes the germline Vk. Accordingly, in another embodiment, the invention is selected monoclonal antibody or its antigennegative part, the antibody includes:

(a) the variable region of the heavy chain, which is a product of or comes from a gene of the human germline VH selected from the group consisting of: VH 3-33, VH 3-30 .3, VH 5-51 and VH 4-61; and

(b) variable region light chain, which is a product of or comes from a gene of the human germline Vk selected from the group consisting of: Vk A27, Vk L15, Vk and Vk L6 L18. Preferably, when the antibody is specific against polypeptide IP-10 person (for example, containing a sequence of Genbank registration No. NP_001556).

The invention also presents antibodies, including preferred combinations of variable regions of the heavy and light chains that represent a product or genes isolated from human germline VH and Vk. For example, in a preferred embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody:

(a) includes the variable region of the heavy chain, which is a product of or derives from gene VH 3-33 person (which encodes the amino acid sequence shown in SEQ I NO:47);

(b) includes the variable region of light chain, which is a product of or derives from A27 gene, L15 or L6 Vk person (which encodes the amino acid sequence shown in SEQ ID NONO:95, 98 and 97, respectively); and

(c) specifically binds to IP-10.

In one embodiment, the antibody comprises the variable region of light chain, which is a product of or derives from gene Vk A27 person. Examples of antibodies having VHand VKVH 3-33 and Vk A27, respectively, include 1D4, 2G1, A, A, A and 10A12S. In another embodiment, the antibody comprises the variable region of light chain, which is a product of or derives from gene Vk L15 person. An example of an antibody having the VHand VKVH 3-33 and Vk L15, respectively, is S. In another embodiment, the antibody comprises the variable region light chain gene Vk L6 person. An example of an antibody having the VHand VKVH 3-33 and Vk L6, respectively, is E.

In another preferred embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody:

(a) includes the variable region of the heavy chain gene VH 3-30 .3 person (which encodes the amino acid sequence shown in SEQ ID NO:48) or separated from it;

(b) who engages variable region light chain gene Vk L6 person (which encodes the amino acid sequence, shown in SEQ ID NO:96) or separated from it; and

(c) specifically binds to IP-10.

Examples of antibodies having VHand VKVH 3-30 .3 and Vk L6, respectively, include V and 8F6.

In another preferred embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody:

(a) includes the variable region of the heavy chain gene VH 5-51 person (which encodes the amino acid sequence shown in SEQ ID NO:49) or separated from it;

(b) includes the variable region of light chain gene Vk L18 person (which encodes the amino acid sequence shown in SEQ ID NO:97) or separated from it; and

(c) specifically binds to IP-10.

An example of an antibody having the VHand VKVH 5-51 and Vk L18, respectively, is S.

In another preferred embodiment, the invention is selected monoclonal antibody or its antigennegative part, and the antibody:

(a) includes the variable region of the heavy chain gene VH 4-61 person (which encodes the amino acid sequence shown in SEQ ID NO:50) or separated from it;

(b) includes the variable region of light chain gene Vk A27 person (which encodes the amino acid sequence shown in SEQ ID NO:95) or separated from it; the

(c) specifically binds to IP-10.

An example of an antibody having the VHand VKVH 4-61 and Vk A27, respectively, is S.

As used in this context, the human antibody comprises variable regions of the heavy and light chain, i.e. the "product" or "isolated from" the sequence of the germ line, if the variable sites of the antibody are obtained from a system in which use genes of human immunoglobulin germline. These systems include the immunization of transgenic mice carrying the genes of the human immunoglobulin, antigen of interest, or the screening of libraries of genes of the human immunoglobulin presented on the phage, using the antigen of interest. Human antibody, i.e. the "product"or "separated from" sequence of human immunoglobulin germline, can be identified as such by comparing the amino acid sequence of human antibodies with the amino acid sequence of human immunoglobulin germline and selection of the sequence of human immunoglobulin germ line, which is closest in sequence (i.e. with the highest % identity) to the sequence of the human antibody. A human antibody that isone "product", or selected from the sequences of immunoglobulin a particular human germline, may contain amino acid differences compared to the germline sequence, due to, for example, natural somatic mutations or targeted introduction of site-directed mutations. However, the selected human antibody, typically, amino acid sequence at least 90% identical to the amino acid sequence encoded by the genome of human immunoglobulin germline, and contains amino acid residues that identify the human antibody as being human when compared with the amino acid sequence of the immunoglobulin germline of other species (e.g., sequences of mouse germ line). In some cases, the human antibody sequence can be at least 95%, or even at least 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by the gene of the immunoglobulin germ line. Typically, a human antibody, selected from the sequence of human germline will discover no more than 10 amino acid differences from the amino acid sequence encoded by the genome of human immunoglobulin germ line is. In some cases, the human antibody can detect no more than 5, or even no more than 4, 3, 2 or 1 amino acid difference from the amino acid sequence encoded by the genome of human immunoglobulin germline.

Homologous antibodies

In yet another embodiment, the antibody corresponding to the invention, includes a variable region heavy and light chain containing amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described in this context, and thus the antibodies retain the desired functional properties of antibodies against IP-10, corresponding to the invention.

For example, the invention is selected monoclonal antibody or its antigennegative portion comprising the variable region of the heavy chain and the variable region of light chain, and:

(a) the variable region of the heavy chain comprises an amino acid sequence that is at least 80% homologous amino acid sequence selected from the group consisting of SEQ ID NONO:35-46;

(b) the variable region of the light chain comprises an amino acid sequence that is at least 80% homologous amino acid sequence selected from the group consisting of SEQ ID NONO:84-94;

(c) the antibody specifically svyazyvaete the IP-10 and (a) antibody demonstrates at least one of the following functional properties:

(i) the antibody inhibits the binding of IP-10 to CXCR3;

(ii) the antibody inhibits induced IP-10 current calcium;

(iii) the antibody inhibits induced IP-10 migration of cells;

(iv) the antibody is cross-reactive with IP-10 rhesus macaques;

(v) the antibody is not cross-reactive with mouse IP-10;

(vi) the antibody is not cross-reactive with human MIG;

(vii) the antibody is not cross-reactive with human ITAC. In various embodiments, the implementation of the antibody may exhibit one or more, two or more, three or more, four or more, five or more, or six or more functional properties that are listed as paragraphs (d)to(j) above. The antibody can be, for example, a human antibody, humanized antibody or chimeric antibody.

In other embodiments, implementation, amino acid sequence of the VHand/or VLcan be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequences. The antibody with the region VHand VLhaving high (i.e. 80% or greater) homology with regions VHand VLSEQ ID NONO:35-46 and 84-94, respectively, can be obtained by mutagenesis (e.g., site-directed or PCR (polymerase chain reaction)-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID NONO:35-46 and/or 84-94, is followed by testing of the encoded altered antibody for retained function (i.e., of the functions listed in paragraphs (c)through(j) above) using the functional assays described in this context.

As used in this context, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions available sequences (i.e.,% homology=number of identical positions/total number of positions×100), taking into account the number of gaps (gaps) and the length of each gap, which need to be entered for optimal alignment two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in non-limiting examples below.

The percent identity between two amino acid sequences can be determined using the algorithm proposed by E. Meyers and W. Miller (Comput see. Appl. Biosci., 4:11-17, (1988)), which is introduced into the ALIGN program (version 2.0) using the table, the weight of the residue of the FRAMES 120, the penalty for the length of the gap 12 and the penalty for gap 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (see J. Mol. Biol., 48:444-453, (1970)), which is first introduced into the GAP program in the GCG software package (available at http://www.gcg.com using matrix Blossum 62 matrix RAM and weight of the gaps 16, 14, 12, 10, 8, 6, or 4 and weight length 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequence corresponding to the present invention, furthermore, can be used as sequence-query" to search the public databases with the purpose of, for example, identify related sequences. This search can be done using the XBLAST program (version 2.0), proposed article Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A search of protein in the BLAST program can be done using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous antibody molecules corresponding to the invention. To obtain alignments with gaps for comparison purposes, you can use the program Gapped BLAST, as described in Altschul et al., (1997), Nucleic acids Res., 25(17):3389-3402. When using the programs BLAST and Gapped BLAST, you can use the default settings of the respective programs (e.g., XBLAST and NBLAST). Cm. http://www.ncbi.nlm.nih.gov.

Antibodies with conservative modifications

In some embodiments, the implementation of the antibody corresponding to the invention, includes a variable region heavy chain comprising sequences of CDR1, CDR2 and CDR3, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3, where one or more of the serial data is of Inesta CDR includes the sequence of amino acids, based on the preferred antibodies described in this context (for example, 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S), or conservative modifications and where the antibodies retain the desired functional properties of antibodies against IP-10, corresponding to the invention.

Accordingly, the invention is selected monoclonal antibody or its antigennegative part, including variable region heavy chain comprising sequences of CDR1, CDR2 and CDR3 and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3, and:

(a) the sequence of CDR3 of the variable region of the heavy chain comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:24-34 and conservative modifications;

(b) sequence of CDR3 of the variable region of the light chain comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NONO:73-83 and conservative modifications;

(c) the antibody specifically binds to IP-10 and (a) the antibody exhibits at least one of the following functional properties:

(i) the antibody inhibits the binding of IP-10 to CXCR3;

(ii) the antibody inhibits induced IP-10 current calcium;

(iii) the antibody inhibits induced IP-10 migration of cells;

(iv) ntitle is cross-reactive with IP-10 rhesus macaques;

(v) the antibody is not Perekrest about-reactive with mouse IP-10;

(vi) the antibody is not cross-reactive with human MIG;

(vii) the antibody is not cross-reactive with human ITAC.

In a preferred embodiment, the sequence With DR2 variable regions of the heavy chain comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:13-23 and conservative modifications; and the CDR2 sequence of the variable region of the light chain comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:62-72 and conservative modifications. In another preferred embodiment, the sequence of CDR3 of the variable region of the heavy chain comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:1-12 and conservative modifications; and the CDR3 sequence of the variable region of the light chain comprises an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NONO:51-61 and their conservative modifications.

In various embodiments, the implementation of the antibody may exhibit one or more, two or more, three or more, four or more, five or more, or W is here or more functional properties, listed as paragraphs (d)to(j) above. These antibodies can be, for example, human antibodies, humanitarianism antibodies or chimeric antibodies.

As used in this context, the term "conservative modification sequence" is intended to refer to modifications of amino acids, which does not substantially affect or do not alter the binding characteristics of the antibody containing the amino acid sequence. Data conservative modifications include substitutions, additions, and deletions of amino acids. Modifications can be introduced into the antibody corresponding to the invention, standard techniques known in the art such as site-directed mutagene and PCR-mediated mutagenesis. Conservative substitutions of amino acids are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the field of technology. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic-basic circuits (for example, aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, series, threonine, tyrosine, cysteine, tryptophan), nonpolar side zip pocket wit and circuits (for example, alanine, valine, leucine, isoleucine, Proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more of amino acid residues at sites CDR of the antibody corresponding to the invention, can be substituted by other amino acid residues from the same family of side chains, and the altered antibody can be tested for retained function (i.e., the features listed in paragraphs (c)through(j) above) using the functional assays described in this context.

Antibodies that bind to the same epitope. what antibodies against IP-10, corresponding to the invention

In another embodiment, the invention provides antibodies that bind to the same epitope that different antibodies against IP-10, corresponding to the invention, represented in this context, such as other human antibodies that bind to the same epitope that an antibody 1D4, E, 2G1, 3C4, A, A, V, S, 8F6, A, 10A12S or S described in this context.

These additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of a statistically significant manner) with other the antibodies, corresponding to the invention, such as 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S in standard analyses of the binding.

The ability of a test antibody to inhibit the binding of, for example, 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S with human IP-10 demonstrates that the test antibody can compete for binding to human IP-10; this antibody may, according to non-limiting theory, to communicate with the same or a related (e.g., structurally close or spatially proximal) epitope of human IP-10, as the antibody with which it competes.

In a preferred embodiment, the antibody that binds to the same epitope on human IP-10, which 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S, is a human monoclonal antibody. Data of the human monoclonal antibodies can be obtained, and to select, as described in the Examples.

Designed and modified antibodies

In addition, the antibody corresponding to the invention can be obtained when using an antibody having one or more sequences of VHand/or VLdescribed in this context as the starting material for constructing a modified antibody, which modified antibody may have altered properties relative what about the original antibody. The antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VHand/or VL), for example, on one or more parcels of CDR and/or in one or more skeletal regions. Additionally or alternatively, the antibody can be constructed by modification of residues in the constant region(s), for example, to change the effector function(s) of the antibody.

One type of design variable regions, which may be implemented, is the grafting of CDR. Antibodies interact with antigens on target mainly through residues of amino acids, which are the six parts of the definition of complementarity heavy and light chain (CDR). For this reason, the sequence of amino acids in the CDR more diverse the individual antibodies than the sequence outside of the CDR. Because the sequences of CDRs are responsible for most interactions, antibody-antigen, it is possible to Express recombinant antibodies that mimic the properties of specific natural antibodies by constructing expression vectors that include CDR sequence of the specific natural antibodies grafted skeletal sequences from a different antibody with excellent properties (see, for example, articles Riechmann L, et al., (1998), Nature, 332:323-327; Jones, P. et al., (1986),Nature, 321:522-525; Queen, C. et al., (1989), Proc. Natl. Acad. Sci. U.S.A., 86:10029-10033; U.S. Patent No. 5225539 issued Winter and U.S. Patent NoNo 5530101; 5585089; 5693762 and 6180370 issued by Queen et al.).

Accordingly, another variant embodiment of the invention relates to the selected monoclonal antibody or its antigennegative part comprising the variable region of the heavy chain comprising sequences of CDR1, CDR2 and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:1-12, SEQ ID NONO:13-23 and SEQ ID NONO:24-34, respectively, and a variable region light chain comprising sequences of CDR1, CDR2 and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NONO:51-61, SEQ ID NONO:62-72 and SEQ ID NONO:73-83, respectively. Thus, these antibodies contain sequences of CDR VHand VLmonoclonal antibody 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S, although may contain various skeletal sequences from these antibodies.

Data skeletal sequence can be obtained from public DNA databases or published references that include the gene sequence of the antibody germ line. For example, the DNA sequence of a germ-line gene variable regions of heavy and light chains can be found in the database sequences of the human germline "VBase" (up to the available on the Internet www.mrc-cpe.cam.ac.uk/vbase), and in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest (Sequences of proteins representing immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson I.M., et al., (1992), "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops" (the Repertoire of sequences VHhuman germline reveals about fifty groups of segments VHwith different hypervariable loops) J. Mol. Biol., 227:776-798; and Mor J.P.L. et al., (1994), "A Directory of Human Germline VH Segments Reveals a Strong Bias in their Usage" (Pointer segments VHhuman germ line indicates an important direction in their application), Eur. J. Immunol., 24:827-836, the contents of each of which are specifically included in this context by reference.

Preferred scaffold sequence for use in the antibodies corresponding to the invention are those that are structurally similar skeletal sequences used by selected antibodies, corresponding to the invention, for example, close sequences VH3-33, 3-30 .3, 4-61 or 5-51 [SEQ ID NOS:47-50], and/or skeletal sequences VkA27, L6, L18 or L15 [SEQ ID NOS:95-98], used in the preferred monoclonal antibodies, corresponding to the invention. The sequence of CDR1, 2 and 3 VHand the sequence of CDR1, 2 and 3 VLyou can graft on scale the data area, which have a sequence identical to that found in the gene for the immunoglobulin germ line, from which there is a skeletal sequence, or sequences of CDRs can be grafted onto the skeletal areas that contain one or more mutations compared to the germline sequences. For example, it is shown that in some cases favorably subjected to mutations in the skeletal remains of the areas with the aim of maintaining or enhancing antigennegative the ability of antibodies (see, for example, U.S. Patents NoNo 5530101, 5585089, 5693762 and 6180370 issued by Queen et al).

Another type of modification of the variable regions is matirovanie of amino acid residues at sites CDR 1, CDR2 and/or CDR3 VHand/or VLin order, therefore, to improve one or more binding characteristics (e.g., affinity) of the antibodies of interest. For introduction of mutation(s) can be site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on the binding of an antibody or other functional property of interest, can be evaluated in in vitro or in vivo, as described in this context and are presented in the Examples. Preferably, when being introduced conservative modifications (as described above). The mutation can be a substitution, addition or deletion of amino acids, FAV is preferably represent a replacement. Moreover, as a rule, change no more than one, two, three, four or five residues.

Accordingly, in another embodiment, the invention is selected monoclonal antibodies against IP-10 or antihistamie part comprising the variable region of the heavy chain, comprising: (a) the site CDR1 VHcomprising an amino acid sequence selected from the group consisting of SEQ ID NONO:1-12, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids compared to SEQ ID NONO:1-12; (b) plot CDR2 VHcomprising an amino acid sequence selected from the group consisting of SEQ ID NONO:13-23, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids compared to SEQ ID NONO:13-23; (C) plot CDR3 VHcomprising an amino acid sequence selected from the group consisting of SEQ ID NONO:24-34, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids compared to SEQ ID NONO:24-34; (d) plot CDR1 VLcomprising an amino acid sequence selected from the group consisting of SEQ ID NONO:51-61, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids comparedwith SEQ ID NONO:51-61; (e) plot CDR2 VLcomprising an amino acid sequence selected from the group consisting of SEQ ID NONO:62-72, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids compared to SEQ ID NONO:62-72; and (f) plot CDR3 VLcomprising an amino acid sequence selected from the group consisting of SEQ ID NONO:73-83, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids compared to SEQ ID NONO:73-83.

Preferred replacement mutation, corresponding to the invention is the replacement of the serine residue by a cysteine residue in position 32 in chain CDR1 VHmAb (monoclonal antibodies) A. This modified form of A mean in this context 10A12S. The amino acid sequence of chain VHA shown in SEQ ID NO:44, and the amino acid sequence of chain VH10A12S shown in SEQ ID NO:45.

Engineered antibodies, corresponding to the invention include those in which modifications in skeletal remains in the VHand/or VLfor example, to improve the characteristics of the antibody. Typically, these skeletal modifications done in order to reduce the immunogenicity of the antibody. For example, one approach is to "reverse matirovanie" one or more skeletal remains in the meet the General sequence of the germ line. More specifically, the antibody, which was subjected to somatic mutations may include skeletal remains, which differ from the germline sequence from which the selected antibody. These balances can be identified by comparing skeletal sequences of the antibodies with the germline sequences from which the selected antibody. For example, for A the balance of amino acids No 2 (FR1) VHrepresents methionine, whereas this residue in the corresponding VHsequence 3-33 germline is a valine (see Figure 23). In order to return a sequence of skeletal region in the configuration of their germ line and somatic mutations can be subject to a "reverse matirovanie" to the germline sequence by, for example, site-directed mutagenesis or PCR-oposredovannogo mutagenesis (e.g., remainder 2 VHA can be "reverse mutation from methionine to valine). It is envisaged that the invention also covers these antibodies, subject to a "reverse mutation".

Another type of skeletal modification includes matirovanie one or more residues in skeletal area or even on one or more parcels CDR in order to remove T-cell epitopes, in order thus to reduce potentialyno the immunogenicity of the antibody. This approach is also called "daimonizomai", and he described in more detail in Patent publication U.S. No. 20030153043 filed Carr et al.

Dopolnenie or alternative to modifications made in the skeletal areas or in areas CDR, antibody, corresponding to the invention can be constructed so that they include modifications in the Fc region, typically to modify one or more of the functional characteristics of the antibodies, such as the period of existence in the serum, the binding of complement fixation, Fc receptor binding and/or an antigen of cellular cytotoxicity. Moreover, the antibody corresponding to the invention, can be modified by chemical means (for example, the antibody can be attached one or more chemical groups) or modified to change its glycosylation also to change one or more of the functional characteristics of the antibody. Each of these embodiments is described in more detail below. The numbering of the residues in the Fc region such as in the index Kabat EU.

