Humanised anti-beta 7 antagonistic antibodies and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-beta 7 antibodies; a composition containing said antibody; a method of inhibiting interaction of human integrin beta 7 subunits with a second integrin subunit using said antibody; as well as a version of use of the listed antibodies.

EFFECT: invention can be used to treat chronic inflammatory diseases such as asthma, Crohn's disease, ulcerative colitis, diabetes, complications during organ transplantation and diseases associated with allotransplantation.

47 cl, 22 dwg, 10 tbl, 3 ex

 

In this application, which is not a provisional application filed under 37 CFR § 1.53(b)(1), priority is claimed in accordance with 35 USC § 119(e) based on provisional application U.S. No. 60/607377, filed September 3, 2004, the full contents of which are incorporated in this description by reference.

The technical field

The present invention generally relates to the field of molecular biology and regulation of growth factors. More specifically, this invention relates to modulators of the biological activity of integrins containing subunit beta, as well as to the use of such modulators.

Background of invention

Integrins are α/β heterodimeric receptors on the cell surface involved in several cellular processes, ranging from cell adhesion to gene regulation (Hynes, R.O., Cell, 1992, 69:11-25; and Hemler, M.E., Annu. Rev. Immunol., 1990, 8:365-368). Some integrins are involved in pathological processes and are of great interest as potential targets for developing drugs (Sharar, S.R. et al., Springer Semin. Immunopathol., 1995, 16:359-378). Integrins are involved in immunological processes, such as transport, adhesion and infiltration of leukocytes in the inflammatory response (Nakajima, H. et al., J. Exp.Med., 1994, 179:1145-1154). Adhesive properties of cells are regulated by changes in the expression of integrins, and RA is different inflammatory responses involved different integrins. Butcher, E.C. et al., Science, 1996, 272:60-66. Integrins beta (i.e. alfaretta (α4β7) and alphabet (αEβ7)) is expressed predominantly on monocytes, lymphocytes, eosinophils, basophils and macrophages, but not neutrophils. Elices on, M.J. et al., Cell, 1990, 60:577-584. The main ligands of integrin α4β7 are endothelial surface proteins, which include cell adhesion molecules - the mucosal adressin (MAdCAM) and adhesion molecules vascular cells (VCAM-I) (Makarem, R. et al., J. Biol. Chem., 1994, 269:4005-4011). The α4β7 binding to MAdCAM and/or VCAM, expressed on the outer endothelial venules (HEV) in the areas of inflammation, leads to strong adhesion of leukocytes to the endothelium, with subsequent infiltration into inflamed tissue (Chuluyan, H.E. et al., Springer Semin. Immunopathol., 1995, 16:391-404). The main ligand of the integrin αEβ7 is a surface protein intraepithelial lymphocytes (IEL) E-cadherin, which provides adhesion αEβ7-bearing cells to epithelial cells. It is shown that the monoclonal antibody against α4β7, MAdCAM or VCAM are effective modulators of chronic inflammatory diseases in animal models, such as asthma (Laberge, S. et al., Am. J. Respir. Crit. Care Med., 1995, 151:822-829), rheumatoid arthritis (RA; Barbadillo, C. et al., Springer Semin. ImmunopathoL, 1995, 16:375-379), colitis (Viney et al, J. Immunol., 1996, 157: 2488-2497) and inflammatory bowel disease (IBD; Podalski, D.K., N. Eng. J. Med., 1991, 325:928-937; Powrie, F. et al., Ther. Immunol., 1995, 2:115-123). P is shown, that monoclonal antibodies against subunit beta associated with the subunit of integrins (Tidswell, M. et al. (1997) J. Immunol. 159:1497-1505), however, since these antibodies are human or humanitarianism, they may not have clinical application.

There is a need for highly specific compounds, such as humanized antibodies or their binding fragments, which are able to inhibit the interaction between integrin alfaretta and its ligands MAdCAM and/or VCAM, as well as the interaction between integrin alphabet and its ligand E-cadherine. Such compounds can be used for the treatment of chronic inflammatory diseases such as asthma, Crohn's disease, ulcerative colitis, diabetes, complications of organ transplantation and diseases associated with allotransplantation.

All cited references, including patent applications and publications, are included in this description by reference in its entirety.

Description of the invention

This invention is based in part on the identification of a number of antagonists of biological pathways involving beta-containing integrins and are in most cases related to biological/cellular processes, represents an important and promising therapeutic target. Such biological processes include all the I, without limitation, inflammation, particularly chronic inflammatory diseases such as asthma, allergies, IBD, diabetes, diseases associated with transplantation, and graft versus host. In this proposed invention compositions and methods directed to the inhibition of cell adhesion and/or cell recruitment mediated by integrin beta, which includes, without limitation, inhibition of binding of MAdCAM and VCAM-1 with the extracellular fragment of the integrin alfaretta and inhibition of the interaction of E-cadherin with integrin alphabet. As described in this document, on the basis of the antagonists according to the invention can be an important therapeutic and diagnostic agents, targeting pathological conditions associated with abnormal or unwanted signal transmission mediated by integrin beta. Accordingly, the present invention methods, compositions, kits and industrial products aimed at the modulation of integrin beta-mediated pathways, including modulation of binding of MAdCAM-Alfama and recruitment of leukocytes in the epithelium of the gastrointestinal tract Allergy, asthma, IBD, such as Crohn's disease and ulcerative colitis), diabetes, inflammation, accompanying the transplantation, graft versus host and/or violations PR is the allotransplantation, and other biological/physiological processes mediated by integrin beta.

In one aspect of the present invention proposed a therapeutic agent against beta suitable for therapeutic applications and can affect various violations of the integrin beta-mediated pathways. For example, in one embodiment of the present invention proposed humanitariannet antibody against beta, where the antibody in the form of a Fab fragment has almost the same affinity binding to human beta as murine Fab fragment, comprising, containing or mainly containing sequences of variable domains of the light chain and heavy chain, as shown in figa and 1B, or figa and 9B. In another embodiment of the present invention proposed humanitariannet antibody against beta, where the affinity of binding of antibodies in the form of a Fab fragment to human beta at least 3 times, at least 5-fold, at least 7 fold or at least 10-fold lower than the affinity of binding of mouse or rat Fab fragment, comprising, containing or mainly containing sequences of variable domains of the light chain and heavy chain, as shown in figa and 1B or figa and 9B. Alternatively, humanitariannet antibody against beta or its fragment, attributed the non beta, according to the invention has the monovalent affinity to human beta, and this affinity is essentially equal to the monovalent affinity to or greater than monovalent affinity to human beta antibodies containing a variable sequence of light chain and heavy chain, as shown in figa (SEQ ID NO: 10) and/or figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12), and/or figv (SEQ ID NO: 13). The affinity of the antibody or its binding fragment with high affinity human beta at least 2 times, at least 5-fold, at least 10 times, at least 50-fold, at least 100 times, at least 500-fold, at least 1000, at least 5000 times, or at least 10,000 times higher than the affinity of the antibody containing the sequence of the light chain and heavy chain, as shown in figa (SEQ ID NO: 10) and/or figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12) and/or figv (SEQ ID NO: 13).

In another embodiment, the invention relates to humanized antibody against beta, where the antibody in the form of a Fab fragment has an affinity binding to human beta, which, for example, at least 3 fold, at least 5-fold, at least 7 fold, at least 9-fold, at least 10 times, at least 15 times, at least 20 times or at least 100-fold higher than the affinity of the binding is of ragment Fab rodents (such as rat or mouse), comprising, containing or mainly containing sequences of variable domains of the light chain and heavy chain, as shown in figa and figv respectively. In one embodiment of the specified fragment Fab rodents has an affinity binding of the Fab fragment containing sequences of variable domains of rat antibodies FIB504.64 produced using hybridoma cell line deposited in the American type culture collection under the number ATCC HB-293. In the following embodiment humanitarianly the Fab fragment according to the invention has an affinity of binding of the Fab fragment containing sequences of variable domains derived from a humanized antibody against beta according to the invention. In this field it is well known that the affinity of binding of the ligand to the receptor can be determined using different assays and expressed as number of quantitative values. Accordingly, in one embodiment the affinity of binding is expressed as the values of Kd and imply their own affinity binding (for example, with minimized avidity). Mainly and preferably the affinity of binding is measured in vitro, in cell-free medium or in medium containing cells. As described herein, the degree of difference in the affinity of binding can quantitative is about to Express as the ratio of the values of the affinity binding gumanitarnogo antibodies in the form of Fab to the value of the affinity binding of Fab antibodies assignment/comparison (for example, mouse antibodies containing donor sequence hypervariable sites)where the values of the affinity of binding is determined in the same conditions. Thus, in one embodiment the degree of difference of the affinity of the binding is defined as the ratio of Kd values gumanitarnogo antibodies in the form of Fab and the Fab antibodies assignment/comparison. To measure the affinity of binding is possible to use any known in the field tests, including those described herein, for example, Biacore® (Biacore International Ab, Uppsala, Sweden) and ELISA.