In one embodiment, the hinge area SN modified so that changed the number of cysteine residues in the hinge region, for example, reduced or increased. This approach is described further in U.S. Patent No. 5677425 issued Bodmeret al. The number of cysteine residues in the hinge region SN treason is about to so, for example, facilitate Assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the hinge region of the Fc antibody is subjected to mutation to reduce the biological half-period of the existence of antibodies. In more detail, one or more mutations of the amino acids being introduced into the region of the domain partition CH2-CH3 hinge Fc fragment, so that the antibody has impaired binding of staphylococcal protein A (SpA) relative to the binding of the native domain of the Fc hinge SpA. This approach is described in more detail in U.S. Patent No. 6165745, issued to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life existence. There are different approaches. For example, you can introductionat one or more of the following mutations: T252L, T254S, T256F, as described in U.S. Patent No. 6277375 issued by the Ward. An alternative for increasing the biological half-time of existence, the antibody can be changed in the field CN or CL so that it contained the epitope binding utilizing receptor, taken from two loops of a CH2 domain of the Fc region of IgG, as described in U.S. Patent NoNo. 5869046 and 6121022 issued by Presta et al.

In the following embodiments, the implementation of the Fc region change by substitution of at least one amino acid residue by another amino acid residue is you to change the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with another amino acid residue such that the antibody has an altered affinity towards effector ligand, but retains antigennegative the ability of the parent antibody. Ligand effector, which changed the affinity may be, for example, Fc receptor or a component of complement C1. This approach is described in more detail in U.S. Patent NoNo 5624821 and 5648260, both of which issued by Winter et al. In another example, one or more amino acids, selected amino acid residues 329, 331 and 322 can be replaced with another amino acid residue so that the antibody had altered C1q binding and/or reduced or eliminated in the presence of complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No 6194551 issued Idusogie et al.

In another case, one or more of amino acid residues in positions of amino acids 231 and 239 are changed to thereby change the capacity of antibodies to bind complement. This approach is described in detail in PCT publication WO 94/29351 filed Bodmer et al.

In one case, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody to the receptor Fey by modificati the one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439.

This approach is described further in PCT Publication WO 00/42072 filed Presta. Moreover, the mapped binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn and described variants with improved binding (see Shields R.L.et al., (2001), J. Biol. Chem., 276:6591-6604). It is shown that specific mutations at positions 256, 290, 298, 333, 334 and 339 improve binding to FcγRIII. In addition, it is shown that the following combination mutants improves binding to FcγRIII: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In another embodiment, modifying the glycosylation of the antibody. For example, you can get deglycosylated antibody (i.e., the antibody does not have glycosylation). Glycosylation can be changed to, for example, to increase the affinity of the antibody to the antigen. Data modification carbohydrates can be done, for example, by modifying one or more centers of glycosylation sequence of the antibody. For example, you can make one or more substitutions of amino acids that will result in the elimination of one or more centers of glycosylation skeletal parts of the variable regions, so as to eliminate glycosylation Yes in the EBM center. This deglycosylation can increase the affinity of the antibody to the antigen. This approach is described in more detail in U.S. Patent NoNo 5714350 and 6350861 issued by et al.

Additionally or alternatively, to obtain an antibody that has an altered type of glycosylation, such as hepatocanalicular antibody with reduced quantities of residues fucosyl or antibody, having the structure of GlcNac with a high level of branching. These types of altered glycosylation, as shown, to increase ADCC ability of antibodies. Such modifications of carbohydrates can be done, for example, by expression of antibodies in the cell host with a modified mechanism of glycosylation. Cells with a modified mechanism of glycosylation are described in the prior art and can be used as host cells for expression of recombinant antibodies, corresponding to the invention, in order thus to obtain an antibody with altered glycosylation. For example, in EP 1176195 filed Hanai et al., described cell line with a functionally destroyed FUT8 gene, which encodes fucosyltransferase, so that antibodies expressed in this cell line, demonstrating hypoglycosylated. In PCT Publication WO 03/035835 filed Presta, describes a variant Cho cell line, Lec13 cells with reduced ability to attach fucose to Asn(297)-linked from the Levada, that also leads to hepatocanalicular antibodies, ExpressionEngine the host-cell (see also Shields R.L et al., (2002), J. Biol. Chern., 277:26733-26740). In PCT Publication WO 99/54342 filed Umana et al. describes cell lines engineered to Express glycoprotein-modifying glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), so that antibodies expressed in the engineered cell lines exhibit increased branching structures GlcNac, which results in increased ADCC activity of the antibodies (see also Umana et al., (1999), Nat. Biotech., 17:176-180).

Another modification of the antibodies in this context that the invention is tahilramani. The antibody can be PageLayout, for example, to increase biological (e.g., serum) half of the existence of antibodies. In order to PageLayout antibody, as a rule, carried out the reaction of the antibody or its fragment with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to antibody or antibody fragment. Preferably, when tahilramani carry out the acylation reaction or an alkylation reaction with a reactive PEG molecules (or similar reaction is an ionic water-soluble polymer). As used in this context, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used for derivatization of other proteins, such as mono(C1-C10)-alkoxy or aryloxyalkyl or polyethylene glycol-maleimide. In some embodiments, the antibody is intended for tahilramani is deglycosylated antibody. Methods tahilramani proteins known in the technical field and can be applied to the antibodies corresponding to the invention. See, for example, EP 0 154 316, filed Nishimura et al. and the EP ON 401 384, filed Ishikawa et al.

The methods of constructing antibodies

As discussed above, antibodies against IP-10, with a sequence VHand VLdescribed in this context, can be used to create new antibodies against IP-10 by modifying the sequences VHand/or VLor attached thereto a constant region(s). Thus, in another aspect of the invention the structural characteristics of the antibodies against IP-10, corresponding to the invention, for example, 1D4, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S, are used to create structurally close antibodies against IP-10, which retain at least one functional property of the antibodies corresponding to the invention, such as binding to human IP-10 and IP-10 poppy and-RH, but not murine IP-10 or human MIG or human ITAC, as well as the inhibition of one or more functional properties of IP-10 (e.g., binding to CXCR3, current calcium, chemotaxis). For example, one or more sections 1D4 CDR, E, 2G1, S, A, A, V, S, 8F6, A, 10A12S or S or mutations can be combined by recombinant with known skeletal sites and/or other CDRs to create additional constructed by recombinant antibodies against IP-10, corresponding to the invention, as discussed above. Other types of modifications include those described in the previous section. The source material for fashion design are one or more of the sequences VHand/or VLin this context, or one or more of their plots CDR. To create the engineered antibody is not really necessary to get (i.e. to Express in the form of protein) antibody having one or more of the sequences VHand/or VLin this context, or one or more of their plots CDR. More precisely, the information contained in the sequence(s), used as starting material to generate the sequence(s) "second generation", derived from the original sequence(s), and then get a sequence(s) "vtoro what about the generation and Express in the form of protein.

Accordingly, in another embodiment, the invention provides a method of obtaining antibodies against IP-10, including:

(a) receiving (i) sequence variable regions of heavy chain antibodies, comprising a sequence of CDR 1, selected from the group consisting of SEQ ID NONO:1-12, a CDR2 sequence selected from the group consisting of SEQ ID NONO:13-23, and/or a CDR3 sequence selected from the group consisting of SEQ ID NONO:24-34; and (ii) the sequence of the variable region of the light chain of the antibody comprising the CDR1 sequence selected from the group consisting of SEQ ID NONO:51-61, a CDR2 sequence selected from the group consisting of SEQ ID NONO:62-72, and/or a CDR3 sequence selected from the group consisting of SEQ ID NONO:73-83;

(b) modifying at least one amino acid residue in the sequence of variable regions of heavy chain antibodies and/or sequence of the variable region of the light chain of the antibody to create at least one altered sequences of the antibodies; and

(C) the expression of the modified sequence of the antibody in the form of protein.

To obtain and expression of the modified sequence of the antibody can be used standard molecular biology techniques.

Preferably, when the antibody encoded by the altered sequence(s) antibodies, p is ecstasy an antibody, retains one, some or all of the functional properties of antibodies against IP-10, is described in this context, with these functional properties include, but are not limited to:

(i) specifically binds to human IP-10;

(ii) inhibits the binding of IP-10 to CXCR3;

(iii) inhibits induced IP-10 current calcium;

(iv) inhibits induced IP-10 migration of cells;

(v) cross-reacts with IP-10 rhesus macaques;

(vi) is not cross-reactive with mouse IP-10;

(vii) is not cross-reactive with human MIG; and (viii) is not cross-reactive with human ITAC. The modified antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or seven or more functional properties, which are listed as items (i)to(viii) above.

Functional properties of the modified antibodies can be assessed using standard assays available in the field of engineering and/or described in this context, such as shown in the Examples (e.g., ELISA, analyses of current calcium, analysis of chemotaxis).

In some embodiments, the implementation of the methods of constructing antibodies, corresponding to the invention, mutations can be randomly or selectively introduced for all or part of the sequence, code the her antibody against IP-10, and the resulting modified antibodies against IP-10 can be subjected to screening for binding activity and/or other functional properties as described in this context. Mutational methods described in the prior art. For example, in PCT Publication WO 02/092780 submitted Short, describes methods for creating and screening of mutations antibodies using saturating mutagenesis, Assembly by ligating synthetic or combinations thereof. Alternative in PCT Publication WO 03/074679 Lazar et al. describe how the use of computerized methods of screening to optimize the physico-chemical properties of antibodies.

Molecules of nucleic acids encoding antibodies, corresponding to the invention

Another aspect of the invention relates to nucleic acid molecules that encode antibodies that correspond to the invention. The nucleic acid may be present in whole cells, cell lysate, or in a partially or substantially pure form. Nucleic acid is "isolated" or "received almost pure"when it is purified from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS (sodium dodecyl sulfate), the separation of the bands in CsCl, column chromatography, electrophoresis in agarose is barely and others, known in the art. Cm. the monograph edited by F. Ausubel et al., (1987), Current Protocols in Molecular Biology (Modern methods in molecular biology), Greene Publishing and Wiley Interscience, New York. Nucleic acid corresponding to the invention may be represented, for example, DNA or RNA, and may contain or not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids corresponding to the invention, can be obtained using standard molecular biological techniques. For antibodies expressed by hybridomas (e.g., hybridomas derived from transgenic mice carrying the genes of human immunoglobulin, as described in detail below), cDNA encoding the light and heavy chains of the antibodies created by hybridoma, can be obtained by standard methods of PCR amplification or cDNA cloning. For antibodies, obtained from the library of immunoglobulin genes (for example, when using the techniques of performance on phage), nucleic acid encoding the antibody can be obtained from the library.

Preferred nucleic acid molecules corresponding to the invention are molecules encoding sequence VHand VLmonoclonal antibody 1D4, E, 2G1, S, A, A, V, S, 8F6, A or S. The DNA sequence encoding the sequence VH1D4, E, 2G1, S, A, A, V, S, 8F6, A and C presented in SEQ ID NONO:99-109, respectively. The DNA sequence encoding the sequence VL1D4, E, 2G1, S, A, A, V, S, 8F6, A and C presented in SEQ ID NONO:110-120, respectively.

After receiving the DNA fragments coding segments Vkand VLwith these DNA fragments can be carried out further manipulations using standard recombinant DNA technologies, for example, with the aim of transforming genes of variable region genes in chain antibodies full-length genes of fragment Fab or scFv gene.

In these manipulations, a DNA fragment encoding the VLor VHfunctionally linked to another DNA fragment, encoding another protein, such as a constant region of the antibody or a flexible linker. The term "functionally linked", as used in this context, is intended to denote that two fragments of DNA are connected so that the amino acid sequence encoded by the two DNA fragments remain in the frame are read.

The selected DNA encoding the V regionHcan be turned into a gene of the heavy chain of the full length through the functional binding of the DNA encoding the VHon the other DNA molecule that encodes a constant region of the heavy chain (CN, CH2 and CH3). P the coherence gene constant region of the heavy chain of the man known in the art (see, for example, the work of Kabat, E. A. et al., (1991), Sequences of Proteins of Immunological Interest (Sequences of proteins representing immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these areas can be obtained using standard PCR amplification. The constant region of the heavy chain may be represented by a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but most preferably is a constant region is IgG1 or IgG4. For gene fragment Fab heavy chain DNA, VH-coding DNA may be functionally linked to another DNA molecule encoding only the constant region SN heavy chain.

The selected DNA encoding the V regionkcan be turned into a gene light chain of the full length (and also in gene Fab light chain) using the functional binding of the DNA encoding the Vkon the other DNA molecule that encodes a constant region of light chain CL. The gene sequence of the constant region of the light chain of a man known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest (Sequences of proteins representing immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these areas can be obtained using standard PCR amplification.

The constant region is easily the circuit can be represented Kappa - or λ constant region, but most preferably is a Kappa constant region.

To create a scFv gene, the DNA fragments encoding the VHand VLfunctionally svyazivaytes another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3so that the sequence VHand VLit was possible to Express as a contiguous single-chain protein with regions VLand VHconnected with a flexible linker (see, for example, article Bird et al., (1988), Science, 242:423-426; Huston et al., (1988), Proc. Natl. Acad. Sci. USA, 85:5879-5883; McCafferty et al., (1990), Nature, 348:552-554).

Obtaining monoclonal antibodies, corresponding to the invention

Monoclonal antibodies (mAbs)corresponding to the present invention can be achieved by many techniques, including accepted methods for generating monoclonal antibodies, for example, the standard method of hybridization of somatic cells proposed by Kohler and Milstein, (1975), Nature, 256: 495. Although the preferred methods of hybridization of cells, in principle, can be used where other methods of obtaining monoclonal antibodies, e.g., viral or oncogenic transformation of b lymphocytes.

The preferred system using animals to produce the hybrid is system in mice. Getting hybridoma in mice is a very well-developed methodology. Duct the crystals immunization and methods of selection of immunized splenocytes for fusion are known in the art. Also known partners to merge (e.g., cells of mouse myeloma), and how the merger.

Chimeric or humanized antibodies, corresponding to the present invention, can be obtained based on a series of murine monoclonal antibodies, obtained as described above. DNA encoding the heavy and light chain immunoglobulin, can be obtained from mouse hybridoma of interest, and to construct, to include sequence Nemchinova (e.g., human) immunoglobulin, using standard molecular biological techniques. For example, to create a chimeric antibody is a murine variable regions can be linked to human constant regions using methods known in the art (see, for example, U.S. Patent No. 4816567 issued by Cabilly et al.). To create gumanitarnogo antibodies murine areas CDR can be introduced into the human skeleton using methods known in the art (see, for example, U.S. Patent No. 5,225,539, issued Winter, and U.S. Patent NoNo. 5530101, 5585089, 5693762 and 6180370 issued by Queen et al.).

In a preferred embodiment, antibodies, corresponding to the invention are human monoclonal antibodies. Data of human monoclonal antibodies against IP-10 may be generating system is to, using transgenic or transamazonic mice carrying parts of the human immune system rather than the mouse system. Data from transgenic or transamazonia mice include mice referred to in this context HuMAb mouse and mouse KM, respectively, and collectively referred to in this context, "mice with human Ig". The HuMAb Mouse® (Medarex, Inc.) is minilogue gene of human immunoglobulin that encode rearranged sequences of the heavy (μ and γ) and Kappa-light chains of human immunoglobulin together with targeted mutations that inactivate the endogenous loci mu - and Kappa-chains (see, for example, article Lonberg et al., (1994), Nature, 368(6474):856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or Kappa, and in response to immunization in the introduced transgenes human heavy and light chains switches class and somatic mutation, aimed at the generation of high-affinity human monoclonal IgG (see article Lonberg, N. et al., (1994), above; review Lonberg N., (1994), Handbook of Experimental Pharmacology (Handbook of experimental pharmacology), 113:49-101; articles Lonberg, N. and Huszar, D., (1995), Intern. Rev. Immunol., 13:65-93, and Harding F. and Lonberg, N., (1995), Ann. N.Y. Acad. Sci., 764:536-546). The preparation and use of HuMab mice, and the genomic modifications, which are mouse data, described in detail in articles L. Taylor et al., (1992) Nucleis Acids Research, 206287-6295; Chen, J. et al., (1993), International Immunology, 5:647-656; Tuaillon et al., (1993), Proc. Natl. Acad. Sci. USA, 90:3720-3724; Choi et al., (1993), Nature Genetics, 4:117-123; J. Chen et al., (1993), EMBO J. 12:821-830; Tuaillon et al., (1994), J. Immunol., 152:2912-2920; Taylor L, et al., (1994), International Immunology, 6:579-591 and Fishwild, D. et al., (1996), Nature Biotechnology, 14:845-851, the contents of all of them so specifically included in this context by reference in their entirety. In addition, see U.S. Patent NoNo 5545806, 5569825, 5625126, 5633425, 5789650, 5877397, 5661016, 5814318, 5874299 and 5770429, all of which issued to Lonberg and KAU; U.S. Patent No. 5545807 issued Suraniet al.; PCT publication NoNo WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all of which are filed Lonberg and KAU, and PCT Publication No. WO 01/14424 filed Korman et al.

In another embodiment, human antibodies, corresponding to the invention, can be obtained by using a mouse that carries a sequence of human immunoglobulin at the transgenes and transpromotional, such as a mouse that carries the transgene, human heavy chain and transfromation human light chain. Data mouse, called in this context "mouse KM", described in detail in PCT Publication WO 02/43478 filed Ishida et al.

In addition, alternative systems of transgenic animals expressing the genes of the human immunoglobulin are available in the prior art and can be used to stimulate the formation of antibodies against IP-10, corresponding to the invention. For example the EP, you can use an alternative transgenic system, called Xenomouse (Abgenix, Inc.), data mouse described, for example, in U.S. Patents NoNo 5939598, 6075181, 6114598, 6150584 and 6162963 issued by Kucherlapati et al.

Moreover, alternative systems transamazonic animals expressing genes of the human immunoglobulin are available in the prior art and can be used to stimulate the formation of antibodies against IP-10, corresponding to the invention. For example, you can use the mice carrying both transfromation human heavy chain and transfromation human light chain, called "mouse TS, mouse data is described in the article Tomizuka et al., (2000), Proc. Natl. Acad. Sci. USA, 97:722-727. Furthermore, cows carrying transhumanity human heavy and light chains (see Kuroiwa et al., (2002), Nature Biotechnology, 20:889-894), described in the prior art and can be used to stimulate the formation of antibodies against IP-10, corresponding to the invention.

Human monoclonal antibodies, corresponding to the invention can also be obtained using the methods of presentation in phage for screening libraries of genes of the human immunoglobulin. These methods present on the phage, to isolate human antibodies developed in the prior art. See, for example: U.S. Patent NoNo. 5223409, 5403484 and 5571698 issued by Ladner et al., U.S. patents NoNo. 5427908 and 5580717 issued by the Dowr et al., U.S. patents NoNo. 5969108 and 6172197 issued by McCafferty et al. and U.S. Patents NoNo. 5885793, 6521404, 6544731, 6555313, 6582915 and 6593081 issued by Griffiths et al.

Human monoclonal antibodies, corresponding to the invention can also be obtained using SCID mice, in which human immune cells restored so that by immunization can generate a response of a human antibody. Data mouse described, for example, in U.S. Patents NoNo 5476996 and 5698767 issued to Wilson et al.

Immunization of mice with human Ig

When mice with human Ig is used for stimulation of the formation of human antibodies, corresponding to the invention, data of mice can immunize purified or enriched preparation of antigen IP-10 and/or recombinant IP-10 or fused protein IP-10, as described in articles Lonberg, N. et al., (1994), Nature, 368(6474):856-859; Fishwild, D. et al., (1996), Nature Biotechnology, 14:845-851 and PCT Publications WO 98/24884 and WO 01/14424. Preferably, when the age of the mice is 6-16 weeks during the first infusion. For example, for intraperitoneal immunization of mice with human Ig you can use purified or recombinant preparation (5-50 μg) of antigen IP-10.