Various aspects and embodiments, antagonistic antibodies against beta according to the invention is reflected in possible claims:

1. Antibody representing the antibody against beta or beta-binding fragment, which includes

(a) at least one, two, three, four or five sequences of hypervariable sites (HVR)selected from the group consisting of

(i) HVR-L1 containing the sequence A1-A11, where A1-A11 means RASESVDTYLH (SEQ ID NO: 1)

(ii) HVR-L2, containing the sequence B1-B8, where B1-B8 means KYASQSIS (SEQ ID NO: 2)

(iii) HVR-L3, containing the sequence C1-C9, where C1-C9 means QQGNSLPNT (SEQ ID NO: 3)

(iv) HVR-H1 containing the sequence D1-D10, where D1-D10 means GFFITNNYWG (SEQ ID NO: 4)

(v) HVR-H2 containing the sequence E1-E17, where E1-E17 means GISYSGSTSYNPSLKS (SEQ ID NO: 5); and

(vi) HVR-H3 containing the sequence F2-F11, where F2-F11 means MTGSSGYFDF (SEQ ID NO: 6).

In the embodiment of the polypeptide or antibody according to claim 1 polypeptide, or antibody has at least one variant HVR, where the variant HVR sequence comprises at least one residue of at least one of the sequences SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 and 9. In another embodiment of claim 1 or claim 2, the invention includes an antibody against beta or beta-binding fragment, containing one, two, three, four, five or six hypervariable sites (HVR)selected from the group consisting of HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,

where (i) HVR-L1 contains the amino acid sequence RASESVDTYLH (SEQ ID NO: 1); RASESVDSLLH (SEQ ID NO: 7), RASESVDTLLH (SEQ ID NO: 8) or RASESVDDLLH (SEQ ID NO: 9);

(ii) HVR-L2 contains the amino acid sequence KYASQSIS (SEQ ID NO: 2), RYASQSIS (SEQ ID NO: 67) or XYASQSIS (SEQ ID NO: 68, where X means any amino acid),

(iii) HVR-L3 contains QQGNSLPNT (SEQ ID NO: 3),

(iv) HVR-H1 contains the amino acid sequence GFFITNNYWG (SEQ ID NO: 4),

(v) HVR-H2 contains the amino acid sequence GYISYSGSTSYNPSLKS (SEQ ID NO: 5) and

(vi) HVR-H3 contains the amino acid sequence MTGSSGYFDF (SEQ ID NO: 6) or RTGSSGYFDF (SEQ ID NO: 66) in the relative positions F2-F11; or this section contains the amino acid sequence F1-F11, where F1-F11 means AMTGSSGYFDF (SEQ ID NO: 63), ARTGSSGYFDF (SEQ ID NO: 64) or AQTGSSGYFDF (SEQ ID NO: 65).

In the following embodiment of claim 1 or in any of its embodiments this invention relates to an antibody against beta or beta-binding fragment, which contains one, two, three, four, five or six hypervariable sites (HVR)selected from the group consisting of HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,

where (i) HVR-L1 contains the amino acid sequence A1-A11, where A1-A11 means RASESVDTYLH (SEQ ID NO: 1); RASESVDSLLH (SEQ ID NO: 7), RASESVDTLLH (SEQ ID NO: 8) or RASESVDDLLH (SEQ ID NO: 9), or a variant of SEQ ID NO: 1, 7, 8 or 9, where the amino acid A2 is selected from the group consisting of A, G, S, T and V, and/or amino acid A3 is selected from the group consisting of S, G, I, K, N, P, Q, R and T, and/or A4 is selected from the group consisting of E, V, Q, A, D, G, H, I, K, L, N and R, and/or amino acid A5 is selected from the group consisting of S, Y, A, D, G, H, I, K, N, P, R, T and V, and/or amino acid A6 is selected from the group consisting of V, R, I, A, G, K, L, M and Q, and/or amino acid A7 is selected from the group consisting of D, V, S, A, E, G, H, I, K, L, N, P, S and T, and/or amino acid A8 is selected from the group consisting of D, G, N, E, T, P and S, and/or amino acid A9 is selected from the group consisting of L, Y, I and M, and/or amino acid A10 is selected from the group consisting of L, A, I, M, and V and/or amino acid A11 is selected from the group consisting of H, Y, F, and S;

(ii) HVR-L2 contains the amino acid sequence B1-B8, where B1-B8 means KYASQSIS (SEQ ID NO: 2), RYASQSIS (SEQ ID NO: 67) or XYASQSIS (SEQ ID NO: 68, where X means any amino acid), or a variant of SEQ ID NO: 2, 67 or 68, where a is inoculate B1 selected from the group consisting of K, R, N, V, A, F, Q, H, P, I, L, Y and X (where X means any amino acid), and/or amino acid B4 is selected from the group consisting of S and D, and/or amino acid B5 is selected from the group consisting of Q and S, and/or amino acid B6 is selected from the group consisting of S, D, L and R, and/or amino acid B7 is selected from the group consisting of I, V, E and K;

(iii) HVR-L3 contains the amino acid sequence C1-C9, where C1-C9 means QQGNSLPNT (SEQ ID NO: 3) or a variant of SEQ ID NO: 3, where the amino acid C8 is selected from the group consisting of N, V, W, Y, R, S, T, A, F, H, I, L, M, and Y;

(iv) HVR-H1 contains the amino acid sequence D1-D10, where D1-D10 means GFFITNNYWG (SEQ ID NO: 4),

(v) HVR-H2 contains the amino acid sequence E1-E17, where E1-E17 means GYISYSGSTSYNPSLKS (SEQ ID NO: 5) or a variant of SEQ ID NO: 5, where E2 amino acid selected from the group consisting of Y, F, V and D, and/or amino acid E6 is selected from the group consisting of S and G, and/or amino acid E10 selected from the group consisting of S and Y, and/or amino acid E12 is selected from the group consisting of N, T, A and D, and/or amino acid E13 selected from the group consisting of P, H, D and A, and/or amino acid E15 motorway selected from the group consisting of L and V and/or amino acid E17 selected from the group consisting of S and G, and

(vi) HVR-H3 contains the amino acid sequence F2-F11, where F2-F11 means MTGSSGYFDF (SEQ ID NO: 6) or RTGSSGYFDF (SEQ ID NO: 66); or contains amino acid sequence F1-F11, where F1-F11 means AMTGSSGYFDF (SEQ ID NO: 63), ARTGSGYFDF (SEQ ID NO: 64) or AQTGSSGYFDF (SEQ ID NO: 65), or a variant of SEQ ID NO: 6, 63, 64, 65 or 66, where the amino acid F2 is a R, M, A, E, G, Q, S, and/or amino acid F11 selected from the group consisting of F and Y.

In one of the embodiments claim 1 or in any of the antibody according to the invention, the amino acid at position 71 (according to the numbering system of Kabat) wireframe plot heavy chain selected from the group consisting of R, A and T, and/or the amino acid at position 73 (according to the numbering system of Kabat) wireframe plot heavy chain selected from the group consisting of N and T, and/or the amino acid at position 78 (according to the numbering system of Kabat) wireframe plot heavy chain selected from the group consisting of F, A and L.

In one embodiment of claim 1 or in any of the antibody according to the invention HVR-L1 antibodies according to the invention contains a sequence of SEQ ID NO: 1. In one embodiment HVR-L2 antibodies according to the invention contains a sequence of SEQ ID NO: 2. In one embodiment HVR-L3 of the antibody according to the invention contains a sequence of SEQ ID NO: 3. In one embodiment HVR-H1 antibodies according to the invention contains a sequence of SEQ ID NO: 4. In one embodiment HVR-H2 antibodies according to the invention contains a sequence of SEQ ID NO: 5. In one embodiment HVR-H3 antibodies according to the invention has the sequence of SEQ ID NO: 6 or 66 in the relative positions F2-F11, or SEQ ID NO: 63, 64 or 65 in the relative positions F1-F11. In one embodiment of the HVR-L1 sod is RIT RASESVDSLLH (SEQ ID NO: 7). In one embodiment of the HVR-L1 contains RASESVDTLLH (SEQ ID NO: 8). In one embodiment of the HVR-L1 contains RASESVDDLLH (SEQ ID NO: 9). In one embodiment the antibody according to the invention containing these sequences (as shown here in combination), is a humanized or human.