The detailed methodology for the generation of fully human monoclonal antibodies against IP-10 is described below in Example 1. Accumulated with different antigens experience has shown that transgenic mice react when their original subjected to immunization in wirebrushing (IP) with antigen in complete Freund's adjuvant followed IP immunization week (up to 6) with antigen in incomplete Freund's adjuvant. However, it is also shown that effective other adjuvants, in addition to blockers. In addition, we discovered that the whole cells in the absence of adjuvant are highly immunogenic. The immune response can be monitored in the course of the immunization Protocol using plasma samples obtained at retroorbital selection of blood. The plasma can be investigated using ELISA (as described below), and mice with sufficient titers of human immunoglobulin against IP-10 can be used for fusions. Mice can do intravenous booster injection of antigen for 3 days before the killing and removal of the spleen. Expect for each immunization may be required to spend 2-3 infusions. For each antigen, as a rule, subjected to immunization from 6 to 24 mice. Usually use the two strains, NSO and NSO. In addition, both the transgene, NSO and NSO, can be hybridized with each other in one mouse bearing two different transgene human heavy chain (NSO/NSO).

Generation of hybridomas producing human monoclonal antibodies, corresponding to the invention

To generate hybridomas producing human monoclonal antibodies, corresponding to the invention, it is possible to allocate splenocytes and/or cells of lymph nodes of immunized mice and drain with a suitable line of immortalized cells, such as cleocin the line of murine myeloma. The resulting hybridoma can be subjected to screening for the production of antigen specific antibodies. For example, a single cell suspensions of lymphocytes in the pancreas immunized mice can be fused to one-sixth the number of " non-secretory cells of mouse myeloma RH-Ag8.653 (ATS, CRL 1580) when using 50% PEG. Cells are placed approximately 2×105in a flat-bottomed Cup for micrometrology followed a two-week incubation in selective medium containing 20% fetal clonal serum, 18% air-conditioned environment 653, 5% Origen (IGEN), 4 mm L-glutamine, 1 mm sodium pyruvate, 5 mm HEPES, 0.055 mm 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin and 1X HAT (Sigma; the HAT is added 24 hours after the fusion). Approximately two weeks later, cells can be grown in an environment in which the HAT is replaced by NT. Can then be carried out screening of individual cells using ELISA on human monoclonal antibodies of IgM and IgG. After going to happen extensive growth hybridoma, environment typically explore 10-14 days. Hybridoma, secreting the antibody can be transferred to other cups, again subjected to screening and, if they are still positive for human IgG, monoclonal antibodies, can be subcloned at least twice by limiting the breeding. Stable subclones then can be grown in vitro to generate small amounts of antibody in the medium for tissue culture and characterization.

For purification of human monoclonal antibodies selected hybridoma can be grown in two-liter roller flasks for the purification of monoclonal antibodies. Supernatant can be filtered and concentrated before affinity chromatography with protein a-separate (Pharmacia, Piscataway, N.J.). Erwerbende IgG can be checked using gel electrophoresis and high performance liquid chromatography in order to ensure their purity. The buffer solution can be replaced with PBS (phosphate buffered saline), and the concentration can be determined by OD280 (optical density), using the extinction coefficient of 1.43. You can take an aliquot of the monoclonal antibody and store at -80°C. the Generation of transfection. producing monoclonal antibodies. corresponding to the invention

Antibodies corresponding to the invention can also be obtained in transfectant host cell, using, for example, a combination of recombinant DNA technologies and methods transfection of genes, as is well known in the art (see, for example, article Morrison, S., (1985), Science, 229:1202).

For example, for the expression of antibodies or their fragments DNA encoding the partial or total length l is hkie and heavy chains, can be obtained using standard molecular biological techniques (e.g., PCR amplification or cDNA cloning using hybridoma, which expresses the antibody of interest), and DNA can be introduced into expression vectors so that the genes were functionally related sequences control transcription and translation. In this context, the term "functionally linked" is intended to indicate that the gene antibodies Legerova in the vector so that the sequence control transcription and translation in the vector carry out intended them to function in the regulation of transcription and translation of genes antibodies. Expression vector and a sequence controlling the expression chosen so that they are consistent with the host-cell gene expression. Gene light chain antibody gene and the heavy chain of the antibody can be entered in a separate vector or, more typically, both the gene is introduced into the same expression vector. Genes of antibodies injected into the expression vector by standard methods (for example, ligation comlementary Restrike valuable sites on the fragment of the antibody gene and a vector or ligation of blunt ends in the absence of any restriction sites). Variable region light and heavy chains of the antibodies described in this context,can be used to create gene full length antibodies of any isotype antibodies by their insertion into expression vectors, already encoding the constant region of the heavy chain and the constant region of the light chain of the desired isotype such that the segment VHwas functionally associated with the segment(s) CLin the vector and segment VLwas functionally associated with the segment CLin the vector. Additionally or alternatively the recombinant expression vector can encode a signal peptide that facilitates secretion of the chain of the antibody from the host cell. Gene chain antibodies can be cloned into a vector so that the signal peptide was linked in reading frame with aminocom.com gene chain antibodies. The signal peptide may be a signal peptide immunoglobulin or a heterologous signal peptide (i.e., the signal peptide of nimmanahaeminda protein).

In addition to genes chain antibodies, recombinant expression vectors, corresponding to the invention carry regulatory sequences that control expression of genes chains of the antibody in the cell host. The term "regulatory sequence" includes the incorporation of promoters, enhancers and other elements controlling the expression (e.g., polyadenylation signals)that control the transcription and translation of genes chains of antibodies. Data regulatory sequences are described, for example, in the monograph Goeddel (Gene Expression echnology. Methods in Enzymology 185 (Technology of gene expression. Methods in Enzymology 185), Academic Press, San Diego, CA (1990)). Experts in the field of technology should be borne in mind that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of host cell targeted for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for expression in the cell of a mammal host include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers selected from cytomegalovirus (CMV), Simian virus 40 (SV40), adenovirus (such as adenovirus major late promoter (AdMLP)) and polyoma. Alternatively can be used non-viral regulatory sequences, such as ubiquitine promoter and promoter, β-globin. In addition, the use of regulatory elements composed of sequences from different sources, such as promoter system SRα, which includes the sequence of the early SV40 promoter and the long terminal repeat of the virus T-cell leukemia human type 1 (see Takebe Y. et al., (1988), Mol. Cell. Biol., 8:466-472).

In addition to genes chains of antibodies and regulatory sequences, the recombinant expression, etc) is ry, corresponding to the invention may carry additional sequences, such as sequences that regulate replication of the vector in the cells of the host (for example, originy replication genes and breeding markers. Gene breeding marker facilitates the selection of host cells in which the introduced vector (see, for example, U.S. Patents NoNo. 4399216, 4634665 and 5179017, all of which issued by Axel et al.). For example, as a rule, breeding gene marker causes resistance to drugs, such as 0418, hygromycin or methotrexate, host cells in which the introduced vector. Preferred genes of breeding markers include gene digidrofolatreduktazy (DHFR) (for use in the cells of the host dhfr - breeding/reproduction on background methotrexate) and the neo gene (for selection on the background of G418).

For the expression of light and heavy chain expression vector(s)encoding the heavy and light chains, transferout cell-host using standard techniques. Provide that various forms of the term "transfection" cover a wide range of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic cell host, for example, electroporation, precipitation of calcium phosphate, transfection using DEAE-dextran (diethylaminoethoxy), etc. Although Theo is eticheski it is possible to Express the antibodies, corresponding to the invention, either prokaryotic or eukaryotic cells-hosts, expression of antibodies in eukaryotic cells, and most preferably in mammalian cells-the masters is the most preferred because data eukaryotic cells, and especially mammalian cells are more likely than prokaryotic cells to assemble and secrete a properly laid and immunologically active antibody. It is shown that prokaryotic expression of antibody genes inefficient in terms of production of active antibodies with high yield (see Boss M.A. and C.R. Wood, (1985), Immunology Today 6:12-13).

Preferred mammalian cells-hosts for expression of recombinant antibodies, corresponding to the invention include cells Chinese hamster ovary cells Cho (including cells of the Cho dhfr is described in the article Uriaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used selectively marker DHFR, for example, as described in article R.J.Kaufman and ..Sharp, (1982), Mol. Biol., 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells another preferred expression system is the GS gene expression, described in WO 87/04462, WO 89/01036 and EP 338841. When recombinant expression vectors encoding antibody genes, being introduced into the cells is Lampedusa hosts antibodies are produced by culturing the host cells for a period of time sufficient to provide for the expression of antibodies in the cells of the host or, more preferably, secretion of the antibody into the culture medium in which to grow cells-owners. Antibodies can be isolated from culture medium using standard methods of protein purification.

Characterization of the binding of an antibody to an antigen

Antibodies corresponding to the invention can be tested for binding to IP-10 using, for example, standard ELISA. Briefly, tablets for micrometrology cover purified IP-10 at a concentration of 0.25 μg/ml in PBS and then blocked with 5% bovine serum albumin in PBS. Breeding antibodies (for example, dilution of plasma obtained from immunized IP-10 mice) are added to each well and incubated for 1-2 at 37°C. the Tablets are washed with PBS/tween and then incubated with secondary reagent (e.g., for human antibodies using goat Fc-specific polyclonal reagent against human IgG), conjugated with alkaline phosphatase for 1 hour at 37°C. After washing tablets are the pNPP substrate (1 mg/ml) and analyzed at OD (optical density) 405-650. Preferably, when to use merge mice, which developed the highest titers.

Analysis of ELISA, as described in the above, can also be used to screen for hybridomas that show positive reactivity with the immunogen IP-10. Hybridoma that bind with high affinity with IP-10, subcloning and additionally charactersbut. One clone from each hybridoma, which retains the reactivity of the parental cells (data ELISA), can be selected to obtain the cell Bank of 5-10 vials, stored at -140°C and for the purification of antibodies.

For the purification of antibodies against IP-10 selected hybridoma can be grown in two-liter roller flasks for the purification of monoclonal antibodies. Supernatant can be filtered and concentrated before affinity chromatography using protein a-sepharose (Pharmacia, Piscataway, NJ). Erwerbende IgG can be checked using gel electrophoresis and high performance liquid chromatography to confirm the purity. The buffer solution can be replaced with PBS, and the concentration can be determined by OD280 using the extinction coefficient of 1.43. You can take aliquots monoclonal antibodies and stored at -80°C.

In order to determine contacted if selected monoclonal antibodies against IP-10 with a unique epitopes, each antibody can be biotinylation using commercially available reagents (Pierce, Rockford, IL). Can be conducted competitive research COI is whether the unlabeled monoclonal antibodies and biotinylated monoclonal antibodies using boards for ELISA, covered with IP-10, as described above. Binding of biotinylated mAb can be determined using a probe arrows (streptomycin)-avidin-alkaline phosphatase.

To determine the isotype of purified antibodies can be carried out ELISA isotypes using reagents specific antibodies against a particular isotype. For example, to determine the isotype of the human monoclonal antibodies holes tablets for micrometrology can be coated with 1 μg/ml of the agent against human immunoglobulin overnight at 4°C. After blocking with 1% BSA (bovine serum albumin) conducting the reaction of tablets with 1 μg/ml or less of test monoclonal antibodies or purified control isotypes at room temperature within one or two hours. Then the holes can be carried out the reaction with either human IgG1 or probes conjugated with alkaline phosphatase-specific human IgM. Tablets are and analyze as described above.

Human IgG against IP-10 can be further tested for reactivity with the antigen of IP-10 by Western blotting. In short, IP-10 may be obtained and subjected to polyacrylamide gel electrophoresis using sodium dodecyl sulfate. After electrophoresis the separated antigens transferred to nitrocellulose membranes, blocked with 10% saw the short calf embryos and probe with the test monoclonal antibodies. The binding of human IgG can be determined by using alkaline phosphatase against human IgG and be using tablets substrate BCIP/NBT (Sigma Chem. Co., St. Louis, Mo.).

Immunoconjugate

In another aspect of the present invention is characterized antibody against IP-10 or a fragment conjugated to a therapeutic molecule, such as a cytotoxin, a drug (e.g., an immunosuppressant) or radiotoxins. These conjugates in this context referred to as "immunoconjugate". Immunoconjugate that include one or more cytotoxins called "immunotoxins". A cytotoxin or cytotoxic agent includes any agent that is detrimental (e.g., kills) cells. Examples include Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Therapeutic agents in key are also, for example, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g. mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustin (CCNU), cyclophos the amide, the busulfan, dibromomannitol, streptozotocin, mitomycin C, and CIS-dichlorodiammineplatinum (II) (DDP)cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin and astromicin (AMC)), and antimitoticescoe agents (e.g. vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated with the antibody corresponding to the invention, include duocarmycin, calicheamicin, maytansine and auristatin and their derivatives. An example of a conjugate calicheamicin-antibody is commercially available Mylotarg™; Wyeth-Ayerst.

Cytotoxins can be conjugated with antibodies corresponding to the invention, using linker technology available in the prior art. Examples of the types of linkers that were used for cytotoxin conjugation with the antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and the peptide-containing linkers. You can select the linker, which, for example, is susceptible to cleavage at low pH value in the lysosomal compartment or sensitive to cleavage by proteases, such as proteases, preferably expressed in tumor tissue, such as cathepsins (such as cathepsins b, C, ).

In further discussion of the types of cytotoxins, linkers and methods of conjugating therapeutic agents to antibodies, see also article G. Saito et al., (2003), Adv. Drug Deliv. Rev., 55:199-215; Trail P.A. et al., (2003), Cancer Immunol. Immunother., 52:328-337; PayneG., (2003), CancerCell, 3:207-212; Alien T.M., (2002), Nat. Rev. Cancer, 2:750-763; Pastan I. and Kreitman R.J., (2002), Curr. Opin. Investig. Drugs, 3:1089-1091; P.D. Senter and C.J. Springer, (2001), Adv. Drug Deliv. Rev., 53:247-264.

Antibodies corresponding to the present invention may also be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals agents, also called radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated with antibodies for use diagnostically or therapeutically include, but are not limited to, iodine131, indium111, yttrium90and Lu177. In the field of technology developed a method of obtaining radioimmunoconjugates. Examples radioimmunoconjugates commercially available, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to obtain radioimmunoconjugates using antibodies corresponding to the invention.

Conjugates of antibodies, corresponding to the invention can be used for modifying a given biological response, and the molecule drug substances should not be considered as the exhaust gas is unionnwu classical chemical therapeutic agents. For example, the molecule drug substance may be a protein or polypeptide possessing a desired biological activity. Data proteins may include, for example, enzymatically active toxin, or active fragment such kakaban, ricin a, Pseudomonas endotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF"), or other growth factors.

Methods of conjugation of this therapeutic molecules to antibodies are well known, see, for example, section Arnonet al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy" (Monoclonal antibodies to immunosupressant drugs in cancer therapy), in the monograph by Monoclonal Antibodies And Cancer Therapy (Monoclonal antibodies and cancer therapy), Ed. by Reisfeld et al., s-56 (Alan R. Liss, Inc. 1985); section Hellstrom et al., "Antibodies For Drug Delivery" Antibodies for drug delivery), in the monograph Controlled Drug Delivery (Controlled drug delivery) (2nd ed.), Ed. by Robinson et al., s.623-53 (Marcel Dekker, Inc. 1987); review Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" Antibody carriers of cytotoxic agents in the treatment of R is SC), in the collection of Monoclonal Antibodies '84: Biological And Clinical Applications of Monoclonal antibodies '84, biological and clinical applications), edited by Pinchera et al., s-506 (1985); "Analysis, Results, and Future Prospective Of therapeutic Use Of Radiolabeled Antibody In Cancer Therapy" Analysis, results and future prospects for therapeutic use of antibodies with radioactive label in cancer therapy), in the monograph Monoclonal Antibodies For Cancer Detection And Therapy (Monoclonal antibodies for the detection and treatment of cancer), edited by Baldwin et al., s-16 (Academic Press 1985), and article Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates" (Obtaining and cytotoxic properties of conjugates of the antibody-toxin), Immunol. Rev., 62:119-58 (1982).

Bespecifically molecules

In another aspect of the present invention is characterized bespecifically molecules, including antibodies against IP-10 or a fragment corresponding to the invention. The antibody corresponding to the invention, or its antigennegative parts can be derivatization or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate bespecifically molecule that binds to at least two different binding sites or by target molecules. The antibody corresponding to the invention, may in fact be derivative or associated with more than one functionality is Inoi molecule to generate multispecificity molecules, associated with more than two different binding sites and/or molecules of the target; provide that the term "bespecifically molecule", as used in this context includes data multispecificity molecules. To create bespecifically molecules corresponding to the invention, the antibody corresponding to the invention, moretraditional (e.g., by chemical coupling, genetic fusion, noncovalent Association or otherwise) with one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, so the result is bespecifically molecule.

Accordingly, the present invention includes bespecifically molecules containing at least one first binding specificity in relation to IP-10 and a second binding specificity for a second epitope of the target. In a specific embodiment of the invention a second epitope of the target is an Fc receptor, e.g., human FcγRI (CD64) or a human Fco receptor (CD89). Consequently, the invention includes bespecifically molecules, the ability to communicate with effector cells expressing how FcfR and FcorR or Cepsilon (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and the target cells, expressing IP-10. Data bespecifically molecules directed to IP-10-expressing cells, effector cells and the switching activity of effector cells, mediated by the Fc receptor, such as phagocytosis IP-10-expressing cells, antibody dependent mediated cell cytotoxicity (ADCC), release of cytokines or generation of anion superoxide.

In the embodiment of the invention, in which bespecifically molecule is multispecific, the molecule can further include a third binding specificity, in addition binding specificity against Fc and binding specificity against IP-10. In one embodiment, the third binding specificity is part against a reinforcing factor (EF), for example, a molecule that binds to a surface protein involved in the cytotoxic activity, and, thus, enhances the immune response against target cells. "Part against the amplifying factor" can be an antibody, a functional fragment of the antibody or ligand that binds to a given molecule, for example, antigen or receptor, and thus, leads to the strengthening effect of the determinants of binding to an Fc receptor or antigen target cells. "Part against the amplifying factor can bind Fc receptor or antigen cells melenie part against the amplifying factor can contact element, which is different from the element, which contact the first and second specificity. For example, the part against the amplifying factor can bind cytotoxic T-cell (for example through CD2, CDS, CD8, CD28, CD4, CD40, SAM-1) or other immune cell, which results in an increased immune response against the target cell.

In one embodiment, bespecifically molecules corresponding to the invention comprise as a binding specificity at least one antibody, or a fragment of this antibody, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv. The antibody may also be a dimer light chain or the heavy chain or any minimal fragment such as Fv or single-chain design, as described in Ladner et al. in U.S. Patent No. 4946778, the contents of which are specifically introduced in the form of links.

In one embodiment, the binding specificity for the receptor is provided a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used in this context, the term "IgG receptor" refers to any of the eight genes γ-chain, located on chromosome 1. These genes encode in General twelve isoforms of the transmembrane or soluble receptor, which are divided into three classes of Fcγ receptors: FcγRI (CD64), FcγRII(CD32) and FγRIII (CD16). In one preferred embodiment, the receptor Feγ represents a molecule of molecular weight of 72 KD, which shows high affinity for Monomeric IgG (108-109 M-1).

Obtaining and characterization of some preferred monoclonal antibodies against Fey presents Fangeret al. in PCT Publication WO 88/00052 and in U.S. Patent No 4954617, the description of which is fully included in this context by reference. These antibodies bind to an epitope FcγRI, FcγRII or FcγRIII in the centre, other than the binding site of Fcγ receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific antibodies against FcγRI antibodies used in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Hybridoma producing mAb 32 in the American type culture collection (American Type Culture Collection, ATCC Registration No. HB9469. In other embodiments, implementation of the antibody against the receptor Fey is humanitarian form of monoclonal antibody 22 (n). Obtaining and characterization of antibodies n presents with article Graziano R.F. et al., (1995), J. Immunol, 155(10):4996-5002 and PCT Publication WO 94/10332. Cell line producing the antibody n, deposited in the American type culture collection (American Type Culture Collection) with the designation HA022CL1 and has a registration no. CRL 11177.