In one aspect of the present invention proposed the antibody contains one, two, three, four, five or six HVR, where each HVR comprises, contains or mainly contains a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 and 9, and where SEQ ID NO: 1, 7, 8 or 9 correspond to the HVR-L1, SEQ ID NO: 2 corresponds to the HVR-L2, SEQ ID NO: 3 corresponds to the HVR-L3, SEQ ID NO: 4 corresponds to the HVR-H1, SEQ ID NO: 5 corresponds to the HVR-H2, and SEQ ID NO: 6 corresponds to the HVR-H3. In one embodiment the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, which have the sequence SEQ ID NO: 1, 2, 3, 4, 5 and 6 respectively. In one embodiment the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, which have the sequence SEQ ID NO: 7, 2, 3, 4, 5 and 6 respectively. In one embodiment the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, which have the sequence SEQ ID NO: 8, 2, 3, 4, 5 and 6 respectively. In one embodiment the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, which have the sequence SEQ ID NO: 9, 2, 3, 4, 5 and 6, respectively, In one embodiment the antibody according to the invention contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, which have the sequence SEQ ID NO: 9, 2, 3, 4, 5 and 66, or SEQ ID NO: 9, 2, 3, 4, 5, 63, or SEQ ID NO: 9, 2, 3, 4, 5, 64, or SEQ ID NO: 9, 2, 3, 4, 5 and 65, or SEQ ID NO: 9, 67, 3, 4, 5, 64, or SEQ ID NO: 9, 68, 3, 4, 5, 64.

Variant HVR included in the antibodies according to the invention may include modifying one or more residues, in addition, the HVR and/or frame parts can be humanitarianism. Embodiments according to the invention, bearing modifications in the HVR and/or frame the site, include, without limitation, the following possible claims:

2. The antibody according to claim 1 or any of his incarnations, where A8 in a variant HVR-L1 mean S, D or T and A9 means L.

3. The antibody according to claim 1 or any one of its embodiments where the antibody is humanized.

4. The antibody according to claim 1 or any one of its embodiments, where at least part of the frame sequence is a consensus sequence of the human frame section.

5. The antibody according to claim 1 or any of his incarnations, where this modification is a substitution, insertion or deletion.

6. The antibody according to claim 1 or any of his incarnations, where a variant HVR-L1 contains 1-10(1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitutions in any combination of the following positions: A2 (G, S, T or V); A3 (G, I, K, N, P, Q, R or T), A4 (A, D, G, H, I, K, L, N, Q, R, or V), A5 ( A, D, G, H, I, K, N, P, R, T, V or Y), A6 (A, G, I, K, L, M, Q or R), A7 (A, E, G, H, I,K, L, N, P, S, T or V), A8 (S, D, E, G, P or N) and A9 (L, I or M), A10 (A, I, M or V) and A11 (F, S, or Y).

7. The antibody according to claim 1 or any of his incarnations, where a variant HVR-L2 contains 1-4 (1, 2, 3, or 4) substitutions in any combination of the following positions: B1 (N), B5 (S), B6 (R or L), and B7 (T, E, K, or V).

8. The antibody according to claim 1 or any of his incarnations, where a variant HVR-L3 comprises at least one substitution at position C8 (W, Y, R, S, A, F, H, I, L, M, N, T or V).

9. The antibody according to claim 1 or any of his incarnations, where a variant HVR-H2 contains 1-7(1, 2, 3, 4, 5, 6 or 7) substitutions in any combination of the following positions: E2 (V, D, or F), E6 (G), E10 (Y), E12 (A, D, or T), E13 (D, A, or H), E15 (V), E17 (G).

10. The antibody according to claim 1 or any of his incarnations, where a variant HVR-H3 contains 1 or 2 substitutions in any combination of the following provisions: (a) F2 (A, E, G, Q, R or S) and F11 (Y).

11. The antibody according to claim 1 or any of his incarnations, which contains HVR-L1 having the sequence of SEQ ID NO: 7.

12. The antibody according to claim 1 or any of his incarnations, which contains HVR-L1 having the sequence of SEQ ID NO: 8.

13. The antibody according to claim 1 or any of his incarnations, which contains HVR-L1 having the sequence of SEQ ID NO: 9.

14. The antibody according to any one of PP-13, a heavy chain which contains a consensus sequence frame area of the human heavy chain subgroup III, carrying a substitution at position 71, 73 and/or 78.

15. The antibody according to 14, where this replacement includes R71A, N73T and/or N78A.

<> 16. The antibody according to claim 1 or any of his incarnations, which contains HVR-L3 having the sequence of SEQ ID NO: 3.

17. The antibody according to claim 1 or any of his incarnations, where A8 in a variant HVR-L1 mean S.

18. The antibody according to claim 1 or any of his incarnations, where A8 in a variant HVR-L1 means D.

19. The antibody according to claim 1 or any of his incarnations, where A9 in a variant HVR-L1 means L.

20. The antibody according to claim 1 or any of his incarnations, where between sequence E1-E17, and F1-F11 is a sequence of frame section HFR3-1-HFR3-31, and where HFR3-6 means A or R, HFR3-8 mean N or T, and HFR3-13 means L or A or F.

21. Humanitariannet antibody against beta, where the monovalent affinity of the antibody to human beta essentially identical to the monovalent affinity of rat antibodies containing a variable sequence of the light chain and heavy chain, as shown in Fig.9.

22. Humanitariannet antibody against beta, where the monovalent affinity of the antibody to human beta at least 3 times higher than the monovalent affinity of rat antibodies containing a variable sequence of the light chain and heavy chain, as shown in Fig.9.

23. Humanitariannet antibody according to item 21 or 22, where the rat antibody is obtained using hybridoma cell line deposited in the American type culture collection under the number ATCC HB-293.

24. The antibody according to any one of PP-23, where the affinity of binding is expressed as Kd values.

25. The antibody according to any one of PP-24, where the affinity of binding is measured by the method of Biacore™ or radioimmuno analysis.

26. The antibody according to claim 1, containing the consensus sequence of frame area of the human light chain κ subgroup I.

27. The antibody according to claim 1, containing the consensus sequence of frame area of the human heavy chain subgroup III.

28. The antibody according to item 27, where the sequence of frame section contains a substitution at position 71, 73 and/or 78.

29. The antibody according p where the specified replacement includes R71A, N73T and/or N78A, or where the substituted amino acid at position 71 is R or A, and/or the amino acid substitution at position 78 includes N or T, and/or the amino acid substitution at position 78 includes an L or A or F.

30. The antibody according p where the specified replacement includes L78F, or A78F, or A78L, or L78A.

31. Method of inhibiting the interaction of subunit beta human integrin with a second integrin subunit and/or ligand by bringing into contact of the antibody according to any one of items 1 to 30 with a second integrin subunit and/or ligand.

32. The method according to p, where the second integrin subunit is alpha-subunit of integrin, and where the ligand is a MAdCAM, VCAM or fibronectin is.

33. The method according to p where alpha-integrin subunit is a human.

34. The method according to p, where the ligand is a human.

35. The method according to p, where the second integrin subunit is algae-subunit of integrin, and where the ligand is an E-cadherin.

36. The method according to p, where algae-integrin subunit is a human.

37. The method according to p, where the ligand is a human.

38. The method according to p, where inhibition reduces or alleviates symptoms of a disease selected from inflammation, asthma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, diabetes, inflammation accompanying organ transplantation, graft versus host and inflammation associated with violations of allotransplantation.

Other embodiments according to the invention include, without limitation, the following.