In the following the x preferred embodiments implement a binding specificity for an Fc receptor is due to antibody which binds to the receptor of human IgA, for example, Fc receptor-α (FcorRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin a (IgA). Provide that the term "IgA receptor" includes a gene product of a single α-gene (FcαRI), located on chromosome 19. It is known that this gene encodes a number of alternative playerowner transmembrane isoforms molecular weight 55-110 KD. FcaRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on the populations refectory cells. FarRI has a high affinity (~5×107M-1) for both IgA1 and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSP (see Morton H.C. et al., (1996), Critical Reviews in Immunology, 16:423-440). Describes the four FcoRI-specific monoclonal antibodies, identified as A3, A59, A62 and A that bind FcorRI outside of the binding domain of the IgA ligand (see Monteiro R.C. et al., (1992), J. Immunol., 148:1764). FcαRI and FcγRI are preferred switching receptors for use in bespecifically molecules corresponding to the invention because they are (1) expressed primarily on immune effector cells, such as monocytes, PMN (mononuclear peripheral blood cells), macrophages and dendritic cells; (2) expressed at high levels (for example, 5000-10000/cell); (3) are mediators of zitotoksicescoe (e.g., ADCC, phagocytosis); (4) mediate enhanced level of presentation of antigens, including native antigen directed at them.

Although the preferred human monoclonal antibodies other antibodies that can be used in bespecifically molecules corresponding to the invention are murine, chimeric and humanized monoclonal antibodies.

Bespecifically molecules relevant to the present invention can be obtained by conjugating the constituent specificdate binding, for example, specificdate binding against FcR and against IP-10, using methods known in the art. For example, each binding specificity bespecifically molecules can be generated separately and then konjugierte with each other. When the binding specificity are proteins or peptides, for covalent conjugation can be used many linking or cross-linking agents. Examples of cross-linking agents include protein a, carbodiimide, N-Succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoyl acid) (DTNB), o-phenylenedimaleimide (oPDM), N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carb is kilat (sulfo-MS) (see, for example, article Karpovsky et al., (1984), J. Exp. Med., 160:1686; M.A. Liu et al., (1985), Proc. Natl. Acad. Sci. USA, 82:8648). Other options are described in the articles Paulus, (1985), Behring Ins. Mitt., No. 78, 118-132; Brennan et al., (1985), Science, 229:81-83 and Glennie et al., (1987), J. Immunol., 139: 2367-2375). The preferred conjugation agents are SATA, sulfo-SMCC, both of which are manufactured by the firm of Pierce Chemical Co. (Rockford, IL).

When the binding specificity are antibodies, they can be conjugated via sulfhydryl binding C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is altered so that it is before conjugation included an odd number of sulfhydryl residues.

Alternative both binding specificity can be encoded in the same vector and expressed and collected in the same cell as the host. This method is particularly effective when bespecifically molecule is a protein mAb x mAb, mAb x Fab, Fab x F(ab')2or ligand x Fab. Bespecifically molecule corresponding to the invention may be single-stranded molecule comprising one single-chain antibody and the determinants of binding or single-stranded bespecifically molecule containing one single-chain antibody and two determinants of binding. Bespecifically molecules can include ENISA least two single-stranded molecules. Methods of obtaining bespecifically molecules described, for example, in U.S. Patent No 5260203; U.S. Patent No 5455030; U.S. Patent No 4881175.

U.S. patent No 5132405; U.S. Patent No 5091513; U.S. Patent No 5476786; U.S. Patent No 5013653; U.S. Patent No 5258498 and U.S. Patent No 5482858.

Linking bespecifically molecules to their specific targets can be confirmed by, for example, using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis (using fluorescent cell sorting device), bioanalysis (e.g., growth inhibition), or Western blotting. Each of the published analyses mainly determines the presence of complexes of protein-antibody of particular interest when using the labeled reagent (e.g., antibodies)that are specific in relation to the complex of interest. For example, complexes FcR-antibody may be determined using, for example, associated with the enzyme, antibody, or antibody fragment, which recognizes and specifically binds to complexes of antibody-FcR. Alternative complexes can be determined using a variety of other immunoassays. For example, the antibody can be marked radioactive label and use in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, (Principles radioimmune the analyses, Seventh course on methods of analysis using radio-Endocrine society, March, 1986), which is included in this context by reference). The radioactive isotope can be defined such means as the use of γ-counter or a scintillation counter or by using autoradiography.

The pharmaceutical composition

In another aspect of the present invention is a composition, e.g. a pharmaceutical composition, containing one or a combination of monoclonal antibodies or yanigasawa part(s)of the present invention, prepared together with a pharmaceutically acceptable carrier. The song data may include one or a combination (for example, two or more different antibodies or immunoconjugates or bespecifically molecules corresponding to the invention. For example, the pharmaceutical composition corresponding to the invention, can include a combination of antibodies (or immunoconjugates or bespecifically molecules)that bind to different epitopes on the target antigen or that have complementary activities.

Pharmaceutical compositions corresponding to the invention can also be used in combination therapy, i.e. combined with other agents. For example, the combination therapy can include titulo against IP-10, the corresponding present invention, in combination with at least one other anti-inflammatory or immunosuppressive agent. Examples of therapeutic agents that may be used in combination therapy, are described in more detail below in the section devoted to the use of antibodies, corresponding to the invention.

As used in this context, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and slowing down the absorption of the agents and the like that are physiologically compatible. Preferably, when the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the method of administration of the active compound, i.e. the antibody immunoconjugate or bespecifically molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

Pharmaceutical compounds corresponding to the invention, can include one or more pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biologicallyactive parent compound and does not give any undesired Toxicological effects (see, for example, article S.M. Berge et al., (1977), J. Pharm. Sci., 66:1-19). The examples of these salts include acid additive salts and basic additive salt. Acid additive salts include salts derived from nontoxic inorganic acids, such kecharitomene, nitric, phosphoric, sulfuric, Hydrobromic, uudistoodetena, phosphorous and the like, as well as non-toxic organic acids such as aliphatic mono - and dicarboxylic acids, phenylsilane alcamovia acid, hydroxyalkanoate acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. basically additive salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-the dibenziletilendiaminom, N-methylglucamine, chloroprocaine, choline, diatomeen, Ethylenediamine, procaine and the like

The pharmaceutical composition conforming to the invention may also include pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, metabisulfite sodium, sodium sulfite and the like; (2) soluble in oil; antioxidants, such as ascorbyl palmitate, bottled hydroxyanisol (BHA), b is melirovanie hydroxytoluene (EIT), lecithin, propylgallate, α-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

Examples of suitable aqueous and nonaqueous carriers which may be used in pharmaceutical compositions corresponding to the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures of vegetable oils, such as olive oil, and injectable organic esters, such as etiloleat. The proper fluidity can be maintained, for example, when using coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surface-active substances.

These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents and dispersing agents. Warning of the presence of microorganisms may be ensured both by sterilization processes, see above, and the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, etc. May also be desirable inclusion in the composition isotonic agents such as sugars, sodium chloride into three and so on In addition, prolonged absorption of the injectable pharmaceutical form may be effected by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation for immediate introduction of sterile injectable solutions or dispersion. The use of these media and agents for pharmaceutically active substances is well known in the technical field. Excluding any adopted environment or the agent because of their incompatibility with the active compound, stipulate their use in pharmaceutical compositions corresponding to the invention. The composition may also include additional active compounds.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be prepared in the form of a solution, microemulsion, liposome, or other ordered structure suitable for high concentration of the drug. The media can be represented by a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures. Proper fluidity mo is but to support, for example, when using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases it will be preferable to include in the composition isotonic agents such as sugars, polyols, such as mannitol, sorbitol, or sodium chloride, etc. Prolonged absorption of injectable compositions can be achieved by incorporating in the composition an agent that slows the absorption of, for example salts of monostearate and gelatin.

Sterile injectable solutions can be prepared by introduction of active compounds in the required amount in the appropriate solvent with one or a combination of the above ingredients as required, followed by sterilization microfiltration. Generally, dispersions are prepared by introduction of the active compound into a sterile vehicle, which contains a basic dispersion medium and other necessary ingredients from above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of obtaining are vacuum drying and freeze-drying (lyophilization)that yield a powder of the active ingredient with any additional required ingredient of its previously about sterilizating filtering solution.

The amount of active ingredient which can be combined with the material of the carrier to obtain a single dosage form will vary depending on the subject, you expect to be treated, and the particular route of administration. The amount of active ingredient which can be combined with the material of the carrier to obtain a single dosage form will, as a rule, such a quantity of a composition which provides a therapeutic effect. Usually one hundred percent, this amount will be in the range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent of active ingredient in combination with pharmaceutically acceptable carrier.

Dosing schedules choose to provide the optimum desired response (e.g., a therapeutic response). For example, there may be a single bolus, there may be several separate doses over time or the dose may be proportionally reduced or increased, as determined by the requirements of therapeutic situation. Particularly beneficial to prepare parenteral compositions in dosage uniform dose to the area is and the introduction and unification of dosing. The term metered uniform dose, as used in this context, refers to physically discrete units corresponding to single doses intended for entities which intend to treated; each unit contains a specified quantity of active compound calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification for the dosage of unified forms, relevant to the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect, which are designed to achieve, and (b) restrictions in the field of receptionarea such an active compound for the treatment of sensitivity in the regions.

For the introduction of antibodies dosages are in the range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of body weight of the host. For example, the dosage can be 0.3 mg/kg of body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg of body weight or to lie in the range of 1-10 mg/kg Illustrative scheme of treatment involves the introduction of once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months or one Cascadia 6 months. Preferred dosing schedules antibodies against IP-10, corresponding to the invention include 1 mg/kg body weight or 3 mg/kg body weight by intravenous administration, with the antibody yield, using one of the following dosing schemes: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg of body weight every three weeks.

In some methods, two or more monoclonal antibodies with different specificnosti bind enter at the same time, in this case the dosage of each input antibodies decreases within the specified interval. The antibody is typically administered for many techniques. Intervals between single dosages can be, for example, week, month, every three months or year. Intervals can also be irregular, as shown by measuring levels of antibodies to the target antigen in the patient's blood. In some ways doses picked to achieve the concentration of antibodies in the plasma of about 1-1000 μg/ml and in some methods about 25-300 μg/ml

An alternative antibody can be entered as a drug with a slow release, and in this case, requires less frequent administration. Dosage and frequency vary depending on the half-period of the existence of antibodies in the patient's body. As a rule, man is ical antibodies show the longest half-life existence, followed by humanized antibodies, chimeric antibodies and human antibodies. Dose and frequency of injection can vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use a relatively low dose at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of your life. Therapeutic application sometimes requires a relatively high dosage at relatively short intervals up until the disease will not decrease or will not end, and preferably as long as the patient does not detect partial or complete attenuation of the symptoms of the disease. After that the patient can make the introduction of preventive scheme.

The actual dose levels of active ingredients in the pharmaceutical compositions of the present invention, can be varied to obtain an amount of active ingredient which is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of introduction without showing toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors including the activity specific to the position, relevant to the present invention, or a complex ester, salt or amide, the method of administration, time of administration, rate of excretion of the specific compound, the duration of the treatment, other drugs, compounds and/or materials used in combination with the specific composition, age, sex, weight, condition, General health and prior medical history of the patient, which is suppose to be treated, and like factors well known in medicine.

"Therapeutically effective dose" antibodies against IP-10, corresponding to the invention preferably results in reducing the severity of disease symptoms, an increase in the frequency and duration of periods without symptoms of the disease or to prevent injury or disability due to physical disability associated with the disease. In the case of rheumatoid arthritis (RA) preferably, when a therapeutically effective dose prevents further aggravation of physical symptoms associated with RA, such as, for example, pain, fatigue, morning stiffness (continued more than one hour), diffuse muscle pain, loss of appetite, weakness, joint pain, accompanied by fever, swelling, tenderness and stiffness of the joints after a period of still is. Preferably, when a therapeutically effective dose also prevents or delays the development of RA, as this may be desirable when there are early or previous signs of illness. It likewise includes a delay chronic progression, associated with RA. Laboratory tests used for the diagnosis of RA include chemical analysis (including measurement of the levels of IP-10), hematological, serological and radiological studies. Accordingly, any clinical or biochemical analysis, which monitorium any of the above options can be used to determine whether a particular treatment a therapeutically effective dose for the treatment of RA. Ordinary specialist in the field of technology would be able to determine these amounts based on such factors as the size of the subject, the severity of symptoms in the subject and selected a particular composition and method of administration.

The composition corresponding to the present invention, it is possible to introduce one or more routes of administration using methods known in the art. Both must be in the form of a competent specialist, method and/or method of introduction will vary depending on the required results. Preferred methods of administration of antibodies, corresponding izaberete the Oia, include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal and other parenteral routes of administration, e.g. by injection or infusion. The phrase "parenteral administration"as used in this context, means a technique of administration, other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, vnutriglaznogo, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and vnutrigrudne injection and infusion.

An alternative antibody corresponding to the invention, it is possible to enter aparentally way, such as the outer, epidermal and administration through the mucosa, for example, intranasally, orally, vaginally, rectally, sublingual and externally.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as drug controlled release, including implants, transdermal patches and microencapsulation delivery system. Can be used biodegradable, biocompatible polymers, such as ethylene is unilateral, polyanhydride, polyglycolic acid, collagen, polyarteritis and polylactic acid. Many methods of obtaining such drugs are patented and, as a rule, well-known specialists in the field of technology. See, for example, the monograph Sustained and Controlled Release Drug Delivery Systems drug delivery with a slow and controlled release), Ed. by J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be entered with medical devices known in the prior art. For example, in a preferred embodiment, therapeutic composition corresponding to the invention, can be entered using without needle hypodermic injection device, such as the device disclosed in U.S. Patent NoNo 5399163, 5383851, 5312335, 5064413, 4941880, 4790824 or 4596556. Examples of well-known implants and modules used in the present invention include: U.S. Patent No. 4487603, which discloses an implantable pump for micropose for the distribution of medicines controlled rate; U.S. Patent No. 4486194, which discloses a therapeutic device for the administration of drugs through the skin; U.S. Patent No. 4447233, which discloses a pump for infusion drugs for drug delivery with a precise infusion rate; U.S. Patent No. 4447224,which discloses an implantable infusion device with changing flow for continuous delivery of a drug; U.S. patent No. 4439196, which discloses an osmotic delivery system of a drug having multi-chamber compartments, and U.S. Patent No. 4475196, which discloses an osmotic delivery system of the drug. These patents are included in this context by reference. Professionals known in the art, many other such implants, delivery systems, and modules.

In some embodiments, the implementation of human monoclonal antibodies, corresponding to the invention, can be manufactured to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many connections with high hydrophilicity. To ensure that therapeutic compounds corresponding to the invention, overcome the BBB (if required), they can be manufactured, for example, in liposomes. On ways of obtaining liposomes, see, for example, U.S. Patents 4522811, 5374548 and 5399331. Liposomes can contain one or more groups, which are selectively transported into specific cells or organs, increasing, thus, targeted delivery of a drug (see, for example, article V.V. Ranade, (1989), J. Clin, Pharmacol., 29:685). Illustrative guides groups include folate and Biotin (see, for example, U.S. Patent 5416016 issued Lowet al.); mannoside (see Umezawa et a., (1988), Biochem. Biophys. Res. Commun., 153:1038); antibodies (see article P.O.Bloeman et al., (1995), FEBS Lett., 357:140; M.Owais et al., (1995), Antimicrob. Agents Chemother., 39:180); the receptor for surfactant protein A (Briscoe et al., (1995), Am. J. Physiol., 1233:134); p120 (see Schreier et al., (1994), J. Biol. Chem., 269:9090), see also article K.Keinanen, M.L.Laukkanen, (1994), FEBS Lett., 346:123; J.J.Killion; I.J.Fidler, (1994), Immunomethods, 4:273. Applications and methods, corresponding to the invention

Antibodies (and immunoconjugates and bespecifically molecule)corresponding to the present invention have diagnostic and therapeutic applications in vitro and in vivo. For example, these molecules can be introduced into cells in culture, e.g. in vitro or ex vivo, or to a subject, for example in vivo, to treat, prevent or diagnose a number of violations. The term "subject"as used in this context, involves the inclusion of humans and animals other than man. Animals, other than humans, include all vertebrates, such as mammals and insects memleketim, such as primates, non-human, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles. The methods are particularly suitable for the treatment of patients-people with disorders associated with abnormal expression of IP-10. When antibodies against IP-10 are administered together with another agent, the two components can be entered in any order or simultaneously.

Water variant implementation of the Oia, antibodies (and immunoconjugates and bespecifically molecule)corresponding to the invention can be used for detecting levels of IP-10 or levels of cells which contain IP-10. This can be achieved, for example, by contacting a sample (such as a sample in vitro) and the control sample with the antibody against IP-10 in conditions which ensure the formation of a complex of the antibody and IP-10. Any complexes formed by the antibody and IP-10, determined and compared in the sample and in the control. For example, standard methods of detection are well known in the prior art, such as ELISA and flow sicomedicina analyses can be carried out using the compositions corresponding to the invention.

Accordingly, in one aspect the invention further presents the methods of detecting the presence of IP-10 (for example, antigen human IP-10) in the sample, or measuring the amount of IP-10, which consists in contacting of the sample and the control sample with the antibody corresponding to the invention, or its antigennegative part, which specifically binds to IP-10 in conditions which ensure the formation of a complex of the antibody or portion, and IP-10. Then determine the formation of the complex, and the difference in complex formation between the sample compared to the control sample is indicative of the presence of IP-10 Varazze.

In addition, the scope of the invention includes kits containing a composition (e.g., antibodies, human antibodies, immunoconjugates and bespecifically molecules), corresponding to the invention, and instructions for use. The kit may further include at least one additional reagent, or one or more additional antibodies corresponding to the invention (for example, the antibody having a complementary activity which binds to an epitope on the target antigen, different from the first antibody). The kits typically include a label indicating the intended application of the set's contents. The term label includes any inscription or written material supplied to or together with the set, or otherwise accompanies the set.

It is known that IP-10 has the effect of chemoattractant against activated T-cells and MC-cells and recruits these cells in inflammation and autoimmune responses. Accordingly, antibodies against IP-10 (and immunoconjugate and bespecifically molecules), corresponding to the invention, can be used to suppress inflammatory or autoimmune response mediated by activated T-cells and/or NK-cells in a number of clinical indications. The invention therefore provides a method of suppressing an inflammatory and or Tomonaga response mediated by activated T-cells and/or MK-cells, which consists in contacting the T-cells or NK-cells with the antibody or antigennegative part corresponding to the invention (or immunoconjugates or bespecifically molecule corresponding to the invention) so that there is a suppression of the inflammatory or autoimmune response. Specific examples of inflammatory or autoimmune conditions that can be used antibodies corresponding to the invention, include, but are not limited to, the following:

A. Multiple sclerosis and other demyelinating diseases

It is shown that the mRNA expression of IP-10 increased in murine experimental allergic encephalomyelitis (EAE)model of multiple sclerosis in mice (see Godiska, R., et al., (1995), J. Neuroimmunol., 58:167-176). Moreover, elevated levels of IP-10 was detected in the cerebrospinal fluid of patients with PC (multiple sclerosis) during acute demyelinating event (see article SorensenT.L et al., (1999), J. Clin. Invest., 103:807-815; Franciotta et al., (2001), J. Neuroimmunol., 115:192-198). It is also shown that IP-10 is expressed by astrocytes in the lesions PC, but not in the unaffected white matter (see Balashov, K.E. et al., (1999), Proc. Natl. Acad. Sci. USA, 96:6873-6878) and is expressed by macrophages in plaques PC and reactive astrocytes in the surrounding parenchyma (see J.E. Simpson et al., (2000), Neuropathol. Appl. eurobiol., 26:133-142). In Patent publication PCT WO 02/15932 shown that antibodies against IP-10 on the PC using the mouse hepatitis virus (MHV) results in reduced invasion of T-lymphocytes and macrophages, suppressed the development of demyelination, increased remyelination and improved neurological function (see also M.L. Liu et al. (2001) J. Immunol. 167:4091-4097). Introduction mouse antibodies against IP-10, as shown, reduces the proportion of clinical and histological cases and the severity of disease in mice (EAE) (see Fife V.T. et al., (2001), J. Immunol., 166:7617-7624).

In light of the foregoing antibodies against IP-10, corresponding to the invention can be used in the treatment of PC and other demyelinating diseases by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents against PC, such as interferon β-1A (e.g., Avonex®, Rebif®), interferon β-1b (e.g., Betaseron®), glatiramer acetate (e.g., Sorachai®) and/or mitoxantrone (e.g., Novantrone®).

Century Rheumatoid arthritis

It is shown that the levels of IP-10 significantly increased in the synovial fluid, synovial tissue and serum of patients with rheumatoid arthritis (RA) (see article D.D. Patel et al., (2001), Clin. Immunol., 98:39-45; R. Hanaoka et al., (2003), Arthritis Res. and Therapy, 5:R74-R81). The receptor for IP-10, CXCR3, as shown, predpochtite the flax is expressed on mast cells (mastocyte) in synovial tissue, obtained from patients with RA (see Ruschpler P. et al., (2003), Arthritis Res. Ther., 5:R241-R252). In the model of arthritis induced by adjuvant, rats (AA) describes the designated response of antibodies against IP-10 (see Salomon I. et al., (2002), J. Immunol., 169:2685-2693). Moreover, the introduction of DNA vaccines encoding IP-10, increased the level of production of neutralizing antibodies against IP-10 in rats, and these autoantibodies IP-10 could adoptive transfer resistance Kaa nepilirovanny (naive) rats (see Salomon I. et al., above).

In light of the foregoing antibodies against IP-10, corresponding to the invention can be used in the treatment of rheumatoid arthritis by injecting antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents against RA, such as non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (eg, prednisone, hydrocortisone), inhibitors of TNF (tumor necrosis factor) (including adalimumab (Humira®), etanercept (Enbrel®) and infliximab (Remicade®), disease modifying Antirheumatic drugs (including methotrexate, cyclophosphamide, cyclosporine, auranofin, azathioprine, thiomalate gold sodium, hydroxychloroquine sulfate, malonamide, minocycline, penicillamine and sulfasalazin), drug the means against fibromyalgia, drugs against osteoporosis and medicines for treating gout.

C. Inflammatory bowel disease

It is shown that the expression of IP-10 significantly increased in the cells, infiltrate own record mucosal biopsies of the colon taken from patients with ulcerative colitis (see Uguccioni M. et al., (1999), Am. J. Pathol., 155:331-336). In addition, it is shown that neutralization of IP-10 protects mice from ulceration of the epithelium in acute colitis and increases the survival of cells of the crypts (see S. Sasaki et al., (2002), Eur. J. Immunol., 32:3197-3205). In addition, in mice IL-10 -/-, have developed colitis similar to Crohn's disease in humans, treatment with antibodies against IP-10 resulted in the improvement of inflammation (see Singh up works et al., (2003), J. Immunol., 171:1401-1406).

In light of the foregoing antibodies against IP-10, corresponding to the invention can be used in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents against IBD, such as drugs, containing mesalamine (including sulfasalazin and other agents, including 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide), nonsteroidal anti-inflammatory Les is artenie drugs (NSAIDs), analgesics, corticosteroids (eg, prednisone, hydrocortisone), TNF inhibitors (including adalimumab (Humira®), intercept (Enbrel®) and infliximab (Remicade®), immunosuppressants (such cacb-mercaptopurine, azathioprine and cyclosporine A) and antibiotics.

D. Systemic lupus erythematosus

It is shown that the serum levels of IP-10 significantly increased in patients with systemic lupus erythematosus (SLE) and shown that these levels correlate with disease activity (see, for example, article Narumi S. et al., (2000), Cytokine, 12:1561-1565). Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of SLE by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents against SLE, such as non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (eg, prednisone, hydrocortisone), immunosuppressants (such Castiglione, azathioprine and methotrexate), antimalarial drugs (such as hydroxychloroquine) and biological drugs that inhibit the production of antibodies to ds (for example, UP 394).

E. Mellitus type I

It is shown that the serum levels of IP-10 was increased in patients with diabetes type I, in particular, with the recent onset of the illness, and p is shown, these levels correlate with the number of reactive with GAD producing γ-interferon T-kitoku patients positive for autoantibodies to GAD (see A. Shimada et al., (2001), Diabetes Care, 24:510-515). In a separate study found that serum levels of IP-10 was increased in patients with newly diagnosed disease and in patients with high risk of disease and the concentration of IP-10 correlated with the levels of IFN-γ (see Nicoletti F. et al., (2002), Diabetologia, 45:1107-1110). Moreover, it is demonstrated that β-cells secrete IP-10, leading to chemoattract T cells, and in mice with deficiency of CXCR3, as shown, it delayed the development of diabetes type I (see S. Frigerio et al., (2002), Nature Medicine, 8:1414-1420).

Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of type I diabetic by injecting antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other antidiabetic agents such as insulin.

F. Inflammatory skin disorders

It is shown that the expression of IP-10 is associated with a number of inflammatory skin disorders. For example, IP-10 determined in keratinocytes and dermal infiltration of active psoriatic plaques (see Gottlieb V. et al., (1988), J. Exp. Med., 168:941-948). Moreover, CXCR3 is expressed skin the lymphocytes CD3+, giving the opportunity to suggest that CXCR3 is involved in the transport of T-lymphocytes in psoriatic dermis (see Rottman J.B. et al., (2001), Lab. Invest., 81:335-347). Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of psoriasis by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents or treatments, such as external agents (e.g., steroids, tar, calcipotriene, tazarotene, anthralin, salicylic acid), phototherapy, systemic medications (e.g., methotrexate, oral retinoids, cyclosporine, esters of fumaric acid and/or biological drugs (for example, alefacept, efalizumab).

It is shown that lichen planus, a chronic inflammatory disease of skin and mucous membranes of the oral cavity, associated with infiltration of T cells CD4+ and CD8+that Express CXCR3 and, moreover, it is shown that infilterate cytolytic T-cells CD8+ are IP-10 in their cytolytic granules, and keratinocytes in the area of the lesion, as shown, are characterized by overexpression of IP-10 (see lijima W. et al., (2003), Am. J. Pathol., 163:261-268). Accordingly, in another embodiment, antibodies against IP-10, corresponding to izaberete the Oia, can be used in the treatment of flat shingles by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents or drugs, such as anti-inflammatory agents, antihistamines, corticosteroids and phototherapy.

It is shown that the expression of IP-10 are elevated in other inflammatory skin disorders, such as chronic discoid lupus erythematosus and lymphocytic infiltration of the skin Jessner (see Flier J. et al., (2001), J.Pathol., 194:398-405). Accordingly, antibodies against IP-10, corresponding to the invention can be used in the treatment of these inflammatory skin disorders by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents or drugs, as described above.

G. Autoimmune disease of the thyroid gland

It is shown that both IP-10 and CXCR3 are expressed in the thyroid gland of patients suffering from graves ' disease (GD), but not expressed (or weakly expressed in normal thyroid tissue, and expression is at its highest level in patients with recent beginning of GD (see Romagnani P. et al., Am. J. Pathol., 161:195-206). IP-10, as also shown, is expressed in the thyroid gland of patients, suffering is from Hashimoto thyroiditis (see article E.H. Kemp et al., (2003), Clin. Endocrinol., 59:207-213). Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of autoimmune thyroid disease, including graves ' disease and Hashimoto's thyroiditis, by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents or treatments, such as medicines against the thyroid gland, radioactive iodine and Subtotal thyroidectomy.

N. Sjogren syndrome

It is shown that the expression of mmcr-10 largely regulated by increases in salivary glands of patients with Sjogren syndrome (S3), and the expression is most prominent in ducktales epithelium, adjacent limfoidnymi the infiltrates (see, for example, article N. Ogawa et al., (2002), Arthritis Rheum., 46:2730-2741). Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of Sjogren syndrome by injecting antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents against the school, such as synthetic lubricating components (for example, does not contain a preservative artificial tears, artificial saliva, neuronitis rowanne skin lotions, saline sprays for the nose and vaginalnye lubricating agents), Lacriserts® for the treatment of dry eye, pilocarpine hydrochloride (Salagen®) and Caemlyn (Eukas®) for the treatment of dry mouth, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids and immunosuppressive drugs

drugs.

I. Pulmonary inflammation

The expression of IP-10 was investigated in models of allergic asthma in mice, and the results demonstrate that IP-10 is regulated by the increase in lung after injection of allergen, and that overexpression of IP-10 is associated with increased hyperactivity of the Airways, eosinophilia, elevated levels of IL-4 and recruitment of lymphocytes CD8+ (see Medoff B.D. et al., (2002), J. Immunol., 168:5278-5286). Moreover, it is shown that in bronchiolar epithelium of smokers who develop chronic obstructive pulmonary disease (COPD), is expressed IP-10 (see Saetta M. et al., (2002), Am. J. Respir. Crit. Care Med., 165:1404-1409). Moreover, high levels of IP-10 was demonstrated in bronchoalveolar lavage fluid of patients with pulmonary sarcoidosis and the lymphocytic alveolitis (see S. Agostini et al., (1998), J. Immunol., 161:6413-6420).

Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of diseases characterized by inflammation of the lungs, such as asthma is, COPD, pulmonary sarcoidosis or lymphocytic alveolitis, by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents to reduce inflammation of the lungs, such as cromolyn sodium, nedocromil sodium, inhaled corticosteroids, systemic (e.g., oral) corticosteroids, β-antagonists, short-acting bronchodilators short-acting β-antagonists or agonists long-term actions (oral and inhaled), leukotriene modifiers, theophylline, and oxygen therapy.

J. graft Rejection

It is shown that IP-10 plays a role in the rejection transplantirovannam tissue. For example, treatment of mice with neutralizing antibodies against IP-10 increases the survival rate of small intestinal allografts and reduces the accumulation of T-cells and NK-cells are masters in our own vinyl mucosa (see Zhang Z. et al., (2002), J. Immunol., 168:3205-3212). In addition, mice receiving allografts islets of the pancreas, treatment with antibody against IP-10 also results in increased allograft survival and reduced infiltration of lymphocytes in the graft (see Baker M.S. et al., (2003), Surgery, 134:126-133). In addition, it is shown that cardiac allografts, but not the normal heart, ek is pressedout IP-10 and CXCR3, and elevated levels of IP-10 is associated with a disorder of vessels of heart allografts (see D.X. Zhao et al., (2002), J. Immunol., 169:1556-1560). It is also shown that CXCR3 and IP-10 are expressed by inflammatory cells, infiltrirujushchim in lung allografts (see Agostini, S. et al., (2001), Am J. Pathol., 158:1703-1711). Neutralization of CXCR3 and IP-10 in vivo, as shown in a murine model of lung transplant weakens syndrome obliterating bronchiolitis (BOS), a main limiting factor for survival in recipients of lung transplant (see J.A. Belperio et al., (2002), J. Immunol., 169:1037-1049).

In light of the foregoing, the invention also presents a method of suppressing rejection transplant by introducing antibodies against IP-10, corresponding to the invention, the transplant recipient in need of treatment. Examples of tissue grafts, which can be treated include, but are not limited to, liver, lung (for example, treatment of BOS), kidney, heart, small intestine and cells of the pancreatic islets. The antibody may be used as monotherapy or in combination with other agents for suppressing transplant rejection, such as immunosuppressants (eg, cyclosporine, azathioprine, methylprednisolone, prednisolone, prednisone, mycophenolate mofetil, sirolimus, rapamycin, tacrolimus), anti-infective agent is (for example, acyclovir, clotrimazole, ganciclovir, nystatin, trimethoprimsulfamethoxazole), diuretics (for example, bumetanide, furosemide, metolazone) and antiulcer tools (e.g., cimetidine, famotidine, lansoprazole, omeprazole, ranitidine, sukralfat).

K. spinal cord Injury Traumatic spinal cord injury leads to infiltration of inflammatory cells. It is shown that IP-10 plays a major role in the secondary degeneration after spinal cord injury (see Gonzalez et al., (2003), Exp. Neurol., 184:456-463; see also Patent PCT publication WO 03/06045). The level of IP-10, as shown, is substantially increased when injuries of the spinal cord of rats after 6 and 12 hours after injury (see McTigue D.M. et al., (1998), J. Neurosci. Res., 53:368-376) and in the injured spinal cord of mice 6 hours after injury (see Gonzalez et al., (2003), above). Accordingly, inhibition of the activity of IP-10 after injury to the spinal cord, as shown, is effective in reducing the infiltration of inflammatory cells and, thus, reduce secondary tissue damage due to inflammation. Inhibition can also reduce the infiltration of inflammatory cells, reduce secondary degeneration of uluchshit recovery after traumatic brain injury and stroke. Thus, the invention also presents a method of treating spinal cord mo the ha and brain damage (for example, stroke) in need of treatment of a subject, which consists in the introduction to the subject antibodies against IP-10, corresponding to the invention. The antibody may be used as monotherapy or in combination with other agents, such as other anti-inflammatory agents.

L Neurodegenerative diseases

Found that the expression of IP-10 and CXCR3 in the Central nervous system is regulated by increasing way in connection with pathological changes associated with Alzheimer's disease (AD) (see Xia M.Q. and Hyman D.T., (1999), J. Neurovirol., 5:32-41). In the brain, diseased (AD), CXCR3, as shown, is expressed constitutively in neurons and neuronal processes in various cortical and subcortical regions, and IP-10, as shown, is expressed in astrocytes, and its level is significantly elevated compared to normal brain (see Xia M.Q. et al., (2000), J. Neuroimmunol., 108:227-235). Accordingly, antibodies, corresponding to the invention can be used in the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's disease, by introducing the subject in need of treatment with an antibody against IP-10, in monotherapy or in combination with other therapeutic agents. Examples of agents that can be combined antibody against IP-10 for the treatment of Alzheimer's disease, on the receive cholinesterase inhibitors (donepezil, rivastigmine, galantamine, taken) and vitamin E. an Example of the agent, which can be combined antibody against IP-10 for the treatment of Parkinson's disease is levodopa.

M Gingivitis

Edge periodontitis is associated with inflamed tissue of the gums. In inflamed tissue of the gums of the person detected cells that produce IP-10, as well as cells expressing the receptor CXCR3 (see Kabashima N. et al., (2002), Cytokine, 20:70-77). Accordingly, in another embodiment, antibodies against IP-10, corresponding to the invention can be used in the treatment of gingivitis by introducing antibodies to a subject in need of treatment. The antibody may be used as monotherapy or in combination with other agents or treatments, such as a gargle for mouth against gingivitis (for example, rinse mouth with antibiotics), removing plaque from periodontal and alignment roots and periodontal surgery.

N. Inflammation associated with gene therapy

Replication-deficient adenoviruses used as adenoviral vectors in gene therapy, can cause acute impairment and inflammation in tissues infected with viral vectors. Data adenoviral vectors, as shown, induce the expression of IP-10 by the capsid-dependent activation of NFkB (see become Borgland S.L et al., (2000), J. Virol., 74:3941-3947). Relevant to the military, antibodies against IP-10, corresponding to the invention can be used for inhibition induced IP-10 violations and/or inflammation during a gene therapy treatment that uses a viral vector such as an adenoviral vector, which stimulates unwanted products IP-10.

O. Diseases associated with angiogenesis

It is shown that IP-10 inhibits angiogenesis in vitro and in vivo (see article Stricter et al., (1995), Biochem. Biophys. Res. Commun., 210:51-57; Angiolillo et al., (1995), J. Exp. Med., 182:155-162; Luster et al., (1995), J. Exp. Med., 182:219-231). Angiogenesis plays a crucial role in many disease processes, such as reaction heal the injury. For example, the vascular network in the injured spinal cord remains in the active state remodeling until at least 28 days after injury (see Popovich et al., (1997), J. Tech. Neurol., 377:443-464).

IP-10 is believed to manifest its angiostatic effects by inhibiting the growth and chemotaxis of endothelial cells. It accomplishes this through its heparin-binding motif, as well as by receptor-mediated mechanism. Through its heparin-binding motif it prevents the binding of angiogenic factors FGF-2 and VEFG165 with their receptors. It also exerts its effects through a receptor-mediated process. The receptor for IP-10, CXCR3, alternative spiceroads for education the Oia two known variants CXCR3A and CXCR3B. The binding of IP-10 receptor CXCR3A leads to proliferation and chemotaxis of target cells, whereas binding of IP-10 receptor CXCR3B has the opposite effect of inhibiting the growth and chemotaxis. IP-10 is acting through a receptor CXCR3B changestates factor (see Lasagni et al., (2003), J. Exp. Med., 197:1537-1549).

In light of the foregoing antibodies against IP-10, corresponding to the invention can be used in the treatment of diseases involving angiogenesis, for example, when angiostatic the behavior of IP-10 inhibits or prevents healing or exacerbate the disease process. These diseases include: 1) aberrant physiological neovascularization, which may affect the healing of wounds, the estrus cycle in women, pregnancy, hypertrophy, caused by the physical exertion and the like; 2) indications, which may require stimulation of neovascularization, including induction of the formation of collateral vessels (including myocardial ischemia, peripheral ischemia, cerebral ischemia), disease of the coronary arteries, peripheral vascular disease, stroke, wound healing, engraftment of the graft after transplantation of an organ, such cantranslate cells of the islets, fracture and damage to the ligaments, reconstructive surgery, tissue engineering, restenosis, hair loss that is roline and varicose ulcers, gastrointestinal ulcers, placental insufficiency, aseptic necrosis, pulmonary and systemic hypertension, vascular dementia, Alzheimer's disease, cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL); pancreatic pseudocyst of the thyroid gland and lymph edema, and 3) readings, which may require vascular remodeling, including malignancy vessels, psoriasis and pre-eclampsia. Antibodies corresponding to the invention, can be used as monotherapy or in combination with other agents that cause angiogenesis.

R. Inflammatory kidney disease

It is shown that the receptor CXCR3 is expressed mesangial cells of patients with IgA nephropathy, membranoproliferative glomerulonephritis or rapidly progressive glomerulonephritis (see Romagnani P. et al., (1999), J. Am. Soc. Nephrol., 10:2518-2526). In addition, on the model of nephrotoxic nephritis in mice, the mRNA levels of IP-10 were increased six times to the cortical substance affected nephritis kidney 7 days after induction of nephritis (see Schadde E. et al., (2000), Nephrol. Dial. Transplant., 15:1046-1053). Moreover, high levels of expression of IP-10 observed in samples of kidney biopsies patients with glomerulonephritis compared to normal kidneys (see Romagnani P. et al., (2002), J. Am. Soc. Nephrol., 13:53-64). Matched with the public, antibodies against IP-10, corresponding to the invention can be used in the treatment of inflammatory renal diseases, including IgA nephropathy, membranoproliferative glomerulonephritis or rapidly progressive glomerulonephritis. Antibodies corresponding to the invention, can be used as monotherapy or in combination with other agents or therapies used in the treatment of glomerulonephritis, such as antibiotics, diuretics, medicines against high blood pressure and dialysis.

Q. Atherosclerosis Shown that IP-10 is a mitogenic and chemotactic factor for vascular smooth muscle, which is an important property of smooth muscle cells in terms of their participation in the pathogenesis of atherosclerosis (see article X. Wang et al., (1996), J. Biol. Chem., 271:24286-24293). Induction of IP-10, as shown, is in the smooth muscle cells after treatment with LPS (LPS) or interferon, as well as in the carotid artery of rats after balloon angioplasty (see article X. Wang et al., (1996), above). Moreover, it is demonstrated that IP-10 is expressed in endothelial cells, smooth muscle cells and macrophages associated with atheroma, allowing you to assume the role of IP-10 in the recruitment and retention of activated T-cells, which were noted in the areas of damage to blood vessels in the essays of atherogenesis (see article F. Mach et al., (1999), J. Clin. Invest., 104:1041-1050). Accordingly, antibodies against IP-10, corresponding to the invention, can be used to treat or prevent atherosclerosis. Antibodies can be used in monotherapy or in combination with other agents or therapies used in the treatment of atherosclerosis, such as medicines against high blood pressure and drugs that reduce cholesterol levels.