In one embodiment of the HVR-L1 represents SEQ ID NO: 1, 7, 8, or 9, or HVR-L1 is a variant of SEQ ID NO: 1, 7, 8, or 9, which contains 1-10(1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitutions at relative positions A1-A11 with any combination of the following positions: A2 (A, G, S, T or V); A3 (S, G, I, K, N, P, Q, R or T), A4 (E, A, D, G, H, I, K, L, N, Q, R, or V), A5 (S, A, D, G, H, I, K, N, P, R, T, V or Y), A6 (V, A, G, I, K, L, M, Q or R), A7 (D, A, E, G, H, I, K, L, N, P, S, T or V), A8 (T, S, D, E, G, P or N) and A9 (Y, L, I or M), A10 (L, A, I, M or V) and A11 (H, F, S, or Y). In one embodiment of the HVR-L represents SEQ ID NO: 2, 67 or 68, or HVR-L2 is a variant of SEQ ID NO: 2, 67, or 68, and this variant HVR-L2 contains 1-4 (1, 2, 3, 4 or 5) substitutions at relative positions B1-B8 with any combination of the following positions: B1 (K, R, N, V, A, F, Q, H, P, I, L, Y or X (where X means any amino acid)), B4 (S), B5 (Q or S), B6 (S, R or L), and B7 (I, T, E, K, or V). In one embodiment HVR-L3 represents SEQ ID NO: 3, or HVR-L3 is a variant of SEQ ID NO: 3, which contains at least one substitution relative clauses C1-C8, for example, at position C8 (W, Y, R, S, A, F, H, I, L, M, N, T or V). In one embodiment HVR-H1 represents SEQ ID NO: 4. In one embodiment HVR-H2 represents SEQ ID NO: 5, or HVR-H2 is a variant of SEQ ID NO: 5, and this variant HVR-H2 contains 1-7(1, 2, 3, 4, 5, 6 or 7) substitutions at relative positions E1-E17 with any combination of the following positions: E2 (Y1 V, D, or F), E6 (S or G), E10 (S or Y), E12 (N, A, D, or T), E13 (P, D, A, or H), E15 (L or V), E17 (S or G). In one embodiment HVR-H3 represents SEQ ID NO: 6, 63, 64, 65 or 66, or HVR-H3 is a variant of SEQ ID NO: 6, 63, 64, 65 or 66, which contains 1 or 2 substitutions in the relative positions F1-F11 in the case of SEQ ID NO: 63, 64 and 65, or in the relative positions F2-F11 in the case of SEQ ID NO: 6 and 66, with any combination of the following provisions: F2 (M, A, E, G, Q, R or S) and F11 (F or Y). The letter (s) in parenthesis following each position indicates an example of replacement (i.e. substitutions) amino acid at a consensus or others is the GUI amino acid, obvious to the person skilled in the art, suitability of other amino acids for substitution in the context of this description, it can be determined routinely using methods known in this field and/or described in this document.

In one embodiment of the HVR-L1 contains the sequence of SEQ ID NO: 1. In one embodiment of the A8 in a variant HVR-L1 means D. In one embodiment of the A8 in a variant HVR-L1 mean S. In one embodiment A9 in a variant HVR-L1 means L. In one embodiment of the A8 in a variant HVR-L1 means D and A9 in a variant HVR-L1 means L. In one embodiment of the A8 in a variant HVR-L1 means S and A9 in a variant HVR-L1 means L. In some embodiments according to the invention these options relate to the HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 include or contain, or predominantly contain sequences SEQ ID NO: 2, 3, 4, 5 and 6 respectively. In some embodiments HVR-H3 comprises or consists of, or primarily contains SEQ ID NO: 6 or 66 (in relative positions F2-F11), or SEQ ID NO: 63 or 64 or 65 (in relative positions F1-F11).

In one embodiment of the A8 in a variant HVR-L1 means I and A9 in a variant HVR-L1 means L, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6, respectively.

In one embodiment A8, A9 and A10 in a variant HVR-L1 mean the D, L and V, respectively, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6, respectively.

In one embodiment of the A8 and A9 in a variant HVR-L1 mean N and L, respectively, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6, respectively.

In one embodiment of the A8 and A9 in a variant HVR-L1 mean P and L, respectively, and B6 and B7 in a variant HVR-L2 mean R and T, respectively, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 3, 4, 5 and 6, respectively.

In one embodiment A2, A4, A8, A9 and A10 in a variant HVR-L1 mean S, D, S, L and V, respectively, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6, respectively.

In one embodiment A5 and A9 in a variant HVR-L1 mean D and T, respectively, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6, respectively.

In one embodiment A5 and A9 in a variant HVR-L1 mean N and L, respectively, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6, respectively.