R. Viral infection

IP-10 may be subject to increase, when various viral infections and can play a positive role in recruiting aktivirovannyh T cells in controlling viral infection. However, in some cases, the production of IP-10 during viral infection can lead to harmful effects and, thus, the activity of IP-10 may be undesirable and may need to inhibit the activity of IP-10 in these viral infections using antibodies against IP-10, corresponding to the invention.

For example, it is shown that IP-10 stimulates the replication of human immunodeficiency virus (HIV) in macrophages macrophage nature and peripheral blood lymphocytes (see Lane, B.R. et al., (2003), Virology, 307:122-134). In addition, levels of IP-10 increased in the cerebrospinal fluid and brain of HIV-infected patients and in the Central nervous system is e mice, transgenic for Dr HIV (see Asensio V.. et al., (2001), J. Virol., 75:7067-7077).

It is also shown that the levels of IP-10 was increased in patients with chronic persistent hepatitis C virus (HCV) and in patients with chronic active hepatitis (see Narumi S. et al., (1997), J. Immunol., 158:5536-5544). In HCV infected liver IP-10, as shown, is expressed by hepatocytes, but not in other cell types, and in the liver detected a significantly higher proportion SHSR-polozhitelnyh-cells than in the blood (see Harvey S.E. et al., (2003), J. Leukoc. Biol., 74:360-369). It is shown that an increased level of secretion of IP-10 is associated with the inflammatory response to acute eye infection, herpes simplex type I (HSV-1) in mice, treatment of infected HSV-1 mice by antibodies against IP-10, as shown, reduces the infiltration of mononuclear cells in the stroma of the cornea reduces the pathology of the cornea and suppresses the spread of the virus from the stroma of the cornea to the retina during acute infection (see Carr D.J. et al., (2003), J. Virol., 77:10037-10046).

It is also shown that the expression of IP-10 is implemented with viral meningitis. IP-10, as illustrated, is present in the CSF (cerebral spinal fluid) patients with viral meningitis and is responsible for the chemotactic activity of neutrophils, mononuclear peripheral blood cells and activated T-cells (see article Lahrtz F. et al., (1997), Eur.J. Immunol., 27:2484-2489; Lahrtz . et al., (1998), J. Neuroimmunol., 85:33-43).

In light of the foregoing antibodies against IP-10, corresponding to the invention can be used in the treatment of viral infections, including unwanted activity of IP-10 by introducing antibodies to a subject in need of treatment. Non-limiting examples of viral infections that can be treated include HIV (e.g., HIV-induced encephalitis), HCV, HSV-1, viral meningitis and severe acute respiratory syndrome (SARS). The antibody may be used as monotherapy or in combination with other antiviral agents, such as in the case of HIV infection nucleoside/nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase and/or protease inhibitors (and their combination)in the case of HCV infection interferon and 2A, pegylated interferon and 2A and/or ribavirin, and in the case of infection with HSV-1 acyclovir, valacyclovir and/or famciclovir.

S. Bacterial infection.

Bacterial infections induce the formation of IP-10 in infected cells (see Gasper N.A. et al., (2002), Infect Immun., 70:4075-82). Bacterial meningitis, such as, in particular, it is known that promotes the expression of IP-10 (see Lapinet J.A. et al., (2000), Infect Immun., 68:6917-23). IP-10 is also generated somatic cells of the seminiferous tubules of the testis in a model of bacterial infection, strongly indicating the potential role Yes the different chemokines in the accumulation of neutrophils and T-lymphocytes in inflammation of the testicle, which classically occurs in the pathogenesis of bacterial infections (see article F. Aubry et al., (2000), EurCytokine Netw., 11:690-8).

In light of the foregoing antibodies against IP-10, corresponding to the invention can be used in the treatment of bacterial infections, including unwanted activity of IP-10 by introducing antibodies to a subject in need of treatment. Examples of bacterial infections include, but are not limited to, bacterial meningitis and bacterial pneumonia. The antibody may be used as monotherapy or in combination with other antibacterial agents such as antibiotics.

The present invention is further illustrated by the following examples, which should not be construed as further limiting. The content of all shapes and all references, patents and published patent applications cited throughout this application, specially introduced in this context by reference.

Example 1: Generation of human monoclonal antibodies against IP-10

Antigen

Purified recombinant human IP-10, isolated from E.coli (firm PeproTech, Inc., Catalog number: 300-12), or purified recombinant human IP-10 conjugated with hemocyanin saucer (KLH), is used as the antigen.

Mouse. transgenic by HuMab. and mouse KM

Fully human monoclonal and is tetela to IP10 get using line NSO, NSO and NSO mice transgenic for HuMab, and line KM transgenic transamazonic mice, each of which expresses the genes of a human antibody. In each of these lines of mice endogenous gene of the mouse Kappa-light chain destroyed, as described in Chen et al., (1993), EMBO J. 12:811-820 and endogenous gene of the murine heavy chain destroyed, as described in Example 1 Of PCT Publication WO 01/09187. Each of these lines of mice carries the transgene, human Kappa-light chain XO, as described in Fishwild et al., (1996), Nature Biotechnology, 14:845-851. Line NSO carries the transgene So human heavy chain as described in U.S. Patent NoNo 5545806, 5625825 and 5545807. Line VAT 12 carries the transgene So human heavy chain, as described in Example 2 Of PCT Publication WO 01/09187. Line NSO carries the transgene So human heavy chain, as described in Example 8 below.

Line KM contains transfromation SC20, as described in the Publication PCTWO 02/43478.

Immunization HuMab and KM:

To generate fully human monoclonal antibodies to R mice HuMab mice and KM are subjected to immunization with purified recombinant R isolated from E. coli or conjugate IP10-KLH as antigen. The General scheme of immunization for HuMab mice is described in articles Lonberg, N. et al., (1994), Nature, 368(6474):856-859; Fishwild, D. et al., (1996), Nature Biotechnology, 14: 845-851 and PCT Publication WO 98/24884. If the first infusion of antigen-aged mice is 6-16 h is del. To immunize the HuMab mice and mice KM intraperitoneally, subcutaneously (Sc) or by injection into the foot pad using a purified recombinant preparation (5-50 μg) of antigen R (e.g., purified from transfected cells E. coli, expressively R).

Transgenic mice twice subjected to immunization with antigen in complete Freund's adjuvant or Freund, Ribi either intraperitoneally (IP)or subcutaneously (Sc) or via foot pad (FP), followed by immunization IP, Sc or FP through 3-21 day (up to 11 immunization) with antigen in incomplete Freund's adjuvant or Freund, Ribi. Immune response monitorium using retroorbital (zaglaniczny) fences blood. The plasma is subjected to screening using ELISA (as described below), and mice with sufficient titers of human immunoglobulin against R used for mergers. Mice do intravenous booster injection of antigen for 3 and 2 days before the killing and removal of the spleen. Typically, for each antigen spend 10-35 m. For each antigen are subjected to immunization few dozen mice. In General R subjected to immunization 82 mouse strains of mice NSO, NSO, NSO and KM. Breeding mice HuMab or KM. producing antibodies against R:

To select HuMab mice or KM, producing antibodies that bind R, serum immunized mice tested using ELISA, as described in Fishwild, D. et al., (199). Briefly, tablets for micrometrology cover purified recombinant R of E. coli at a concentration of 1-2 µg/ml in PBS (phosphate buffered saline), 50 μl/well, incubated at 4°C overnight, then blocked with 200 µl/well of 5% chicken serum in PBS/tween (0.05 per cent). Dilution of plasma obtained from immunized R mice, add to each well and incubated for 1-2 hours at room temperature. Tablets are washed with PBS/tween and then incubated with goat polyclonal antibody against the Fc human IgG, conjugated with horseradish peroxidase (HRP)for 1 hour at room temperature. After washing tablets are using ABTS substrate (Sigma, A-1888, 0.22 mg/ml) and analyzed by spectrophotometer at OD 415-495. Mice that have developed the highest titers of antibodies against R use for mergers. The merge is carried out, as described below, and supernatant hybridomas are tested for activity against R using ELISA.

Generation of hybridomas. producing human monoclonal antibodies to IP10:

The mouse splenocytes, isolated from the HuMab mice and mice MILES, merge with Pegs cell line murine myeloma based on standard protocols. Then the resulting hybridoma subjected to screening for the production of antigen-specific antibodies. Unicellular suspension of lymphocytes, pagelogo the second gland from immunized mice merge with one-fourth part of " non-secretory cells of the mouse myeloma SP2/0 (ATS, CRL 1581) with 50% PEG (Sigma). Cells are placed with a density of approximately 1×105/well in flat-bottomed tablet for micrometrology with subsequent incubation for approximately two weeks in selective medium containing 10% calf serum embryos, 10% conditioned medium P388D1 (ATS, CRLTIB-63), 3-5% Origen (IGEN) in DMEM (Mediatech, CRL 10013, with high glucose, L-glutamine and sodium pyruvate)plus 5 mm HEPES, 0.055 mm 2-mercaptoethanol, 50 mg/ml gentamicin and hat (Sigma, CRL P-7185). After 1-2 weeks the cells are cultivated in an environment in which HAT replaces the NT. Then individual wells are screened by ELISA (described above) for human monoclonal IgG antibody against R. After reaching the extensive growth of hybridoma environment usually monitorium 10-14 days. Hybridoma, secreting antibody subcultured again subjected to screening and, if still positive for human IgG, monoclonal antibodies against R subcloning at least twice by limiting dilution. Stable subclones are then cultivated in vitro to generate small amounts of antibody in the medium for tissue cultures for further characterization.

For further analysis of selected hybridoma clones 1D4, E, 2G1, S, A, A, V, S, 8F6, A and S.

Example 2: Structural characterization of human monoclonal the x-antibodies against IP-10

The cDNA sequence encoding the variable region light and heavy chains of a monoclonal antibody 1D4, E, 2G1, S, A, A, V, S, 8F6, A and C receive from the respective hybridomas using standard PCR methods, and is sequenced using standard methods of DNA sequencing. In cases where DNA sequencing is not enough to uniquely determine the structure of antibodies, conduct analysis of the protein (for example, analysis of N-terminal amino acid and mass spectroscopy and the results compared to the analysis of DNA sequences in order to determine the correct structure of antibodies. The following results are obtained structural analysis:

Sequences of nucleotides and amino acids of the variable region of the heavy chain 1D4 presented on Figure 1 and in SEQ ID NONO:99 and 35, respectively. Sequences of nucleotides and amino acids of the variable region light chain 1 D4 presented on Figure 2 and in SEQ ID NONO:110 and 84, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain E presented on Figure 3 and in SEQ ID NONO:100 and 36, respectively. Sequences of nucleotides and amino acids of the variable region light chain 1 E1 presented on Figure 4 and in SEQ ID NONO:111 and 85, respectively.

The sequence of nucleotides and amino acids of the variable region and the heavy chain 2G1 presented on the Figure - SEQ ID NONO:101 and 37, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain 2G1 presented on the Figure - SEQ ID NONO:112 and 86, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain IS presented in Figure 7 and SEQ ID NONO:102 and 38, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain IS presented on Figure 8 and SEQ ID NONO:113 and 87, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain A presented in Figure 9 and SEQ ID NONO:103 and 39, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain A presented in Figure 10 and SEQ ID NONO:114 and 88, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain A presented on Figure 11 and SEQ ID NONO; 104 and 40, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain A presented in Figure 12 and SEQ ID NONO:115 and 89, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain V presented in Figure 13 and SEQ ID NONO:105 and 41, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain V presented in Figure 14 and SEQ ID NONO:116 and 90, respectively.

P the coherence of nucleotides and amino acids of the variable region of the heavy chain IS presented in Figure 15 and SEQ ID NONO:106 and 42, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain IS presented in Figure 16 and SEQ ID NONO:117 and 91, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain 8F6 presented in Figure 17 and SEQ ID NONO:107 and 43, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain 8F6 presented in Figure 18 and SEQ ID NONO:118 and 92, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain A presented in Figure 19 and SEQ ID NONO:108 and 44, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain A presented in Figure 20 and SEQ ID NONO:119 and 93, respectively.

Sequences of nucleotides and amino acids of the variable region of the heavy chain IS presented in Figure 21 and SEQ ID NONO:109 and 46, respectively. Sequences of nucleotides and amino acids of the variable region of the light chain IS presented in Figure 22 and SEQ ID NONO:120 and 94, respectively.

Comparison of sequences of the heavy chain immunoglobulin 1D4, E, 2G1, A, A, S and A with the known sequences of the heavy chains of human immunoglobulin germline demonstrates that the heavy chains of this antibody was used segment VHfrom the human C is radisheva line VH 3-33. Alignment sequences VH1D4, E, 2G1, A, A, S and A sequences of germline VH 3-33 (SEQ ID NO:47) is shown in Figure 23. Subsequent sequence analysis of the VH1D4, E, 2G1, A, A,S and A using the system to identify areas Kabat CDR leads to the construction of the scheme plots CDR1, CDR2 and CD3 heavy chain, as shown in Figures 1, 2, 5, 9, 11, 15 and 19, respectively.

Comparison of sequences of the heavy chain immunoglobulin V and 8F6 with the known sequences of the heavy chains of human immunoglobulin germline demonstrates that in these heavy chains of the antibodies used VHfrom human germline VH 3-30 .3. Alignment sequences Vk6B10 and 8F6 with a sequence of germline VH 3-33 (SEQ ID NO:48) is shown in Figure 24. Subsequent sequence analysis of the VH6B10 and 8F6 using the system to identify areas Kabat CDR leads to the construction of the scheme plots CDR1, CDR2 and CDR3 of the heavy chain as shown in Figures 13 and 17, respectively.

Comparison of the sequences of the heavy chain immunoglobulin S with the known sequences of the heavy chains of human immunoglobulin germline demonstrates that in these heavy chains of the antibodies used segment VHfrom the human germline VH 5-51. Elaine is UNT sequence V HS with a sequence of germline VH 5-51 (SEQ ID NO:49) is shown in Figure 25. Subsequent sequence analysis of the VH S using the system to identify areas Kabat CDR leads to the construction of the scheme plots the heavy chain CDR1, CDR2 and CDR3 as shown in Figure 7.

Comparison of the sequences of the heavy chain immunoglobulin S with the known sequences of the heavy chains of human immunoglobulin germline demonstrates that in these heavy chains of the antibodies used segment VHfrom human germline VH 4-61. Alignment sequence VHS with a sequence of germline VH 4-61 (SEQ ID NO:50) is shown in Figure 26. Subsequent sequence analysis of the VHS using the system to identify areas Kabat CDR leads to the construction of the scheme plots the heavy chain CDR1, CDR2 and CDR3 as shown in Figure 21.

Comparison of sequences of the light chain immunoglobulin 1D4, 2G1, A, A, A and S with the known sequences of the light chains of human immunoglobulin germline shows that the light chains of this antibody was used segment VLfrom human germline Vk A27. Alignment sequences VL1D4, 2G1, A, A, A and S sequence germline VK A27 (SEQ ID NO:95) is shown in Figure 7. Subsequent sequence analysis VL 1D4, 2G1, A, A, A and S using the system to identify areas Kabat CDR leads to the construction of the scheme plots CDR1, CDR2 and CDR3 of light chain, as shown in Figures 2, 6, 10, 12, 20 and 22, respectively.

Comparison of sequences of the light chain immunoglobulin E, V and 8F6 with the known sequences of the light chains of human immunoglobulin germline shows that the light chains of antibodies used segment VLfrom human germline Vk L6. Alignment sequences VL1E1, V and 8F6 with a sequence of germline VK L6 (SEQ ID NO:96) is shown in Figure 28. Subsequent sequence analysis of the VL1 E1, V and 8F6 using the system to identify areas Kabat CDR leads to the construction of the scheme plots CDR1, CDR2 and CDR3 of light chain, as shown in Figures 4, 14 and 18, respectively.

Comparison of sequences of the light chain immunoglobulin S with the known sequences of the light chains of human immunoglobulin germline shows that the light chain IS used segment VLfrom human germline Vk L18. Alignment sequences VL3C4 sequence germline Vk L18 (SEQ ID NO:97) is shown in Figure 29. Subsequent sequence analysis of the VL3C using the system to identify areas Kabat CDR leads to the construction of the scheme plots CDR1, CDR2 and CDR3 of light chain, as shown in Figure 8.

Comparison of sequences of the light chain immunoglobulin S with the known sequences of the light chains of human immunoglobulin germline shows that the light chain IS used segment VLfrom human germline Vk L15. Alignment sequences VLS sequence germline Vk L15 (SEQ ID NO:98) is shown in Figure 30. Subsequent sequence analysis of the VLS using the system to identify areas Kabat CDR leads to the construction of the scheme plots the light chain CDR1, CDR2 and CDR3 as shown in Figure 16.

Example 3: Characterization of binding specificity and binding kinetics of human monoclonal antibodies against IP-10

In this example, the binding affinity of the binding kinetics and binding specificity of antibodies against IP-10 explore using Biacore analysis. In addition, the binding specificity and cross-competition examined using ELISA.

The Biacore analysis

Antibodies against IP-10 is characterized by the affinity and binding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Expressed E. coli purified recombinant human IP-10 (R& D Systems) associated with the sensor chip CM @ 97 RU, using the Protocol binding EDC/NHS, presents Biacore AB. Binding measure is, omitting the antibody in the stream buffer HBS-EP (represented by Biacore AB) at concentrations 33-267 nm at a flow rate of 40 μl/min For the kinetics of Association of the antigen with the antibody watch for 5 minutes and kinetics of dissociation was monitored for 8 minutes. Curves of the Association and dissociation of lead in the model binding Langmuir 1:1, using BIAevaluation (Biacore AB). Data corresponding to the original a few dozen seconds, separately for phases Association and dissociation consider when aligning curves in order to minimize the effect of avidity. Experiments are conducted both at 25°C and at 37°C. values of KD, konand koffthat control is given in Table 1 for binding at 25°C and in Table 2 for binding at 37°C:

/tr>
Table 1
Characterization of binding at 25°C With human IP-10
Clone NoThe affinity of KD×10-9(M)The velocity at the inlet Kon×104(1/Mc)The speed of the output Kon×10-5(1/c)
S0,021,700,04
A0,533,832,02
8F60,8123,319,0
10A12S0,883,683,24
A1,203,11of 3.64
A Sample 2*0,963,203,10
1D41,208,4010,20
W1,789,5017,2
2G11,903,780,75
* A Sample 2 represents an independent sample of purified antibody from the supernatant of hybridoma A relative to the specimen A.

Table 2
Characterization of the binding of n and 37°C With human IP-10
Clone NoThe affinity of KD×10-9(M)The velocity at the inlet Kon×104(1/Mc)The speed of the output Kon×10-5(1/c)
S0,0165,270,08
Clone NoThe affinity of KD×10-9(M)The velocity at the inlet Kon×104(1/Mc)The speed of the output Kon×10-5(1/c)
A0,3411,13,81
8F60,7834,727,0
10A12S0,499,104,43
A1,7010,27,15
A Sample 20,74to 8.416,26
1D41,15 16,619,2
W2,54to 19.950,4
2G10,3414,8equal to 4.97

The period of existence of antibodies (in hours), as determined by the time required for dissociation half of the complex of antibody-antigen during phase dissociation, measured at 25°C and 37°C. Values are determined by the expansion of the dissociation curves before reaching the time required for a 50% reduction in the Y-axis sensogram dissociation. The results are presented below in Table 3:

Table 3
The period of existence of antibodies at 25°C and 37°C
Clone NoThe floor of the lifetime (in hours) at 25°CThe period of existence (in hrs.) at 37°C
2G125,67a 3.87
Aat 9.53of 5.05
10A12S5,944,34
Aof 5.292,69
A Sample 26,21a 3.87
1D41,881,00
W1,120,38
8F61,010,71

The cross-reactivity of antibodies at 25°C With IP-10 rhesus macaques, human MIG, human ITAC and murine IP-10 determined by Biacore analysis, using the same methods as described above for human IP-10. Human MIG, human IP-10 and murine IP-10 receive commercial way (PeproTech, Rocky Hill, NJ), whereas IP-10 rhesus macaques produced by recombinant expression and purified by standard methods. Antigens kongugiruut touch chip SM @ 140 Eng (IP-10 rhesus macaques), 457 (RUs human MIG), Eng 206 (human ITAC) and 150 (RUs murine IP-10). Sensogram Association is produced by passing a stream of antibody with buffer HBS-EP at a concentration of 133 nm for 5 minutes. Then the stream stopped and monitorium dissociation within 5 minutes. Curves of the Association and dissociation of lead in accordance with the Langmuir binding model, ISOE is esua program BIAevaluation (Biacore AB). The results of the experiments on cross-reactivity summarize in Table 4:

Table 4
Cross reactivity against IP-10 with different ligands of CXCR3
Clone NoIP-10 RH-KD×10-9(M)MIG person KD×10-9(M)The ITAC person KD×10-9(M)IP-10 mouse KD×10-9(M)
S4,4105,0No binding464,0
A0,71161,0No bindingNo binding
8F60,81No bindingNo bindingNo binding
10A12S1,21722,0No bindingNo binding
A 1,06No bindingNo bindingNo binding

Clone NoIP-10 RH-KD×10-9(M)MIG person KD×10-9(M)The ITAC person KD×10-9(M)IP-10 mouse KD×10-9(M)
A Sample 2*1,23No bindingNo bindingNo binding
1D40,94No bindingNo bindingNo binding
W2,0115,451,5105,0
2G10,2670,1No bindingNo binding

The ELISA

Additional experiments carried out using the ELISA to study the antigenic cross-reactivity of antibodies against IP-10 in human MIG, IP-10 rhesus macaques or murine IP-10. Techniques used for ELISA, the same as described above in Example 1 except that the tablets for micrometrology cover 1 μg/ml of either recombinant human IP-10, human MIG (PeproTech, No catalog 300-26), murine IP-10 (PeproTech, No No directory 250-16)or recombinant IP-10 rhesus macaques. The results, expressed as EC50(ng/ml) are summarized below in Table 5:

Table 5
Cross reactivity against IP-10 by ELISA data with different ligands of CXCR3
Clone NoIP-10 person (EC50ng/ml)IP-10 monkey (EC50ng/ml)MIG person (EC50ng/ml)IP-10 mouse
A4080180No binding
10A12Sa 4.9the 15.6380No binding
2G13030 45No binding
A3590No bindingNo binding
A Sample 262125No bindingNo binding
W304520No binding
8F69031No bindingNo binding

Clone NoIP-10 person (EC50ng/ml)IP-10 monkey (EC50ng/ml)MIG person (EC50ng/ml)IP-10 mouse
1D42562No bindingNo binding

Studies of cross-competition between different antibodies against IP-10 Ave is lead by using ELISA, using biotinylated form A and 2G1, to define, recognize whether antibodies to different epitopes on IP-10. The technique of competitive ELISA similar to the ELISA described above in Example 1. Briefly, tablets for micrometrology cover purified recombinant IP-10 from E. coli at a concentration of 0.2 μg/ml in PBS, 50 μl/well and incubated at 4°C over night. Then the wells are blocked with 200 µl/well of 5% chicken serum in PBS/tween (0.05 per cent). Reconstitution of purified human antibodies against IP-10 person (2 μg/ml to 3,91 ng/ml) added to each well and incubated for 30 minutes at room temperature. Tablets washed RW/tween and then incubated with 0.1 µg/ml Biotin-A or Biotin-201 for 30 minutes. Then the tablets thrice washed with PBS/tween. After washing each well add phosphatase labeled with streptavidin (KPL, No catalog: 15-30-00), at a dilution of 1:2000 in 5% chicken serum and incubated for 1 hour at room temperature. After washing tablets are substrate p-NPP (Moss, Inc., party 10274021) and analyzed by spectrophotometer at OD 405. As expected, unlabeled 2G1 can compete with Biotin-201 for binding to IP-10 and, moreover, each of the unlabeled A, S, A, 10A12S, V, 8F6 and 1D4 able to compete for binding Biotin-201 with human IP-10. Similarly, unlabeled A can compete with Biotin-A for binding to IP-10 and bol is e, each of unlabeled 2G1, S, A, 10A12S, V, 8F6 and 1D4 able to compete for binding Biotin-A with human IP-10. These results show that each of these antibodies has specificity of binding to the same epitope (or a group of epitopes), human IP-10.

Example 4: Inhibition of binding of IP-10 to CXCR3

In this example examined the capacity of antibodies against IP-10 to inhibit the binding of human IP-10 with its receptor, CXCR3, in expressing the receptor cells. First, analyze Scatchard binding125R-10 with 300.19 cells, transfitsirovannykh so that they are expressed CXCR3. Cells are grown in RPMI media containing 10% FCS and selective background G418. Before use, cells are washed twice with balanced salt solution, Henk (HBSS) at 4°C and down to density of 4×107cells/ml Fiberglass plates (Millipore Multiscreen®, No catalog MAFBNOB50) block 200 μl of 0.1% solution of polyethylenimine one day before the experiment. Daily study of the blocking buffer is sucked off through a Millipore manifold. Tablets washed three times with 200 μl of binding buffer (50 mm HEPES, pH of 7.2, 1 mm CaCl2, 5 mm MgCl2, and 0.5% BSA). To each well add twenty-five microlitres binding buffer, and then either 25 μl of 1000-fold excess unlabeled IP-10, or the linking buffet is. Add twenty-five microlitres125-IP-10 (Amersham, No No directory IM332-25 µci) with increasing concentrations, and then 25 μl of cells with a density of 1×106cells/well. Tablets incubated on a shaking device for a tablet for 60 minutes at room temperature and washed three times with buffer wash (10 mm HEPES, pH of 7.2, 0.5 M NaCl, 0,5% BSA) at a volume of 200 μl/wash. The tablets are dried, add 25 μl of scintillation agent, and count the tablets on the counter Wallac Microbeta Counter. Data analyzed using the program Prism and calculate the KD. For binding of the receptor to determine the average KD0,231 nm.

Then examine the ability of antibodies against IP-10 to inhibit the binding of 100 PM125I-hlP-10 cells expressing CXCR3. Competitive tests conducted similarly to the above experiment. Briefly, fiberglass tablets for filtering add 25 ál of binding buffer, and then 25 μl of increasing concentrations of antibodies against IP-10. Twenty-five microlitres125I-IP-10 added to a final concentration 0,100 nm. Finally, add 25 ál of cells with a density of 1×106the hole and tablets incubated on the shaker for tablets for 60 minutes at room temperature, washed and counted as described above. The values of EC50calculate, using the program Prism. Value is of To, (nm) is determined using the following formula:

Ki=EC501+[L]/KD

The results are summarized below in Table 6:

Table 6
Inhibition of binding of IP-10 to CXCR3
Clone NoKi (nm)
A0,09
10A12S0,06
2G10,09
8F60,16
E0,29
W0,30
S0,41
A0,67
A Sample 20,35
1D40,86

Example 5: Inhibition induced IP-10 flow of calcium

In this when the ore investigate the ability of antibodies against IP-10 to inhibit induced IP-10 current calcium using either 300.19 cells, transfected so that they Express CXCR3, or activated against CD3 human peripheral blood lymphocytes (PBLs)that Express CXCR3. To obtain PBL of normal human blood cleanse the standard division in ficulle. Purified human PBL stimulated by causing cells to plates coated with 3 μg/ml antibodies against CD3 and grown in RPMI medium with addition of 10% FBS. Following three days of incubation cells sustain in the medium for growth, containing 500 units/ml IL-2. On the day of the study, the cells are washed and resuspended in media with a density of 2.5×107cells/ml Cells 300.19, transfetsirovannyh in such a way that they Express CXCR3, grown in RPMI containing 10% FBS. 300.19 cells resuspended in the environment for growth with a density of 2×106cells/ml

For analysis, 100 µl of cell suspension added to 96-well plate with black side walls and a transparent bottom, which is covered with poly-lysine (Corning/Costar, No catalog 3667). 100 microlitres set loading dye Calcium 3 (FlexStation™, Molecular Devices, Inc., Sunnyvale, CA) is added to each well, tablets centrifuged at 1100 rpm./min for 4 minutes and incubated at 37°C for 30 minutes. Using 96-well plate with a reagent of human IP-10 (Peprotech, No catalog 300-12) diluted in balanced salt solution is ENKA 20 mm HEPES and 1% FBS (800 PM for 300.19 cells and 1200 PM for human PBL). Antibody serially diluted in the tablet reagent containing IP-10. Include control wells containing only buffer or only IP-10. In tablets containing dye-labeled cells, add twenty-two microliters of a solution of IP-10/antibody/well and determine the calcium current by monitoring the fluorescence of Calcium 3 when using devices FlexStation™ according to the manufacturer's instructions with a period of 200 seconds. The area under the curve (AUC) is calculated by intellibeam current calcium in the range of 20 to 100 seconds according to standard protocols (see, for example, article Smart D. et al., (1999), Br. J. Pharmacol., 128:1-3). Data analyzed using Prism™ software (Molecular Devices, Inc.) determine the values of the IC50(in nm). The results are summarize in the following Table 7:

Table 7
The inhibition induced IP-10 flow of calcium
Clone NoIC50(nm) for HPBL*IC50(nm) for 300.19 cells
A1,250,18
10A12S2,310,08
2G10,50
8F66,270,30
Eof 3.64Not tested
W4,400,50
S8,150,77
A2,850,61
A Sample 22,180,42
1D44,070,34
* Human peripheral blood leukocytes

Example 6: Inhibition induced IP-10 migration of cells

The ability of antibodies against IP-10 to inhibit the migration of cells induced IP-10, research in the analysis of chemotaxis in vitro. In these experiments use 300.19 cells expressing CXCR3 and stimulate them either: (i) 100 ng/ml recombinant human IP-10 (rhlP-10); (ii) the supernatant of cells TNR-1-stimulated IFN-γ (which induces the secretion of native IP-10 and MIG, and IP-10, isolated from TNR-1, used at a concentration of 16 ng/ml, and AK is Yunosti MIG blocked by adding anti-MIG (R& D Systems) at a concentration of 2.5 µg/ml, or (iii) 100 ng/ml recombinant IP-10 rhesus macaques (rrmlP-10). Inhibition of cell migration assessed using different concentrations of antibodies and determine hemotoxicity index.

In particular, Inhibition of chemotaxis assessed using standard analysis on 96-well plates (Multiscreen MIC plates (Millipore)). For transfected cell lines using a filter of 5 microns and primary cells using the filter 3 µm. Reactive cells (300.19 CXCR3+; MBP-specific human cells RVMS) resuspended in the buffer for chemotaxis (RPMI+1% BSA or FBS) with a density of 1×106cells/ml 100 ál of cell suspension added to the upper well and left for incubation for 30 minutes at 37°C. Before adding to the bottom hole impose 5% CO2.

IP-10 human and rhesus macaques are prepared at a concentration of 100 ng/ml IP-10, obtained from TNR-1, cells TNR-1 stimulate ifn-k (0.2 ng/ml) and collect the supernatant; IP-10 from PC CSF use purified. Ligand receive in 150 ál of buffer for chemotaxis and placed in the lower chamber.

For analysis of inhibition of chemotaxis ligand pre-incubated with various concentrations of the indicated antibodies against IP-10 (5 μg/ml, 2.5 μg/ml to 1.25 mg/ml, 0,613 μg/ml, 0.3 ág/ml of 0.015 µg/ml) for 30 minutes at 37°C in 5% CO2prior to analysis. Ve is hnwu the camera is placed on the wells and incubated in the wells for 2 hours at 37°C in 5% CO 2. At the end of the incubation reactive cells are sucked off from the top of the camera. Upper chamber carefully removed. Cells counted in 4 random fields/well at magnification 400x. Data on average three holes and is presented as a chemotaxis index (i.e. reduced migration in response to ligand relative to one environment).

The results, expressed as the values of the IC50, summarize in Table 8 below:

Table 8
The inhibition induced IP-10 cell migration
Clone NorhlP-10; IC50(ág/ml)IP-10 THP-1; IC50(ág/ml)rrmlP-10; IC50(ág/ml)
A (Sample 2)0,1560,1320,119
8F60,3550,1730,100
W1,10,1930,149
10A12S0,1150,21115,1
1D40,1560,2470,163

Investigate the ability of antibodies against IP-10 to inhibit the migration of expressing CXCR3 300.19 cells in response to cerebrospinal fluid (CSF) of patients with multiple sclerosis (PC). Again the antibody against MIG at a concentration of 2.5 µg/ml added to sample SRDS neutralizing activity MIG. The results of the two experiments, expressed as the values of the IC50as summarized in Table 9:

Table 9
The inhibition induced PC-CSF cell migration
Clone NoExperiment 1 IC50(ág/ml)Experiment 2 IC50(ág/ml)
A (Sample 2)0,1000,236
10A12S0,5620,577

Studies of cell migration is done using expressing CXCR3 300.19 cells and recombinant human MIG (R&D Systems) at a concentration of 200 ng/ml to study the ability of antibodies against IP-10 to inhibit induced MIG migration of cells. Antibodies against IP-10 V, 8F6, 1D4,A sample 2, A and 10A12S tested individually at a concentration of 1 μg/ml and find that they do not inhibit induced MIG migration of cells.

Example 7: the binding of the antibody preparations of the brain of patients with PC

In this example examined the capacity of antibodies against IP-10 painted brain slices obtained from patients with multiple sclerosis (PC). Brain slices sick PC women aged 57 years, get through to 19.8 hours after death, and show the presence of periventricular plaques wrong form (1.3 cm×1.0 cm) with many additional plaques smaller present in the rest of the white matter. LFB staining demonstrated a complete demyelination. Performed immunohistochemical analysis of sections using antibodies A (sample 2) and 10A12S against IP-10, and agents against GFAP and against CD68 as a positive control and the antibody for the negative control.

For staining sections against IP-10 using standard immunohistochemical method. Slices block using 2%-10% serum. Add 100 ál/slice primary antibodies (for example, A), diluted 1:100 in 2% serum, and then incubated for 1 hour at room temperature. The optional sections incubated overnight at 4°C and washed. Then add the secondary biotinylated antibody against human (intelligence is Noah in 2% serum (100 µl/slice)) and incubated for 30-60 minutes at room temperature. Endogenous peroxidase removed by the addition of dilute H2O2in MeOH. Then add a solution of ABC (Vector Labs), 100 μl/slice and incubated for 30 minutes at room temperature. The DAB substrate solution prepared immediately before use. On the subject of glass put 100 ál/slice before incubation in the dark for 10-20 minutes (as required for the manifestation). Then spend a contrast staining slides nuclear dye with hematoxylin for 3 minutes (display check after 1 minute). Finally, slides are incubated in a 2% sodium bicarbonate for 45 seconds for the display of color.

The results show that as A and 10A12S can contact IP-10 in situ in brain slices of the patient PC, and the binding A more intensively than linking 10A12S.

Example 8: Construction of lines of transgenic mice NSO

A line of transgenic mice NSO generate joint injection inserts 80 TPN RNs (see Taylor et al., (1994), Int. Immunol., 6:579-591), inserts 25 KBP pVX6 and fragment artificial chromosomes in yeast ~460 TPN chromosome yIgH24. The construction itself RNs fully capable of rearrangeable in vivo with the formation of functional loci heavy chain of human immunoglobulin; pVX6 and yIgH24 enter to participate with the creation of additional diversity VHembryonic liniile components of the mixture of DNA, used to obtain NSO described below.

The above insert RNs includes four functional gene segment VHthe human germ line: 1-69 (DP-10), 5-51 (DP-73), 4-34 (DP-63) and 3-30 .3 (DP-46). In addition, this design also contains the human genomic sequence, including 15 functional D segments all 6 J segments, and the segments µ and γ1 constant region and functional area switch µ-γ1.

Insert pVX6 contains 3 segments VHhuman germline VH1-18 (DP-14), VH5-51 (DP-73) and VH3-23 (DP-47). The DNA fragment of 8.5 TPN Hindlll/Sall, including gene VH1-18 (DP-14) human germ line, together with approximately 2.5 TPN 5'-flanking and 5 TPN 3'-wandering genomic sequence subcloning in plasmid vector RR (Promega, Madison, Wis.) to generate plasmids R. The DNA fragment of the BamHI/Hindlll 7 TPN containing the gene of the human germline VH5-51 (DP-73), together with approximately 5 TPN 5'-flanking and 1 TPN 3'-wandering genomic sequence, clone in plasmid cloning vector pGP1f based on pBR322 (see Taylor et al., (1992), Nucleic acids Res., 20:6287-6295)to generate plasmids p251f. A new cloning vector derived from pGP1f, pGP1k, cut with EcoRV/BamHI, and are ligated into a DNA fragment 10 TPN EcoRV/BamHI, including gene VH3-23 (DP47) human germ line together with priblizitelen the 4 TPN S'-flanking and 5 TPN 5'-wandering genomic sequence. The resulting plasmid p112.2RR.7 cut BamHI/Sall and are ligated with the purified insert BamHI/Sall p251f 7 TPN the resulting plasmid pVx4 cut with Xhol and are ligated to the insert Xhol/Sall p343.7.16 8,5 TPN Receive clone with the gene VH1-18 in the same orientation that the other two V-gene. This clone, named pVx6, then cut Notl to obtain the inserts.

The original yeast chromosome (YAC)yIgH24 initially identified by screening by PCR using family-specific primers VH3 and VH4 and Carteret on chromosome 14 man on the content of VH. It is established that yIgH24 contains segments VHincluding representatives of the families VH VH1, VH2, VH3, VH4 and VH5 and, in particular, at least VH1-24, VH1-45, VH1-46, VH2-26, VH3-30, V 3-30 .5, VH3-30.3, VH3-33, VH3-43, VH3-48, VH3-49, VH3-53, VH4-28, VH4-30, VH4-30.4, VH4-30.3, VH4-31, VH4-34, 4-39 and VH5-51.

Purified inserts from pVX6 (26 TPN), RNs (80 TPN) and yIgH24 (pribl TPN) are combined in a molar ratio of 1:1:1 and microinjection injected into the pronuclei of half day of F2 embryos (C57BL/6J×DBA/2J), as described by Hogan et al. (see the monograph by C. Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual (Laboratory manual manipulation of mouse embryos), 2nd ed., 1994, Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The original line of transgenic mice containing the sequence of pVx6, HC2 and yIgH24 generated from mice that develop ikinyasiloveniya embryos. This line is called (NSO) 25950.

Then line (NSO) 25950 crossed with mice carrying a mutation CMD (described in Example 1 Of PCT Publication WO01/09187), the JKD mutation (see Chen et al., (1993), EMBO J. 12:811-820) and the transgene (XO)9272 (see Fishwild et al., (1996), Nature Biotechnology, 14:845-851). The resulting mice Express the transgene heavy and Kappa light chains of human immunoglobulin in the background, homozygous for the destruction of the endogenous loci heavy and Kappa light mouse chain.

A BRIEF SEQUENCE LISTING
SEQ ID NO:SequenceSEQ ID NO:Sequence
1amino acids CDR1 VH1D424amino acid CDR3 VH 1D4
2amino acids VH CDR1 1E125amino acid CDR3 VH 1E1
3amino acids VH CDR1 2G126amino acid CDR3 VH 2G1
4amino acids VH CDR1 3C4 27amino acid CDR3 VH 3C4
5amino acids VH CDR1 6A528amino acid CDR3 VH 6A5
6amino acids VH CDR1 6A829amino acid CDR3 VH 6A8
7amino acids VH CDR1 6B1030amino acid CDR3 VH 6B10
8amino acids CDR1 7C10 VH31amino acid CDR3 7C10 VH
9amino acids VH CDR1 8F632amino acid CDR3 VH 8F6

SEQ ID NO:SequenceSEQ ID NO:Sequence
10amino acids VH CDR1 10A1233amino acid CDR3 VH 10A12
11amino acids VH CDR1 10A12S 34amino acid CDR3 VH 13C4
12amino acids VH CDR1 13C435amino acids VH 1D4
13amino acids CDR2 VH 1D436the VH amino acids 1 E1
14amino acids VH CDR2 1E137the VH amino acid 2G1
15amino acids VH CDR2 2G138the VH amino acid 3C4
16amino acids VH CDR2 3C439the VH amino acid 6A5
17amino acids VH CDR2 6A540the VH amino acid 6A8
18amino acids VH CDR2 6A841the VH amino acid 6B10

SEQ ID NO:SequenceSEQ ID NO:Sequence
19amino acids VH CDR2 6B1042amino acids 7C10 VH
20amino acids CDR2 7C10 VH43the VH amino acid 8F6
21amino acids VH CDR2 8F644the VH amino acid 10A12
22amino acids VH CDR2 10A1245the VH amino acid 10A12S
23amino acids VH CDR2 13C446the VH amino acid 13C4
47amino acids germline VH 3-3349amino acids germline VH 5-51
48amino acids germline VH3-30.3 50amino acids germline VH 4-61
51amino acids Vk CDR1 1D473amino acids Vk CDR3 1D4

SEQ ID NO:SequenceSEQ ID NO:Sequence
52amino acids Vk CDR1 1E174amino acids Vk CDR3 1E1
53amino acids CDR1 Vk2G175amino acid CDR3 Vk2G1
54amino acids CDR1 Vk3C476amino acid CDR3 Vk3C4
55amino acids Vk CDR1 6A577amino acid CDR3 Vk6A5
56amino acids Vk CDR1 6A878amino acid CDR3 Vk6A8
57amino is islote Vk CDR1 6B10 79amino acid CDR3 Vk6B10
58amino acids Vk CDR1 7C1080amino acid CDR3 Vk7C10
59amino acids Vk CDR1 8F681amino acid CDR3 Vk8F6
60amino acids Vk CDR1 10A1282amino acids Vk CDR3 10A12
61amino acids Vk CDR1 13C483amino acids Vk CDR3 13C4

SEQ ID NO:SequenceSEQ ID NO:Sequence
62amino acids Vk CDR2 1D484amino acids Vk 1D4
63amino acids Vk CDR2 1E185 amino acids Vk 1E1
64amino acids CDR2 Vk2G186amino acids Vk 2G1
65amino acids CDR2 Vk3C487amino acids Vk 3C4
66amino acids Vk CDR2 6A588amino acids Vk 6A5
67amino acids Vk CDR2 6A889amino acids Vk 6A8
68amino acids Vk CDR2 6B1090amino acids Vk 6B10
69amino acids Vk CDR2 7C1091amino acids Vk 7C10
70amino acids Vk CDR2 8F692amino acids Vk 8F6

SEQ ID NO:SequenceSEQ ID NO:Posledovatelno the ü
71amino acids Vk CDR2 10A1293amino acids Vk 10A12
72amino acids Vk CDR2 13C494amino acids Vk 13C4
95amino acids germline VkA2797amino acids germline VkL18
96Vk L6 amino acids germ line98amino acids germline Vkl15
99nucleotides VH 1D4121amino acids of human IP-10
100nucleotides VH E122amino acids of human CXCR3
101nucleotides VH 2G1123amino acids IP-10 rhesus macaques
102nucleotides VH 3C4124amino acids of murine IP-10

SEQ ID NO:SequenceSEQ ID NO:Sequence
103nucleotides VH 6A5125amino acids of human MIG
104nucleotides VH A126amino acids of human ITAC
105nucleotides VH 6B10
106nucleotides VH S
107nucleotides VH 8F6
108nucleotides VH A
109nucleotides VH S
110nucleotides Vk 1D4
111nucleotides Vk E
112nucleotides Vk 2G1
113nucleotides Vk 3C4
114nucleotides Vk 6A5
115nucleotides Vk A

SEQ ID NO:SequenceSEQ ID NO:Sequence
116nucleotides Vk 6B10
117nucleotides Vk 7C10
118nucleotides Vk 8F6
119nucleotides Vk A
120nucleotides Vk 13C4

Equivalent solutions

Competent professionals in the field of technology know or are able to install using no more than routine experimentation, many equivalent solutions to specific embodiments of the invention described in this context. It is envisaged that subsequent claims includes data equivalent solutions

1. The selected monoclonal antibody or its antigennegative part, with the ability to specifically bind with IP-10, with the indicated antibody or antigennegative part includes:
Variable regions CDR1 heavy chain comprising SEQ ID NO:1,
CDR2 variable region heavy chain comprising SEQ ID NO:13,
CDR3 variable region heavy chain comprising SEQ ID NO:24,
Variable regions CDR1 is egcoa circuit, comprising SEQ ID NO:51,
CDR2 variable region light chain comprising SEQ ID NO:62 and
CDR3 variable region light chain comprising SEQ ID NO:73;
Variable regions CDR1 heavy chain comprising SEQ ID NO:5,
CDR2 variable region heavy chain comprising SEQ ID NO:17,
CDR3 variable region heavy chain comprising SEQ ID NO:28,
Variable regions CDR1 light chain comprising SEQ ID NO:55,
CDR2 variable region light chain comprising SEQ ID NO:66 and
CDR3 variable region light chain comprising SEQ ID NO:77;
Variable regions CDR1 heavy chain comprising SEQ ID NO:9,
CDR2 variable region heavy chain comprising SEQ ID NO:21,
CDR3 variable region heavy chain comprising SEQ ID NO:32,
Variable regions CDR1 light chain comprising SEQ ID NO:59,
CDR2 variable region light chain comprising SEQ ID NO:70 and
CDR3 variable region light chain comprising SEQ ID NO:81;
Variable regions CDR1 heavy chain comprising SEQ ID NO:2,
CDR2 variable region heavy chain comprising SEQ ID NO:14,
CDR3 variable region heavy chain comprising SEQ ID NO:25,
Variable regions CDR1 light chain comprising SEQ ID NO:52,
CDR2 variable region light chain comprising SEQ ID NO:63, and
CDR3 variable region light chain comprising SEQ ID NO:74;
Variable regions CDR1 heavy chain comprising SEQ ID NO:3,
CDR2 variable regions of the heavy chain is, comprising SEQ ID NO:15,
CDR3 variable region heavy chain comprising SEQ ID NO:26,
Variable regions CDR1 light chain comprising SEQ ID NO:53,
CDR2 variable region light chain comprising SEQ ID NO:64, and
CDR3 variable region light chain comprising SEQ ID NO:75;
Variable regions CDR1 heavy chain comprising SEQ ID NO:4,
CDR2 variable region heavy chain comprising SEQ ID NO:16,
CDR3 variable region heavy chain comprising SEQ ID NO:27,
Variable regions CDR1 light chain comprising SEQ ID NO:54,
CDR2 variable region light chain comprising SEQ ID NO:65 and
CDR3 variable region light chain comprising SEQ ID NO:76;
Variable regions CDR1 heavy chain comprising SEQ ID NO:6,
CDR2 variable region heavy chain comprising SEQ ID NO:18,
CDR3 variable region heavy chain comprising SEQ ID NO:29,
Variable regions CDR1 light chain comprising SEQ ID NO:56,
CDR2 variable region light chain comprising SEQ ID NO:67 and
CDR3 variable region light chain comprising SEQ ID NO:78;
Variable regions CDR1 heavy chain comprising SEQ ID NO:7,
CDR2 variable region heavy chain comprising SEQ ID NO:19,
CDR3 variable region heavy chain comprising SEQ ID NO:30,
Variable regions CDR1 light chain comprising SEQ ID NO:57,
CDR2 variable region light chain comprising SEQ ID NO:68
CDR3 variable region light chain comprising SEQ ID NO:79;
Variable regions CDR1 heavy chain comprising SEQ ID NO:8,
CDR2 variable region heavy chain comprising SEQ ID NO:20,
CDR3 variable region heavy chain comprising SEQ ID NO:31,
Variable regions CDR1 light chain comprising SEQ ID NO:58,
CDR2 variable region light chain comprising SEQ ID NO:69, and
CDR3 variable region light chain comprising SEQ ID NO:80;
Variable regions CDR1 heavy chain comprising SEQ ID NO:10 or 11;
CDR2 variable region heavy chain comprising SEQ ID NO:22,
CDR3 variable region heavy chain comprising SEQ ID NO:33,
Variable regions CDR1 light chain comprising SEQ ID NO:60,
CDR2 variable region light chain comprising SEQ ID NO:71, and
CDR3 variable region light chain comprising SEQ ID NO:82;
Variable regions CDR1 heavy chain comprising SEQ ID NO:12,
CDR2 variable region heavy chain comprising SEQ ID NO:23,
CDR3 variable region heavy chain comprising SEQ ID NO:34,
Variable regions CDR1 light chain comprising SEQ ID NO:61,
CDR2 variable region light chain comprising SEQ ID NO:72, and
CDR3 variable region light chain comprising SEQ ID NO:83.

2. The antibody or antigennegative part according to claim 1, which contains:
Variable regions CDR1 heavy chain comprising SEQ ID NO:5,
CDR2 variable region heavy chain comprising SEQ ID NO:17,
CDR3 variable region heavy the ETUI, comprising SEQ ID NO:28,
Variable regions CDR1 light chain comprising SEQ ID NO:55,
CDR2 variable region light chain comprising SEQ ID NO:66 and
CDR3 variable region light chain comprising SEQ ID NO:77.

3. The antibody or antigennegative part according to claim 1, which includes:
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:35 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:84;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:39 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:88;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:43 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:92;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:36 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:85;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:37 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:86;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:38 and
variable region light chain, stereoselectivities amino acids of SEQ ID NO:87;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:40 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:89;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:41 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:90;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:42 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:91;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:44 or 45 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:93;
variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:46 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:94.

4. The antibody or antigennegative part according to claim 2, which includes the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:39 and
the variable region of the light chain containing the amino acid sequence of SEQ ID NO:88.

5. The selected monoclonal antibody or its antigennegative part according to any one of claims 1 to 4, which has at least one of the following properties is:
inhibits the binding of IP-10 to CXCR3;
inhibits induced IP-10 current calcium;
inhibits induced IP-10 migration of cells;
is cross-reactive with IP-10 rhesus macaques;
is not cross-reactive with mouse IP-10;
is not cross-reactive with human MIG;
is not cross-reactive with human ITAC.

6. The antibody or antigennegative part according to claim 1, which is a full length antibody isotype lgG1 or lgG4.

7. The antibody or antigennegative part according to claim 1, which is an antibody fragment or single-chain antibody.

8. Composition for treatment of inflammatory and autoimmune diseases based on the specific binding of IP-10 human, comprising a therapeutically effective dose of the antibody or its antigennegative part according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. Immunoconjugate for the treatment of inflammatory and autoimmune diseases based on the specific binding of IP-10 human, comprising the antibody or antigennegative part according to any one of claims 1 to 7, associated with a therapeutic agent.

10. Immunoconjugate according to claim 9, in which therapeutic agent is a cytotoxin.

11. Immunoconjugate according to claim 9, in which therapeutic agent is a radioactive isotope.

12. the song for the treatment of inflammatory and autoimmune diseases based on the specific binding of IP-10 man comprising therapeutically effective dose immunoconjugate on any of p-11 and a pharmaceutically acceptable carrier.

13. Bespecifically molecule for the treatment of inflammatory and autoimmune diseases based on the specific binding of IP-10 human, comprising the antibody or antigennegative part, with the ability to specifically bind with IP-10 man, according to any one of claims 1 to 7, associated with the second functional group having a different binding specificity than the antibody or antigennegative part.

14. Composition for treatment of inflammatory and autoimmune diseases based on the specific binding of IP-10 human, comprising a therapeutically effective dose bespecifically molecule according to item 13 and a pharmaceutically acceptable carrier.

15. The selected nucleic acid molecule encoding the antibody or antigennegative part according to any one of claims 1 to 7.

16. The expression vector comprising the nucleic acid molecule according to item 15.

17. A host cell to obtain the antibody according to any one of claims 1 to 7, containing the expression vector according to item 16.

18. Hybridoma producing the antibody according to any one of claims 1 to 7, obtained from splenocyte, which is isolated from transgenic mice containing the transgene, or transfromation heavy chains of human rights and the transgene or transfromation light C is PI man and sewn with immortalizing cell.

19. The use of antibodies or antigennegative part according to any one of claims 1 to 7 as a component of a medicinal product for the treatment of inflammatory or autoimmune diseases caused by undesirable activity of IP-10.

20. The application of claim 19, in which the disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease), systemic lupus erythematosus, diabetes type I, inflammatory skin disorders (e.g. psoriasis, flat shingles), autoimmune thyroid disease (e.g., graves disease, Hashimoto thyroiditis, Sjogren syndrome, pulmonary inflammation (e.g. asthma, chronic obstructive pulmonary disease, sarcoidosis lungs, autoimmune alveolitis), transplant rejection, spinal cord injury, brain damage (e.g., stroke), neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, gingivitis, inflammation, caused by gene therapy, disease, angiogenesis, inflammatory diseases of the kidney (e.g., IgA nephropathy, membranoproliferative glomerulonephritis, rapidly progressive glomerulonephritis) and atherosclerosis.

21. The use of antibodies or antigennegative the Asti according to any one of claims 1 to 7 as a component of a medicinal product for the treatment of viral or bacterial infection, signal and undesirable activity of IP-10.

22. Use item 21, in which the viral infection is mediated by human immunodeficiency virus (HIV), hepatitis C virus (HCV), herpes simplex virus type I (HSV-1) or virus severe acute respiratory syndrome (SARS).



 

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Antibodies // 2482131

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. There are presented versions of antibodies specifically bound with amino acid residues of murine 1446-1725 Notch1 or human 1446-1735 Notch1. What is disclosed is a coding polynucleotide, an expression vector based on the polynucleotide, a host cell for antibody expression. What is described is a method for producing the antibody with the use of the vector, as well as using the antibodies as a therapeutic agent or in a method of treating the disorders associated with higher signal transmission or higher Notch1 expression.

EFFECT: use of the invention provides the antibodies which reduces the Notch1 signal transmission that can find application in medicine for treating the disorders associated with higher Notch1 expression.

42 cl, 17 dwg, 5 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses CD19-binding agents representing an antibody or an antigen-binding fragment containing an amino acid sequence of a variable region of a heavy chain, and an amino acid sequence of a variable region of a light chain which are bound with human CD 19 with a dissociation constant equal or less 1x10 -7M. The amino acid sequences are presented in the description. There are disclosed nucleic acid coding the heavy and/or light chain of the antibody or the antigen-binding fragment and a ligand conjugate - a therapeutic agent, or its pharmaceutically acceptable salt or solvate for treating a CD19-associated disorder in a mammal specified in a group involving CD19-expressing cancer, chronic leukaemia, B-cell lymphoma, multiple myeloma and a number of the other oncological diseases. The conjugate for treating the disorder in the mammal is used in an effective amount.

EFFECT: use of the presented antibodies or conjugate enables higher survival rate of the patients with oncological diseases expressing CD19, as well as in treating immunopathological diseases.

18 cl, 26 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention discloses an isolated antibody which selectively binds to the C-end of beta-amyloid (Abeta) and is humanised or fully human. The antibody can be a single-chain antibody (scFv), a Fab fragment or a P(ab')2 fragment. The antibody is capable of preventing oligomerisation of Abeta. The invention discloses a nucleic acid sequence which codes the disclosed antibody, a vector and a host cell for producing the antibody, as well as a pharmacological composition for treating neurological disorders, particularly Alzheimer's disease. The invention provides methods for diagnosis using labelled antibodies disclosed herein and treating neurological disorders associated with abnormal accumulation and/or deposition of Abeta in the central nervous system by administering a therapeutically effective amount of a polynucleotide, vector or host cell to a subject.

EFFECT: invention enables successful application of the disclosed antibodies for therapeutic purposes and clinical application since in contrast to mouse antibodies, said antibodies are low- or nonimmunogenic when used in humans.

25 cl, 23 dwg, 1 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: there are described versions of humanized monoclonal anti-factor D antibodies and their functional fragments. There are offered: a coding nucleic acid, an expression vector, as well as a cell for preparing c the antibody containing the vector. What is described is a method for preparing the anti-factor D antibody by cell culture and expressed antibody purification. Also, there are offered: an antibody composition and use of the antibody for treating disorders mediated by a complement system.

EFFECT: higher clinical effectiveness in the diseases related to excessive or uncontrolled activation of the complement system.

32 cl, 9 dwg, 4 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a human antibody or an antigen-binding antibody fragment which specifically binds human nerve growth factor (NGF) with KD in the order of 5 pM or less and does not cross-react with neurotrophin-3 (NT-3). The antibodies are effective in treating inflammatory pain, postoperative suture pain, neuropathic pain, fracture pain, osteoporotic fracture pain, postherpetic neuralgia, pain associated with osteoarthritis, rheumatoid arthritis, cancer, burns, joint pain in gout, as well as such diseases as hepatocellular carcinoma, breast cancer and hepatic cirrhosis. The invention discloses a nucleic acid coding a human antibody or its antibody-binding fragment, an expression vector containing the nucleic acid, and a method for producing the antibody or its antigen-binding fragment. The antibody or its fragment is used to create a pharmaceutical composition for treating the above diseases, as well as a drug preparation additionally containing IL-1 inhibitor, an antiepileptic drug, a cytokine antagonist and another neurotrophin.

EFFECT: specificity of human NGF binding by the antibody or its fragment according to the invention is 2-10 times higher than a binding ability of the antibody or its fragment in relation to rat's or mouse's NGF.

21 cl, 28 tbl, 13 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a binding protein which is specific to IL-13, having 6CDR sections (three light and three heavy chains). The invention discloses a structure of an antibody based on this protein and a conjugate based on the antibody. The invention describes versions of nucleic acid which codes the antibody or structure, and an expression vector, a replication vector and cells bearing such vectors. Described is a method of producing the protein by culturing cells and proteins obtained using said method. Described are compositions based on versions of the proteins.

EFFECT: invention can be used in medicine to treat and diagnose diseases associated with IL-13 activity which negatively affects health.

50 cl, 23 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: there are described versions of polypeptides specifically binding human DR5 each of which contains CDR versions containing a high-limited variety of amino acid sequences. There are described versions of polypeptides fused with at least a portion of protein, viral envelopes specifically binding human DR5. There are offered: a coding nucleic acid, an expression vector, as well as a cell for polypeptide expression containing the vector. What is also described is a method for producing the polypeptide by cell culture and a method for selecting an antigen-binding variable domain using the polypeptide. Besides, there are presented compositions and methods for using for the purpose of treating a malignant tumour and conditions related to the immune system.

EFFECT: use of the invention can find application in medicine in treating and diagnosing the tumour diseases.

88 cl, 29 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is presented is an antibody neutralising endothelial cell infection by human cytomegalovirus (hCMV) characterised by the presence of three heavy chain CDR and three light-chain CDR. There are described: a nucleic acid for antibody expression containing coding NA and a cell based on such expressing NA. There are disclosed: a composition for preventing and treating hCMV based on the antibody and the use of the antibody or NA for preparing a drug for treating hCMV. The invention provides the antibodies the concentration of which are required for 50% neutralisation of hCMV in endothelial cells makes 0.003 mcg/ml or less that can find further application in methods of screening, as well as in diagnosing and treating the hCMV-mediated diseases.

EFFECT: higher effectiveness of the use of the antibodies.

13 cl, 5 dwg, 2 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: protein complex is proposed with improved activity of long-term action and biostability, containing a physiologically active polypeptide, an Fc-domain of immunoglobulin and a non-peptidyl polymer, having three functional ends. The method is disclosed to produce a protein complex containing a physiologically active polypeptide, an Fc-domain of immunoglobulin and a non-peptidyl polymer, having three functional ends A pharmaceutical composition is proposed, which contains the effective quantity of the specified protein complex, having the improved resistance in-vivo of the physiologically active polypeptide.

EFFECT: invention makes it possible to produce a protein complex with improved activity of long-term action and biostability.

24 cl, 3 dwg, 4 tbl, 9 ex

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