In one embodiment A9 in a variant HVR-L1 means L, this option also applies to the HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 2, 3, 4, 5 and 6.

In one embodiment of the present invention the antibody or binding polypeptide beta contains HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each HVR comprises, contains or mainly contains SEQ ID NO: 9, 2, 3, 4, 5 and 64, respectively. In another embodiment, each HVR comprises, contains or mainly contains SEQ ID NO: 9, 67, 3, 4, 5 and 64. In another embodiment, each HVR comprises, contains or mainly contains SEQ ID NO: 9, 68, 3, 4, 5 and 64. In another embodiment, each HVR comprises, contains or mainly contains SEQ ID NO: 9, 2, or 67, or 68, 3, 4, 5 and 66.

In some embodiments the variant antibodies with the specified variant HVR-L1 also contain HVR-L2, HVR-L3, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 2, 3, 4, 5 and 6. If option contains antibodies HVR-L1 A8(P) and A9(L) and HVR-L2 B6(R) and B7(T), in some embodiments specified HVR-L1, HVR-L2 variant also contains HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 3, 4, 5 and 6.

In some embodiments of these antibodies more the tion contain a consensus sequence of human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In one embodiment the antibody according to the invention contains HVR-L1 having the sequence of SEQ ID NO: 1. In one embodiment, the variant antibody according to the invention contains a variant HVR-L1, where A10 means V. In one embodiment of the specified variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 2, 3, 4, 5 and 6. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies, these antibodies also include a consensus sequence is a frame area of the human κI light chain.

In one embodiment the antibody according to the invention contains HVR-L3 having the sequence of SEQ ID NO: 3. In one embodiment variant antibody according to the invention contains a variant HVR-L3, where C8 means L. In one embodiment of the specified variant antibody further comprises HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 4, 5 and 6. In one embodiment the antibody according to the invention contains a variant HVR-L3, where C8 means W. In one embodiment of the specified variant antibody further comprises HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 4, 5 and 6. In one embodiment of the HVR-L1 has the sequence of SEQ ID NO: 7, 8 or 9. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies, these antibodies also include a consensus sequence frame area of the human κI light chain.

One in which the polishing antibody according to the invention contains HVR-H3, having the sequence of SEQ ID NO: 6. In one embodiment variant of the indicated antibody contains a variant HVR-H3, where F1 denotes Q. In one embodiment of the specified variant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H2, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 3, 4 and 5. In one embodiment the antibody according to the invention contains a variant HVR-H3, where F1 denotes R. In one embodiment of the specified variant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H2, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 3, 4 and 5. In one embodiment of the HVR-L1 has the sequence of SEQ ID NO: 7, 8 or 9. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In one embodiment the antibody according to the invention contains HVR-L1 having the sequence of SEQ ID NO: 1. In the bottom of the embodiment, the antibody contains a variant HVR-L1, where A4 means Q. In one embodiment of the specified variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 2, 3, 4, 5 and 6. In one embodiment the antibody according to the invention contains a variant HVR-L1, where A6 means I. In one embodiment of the specified variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 2, 3, 4, 5 and 6. In one embodiment the antibody according to the invention contains a variant HVR-L1, where A7 means S. In one embodiment of the specified variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 2, 3, 4, 5 and 6. In one embodiment the antibody according to the invention contains a variant HVR-L1, where A8 means D or N. In one embodiment of the specified variant antibody further comprises HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 2, 3, 4, 5 and 6. In some embodiments, these antibodies also include a consensus sequence frame plot heavy chain human subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/who do 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In one embodiment the antibody according to the invention contains HVR-L2 having the sequence of SEQ ID NO: 2. In one embodiment the antibody according to the invention contains a variant HVR-L2, where B1 denotes N. In one embodiment the antibody according to the invention contains a variant HVR-L2, where B5 means S. In one embodiment the antibody according to the invention contains a variant HVR-L2, where B6 means L. In one embodiment the antibody according to the invention contains a variant HVR-L2, where B7 means V. In one embodiment the antibody according to the invention contains a variant HVR-L2, where B7 means E or K. In some embodiments, the specified variant antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 1, 3, 4, 5 and 6. In some embodiments HVR-L1 has the sequence of SEQ ID NO: 7, 8 or 9. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence CT is the ACLs section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In one embodiment the antibody according to the invention contains HVR-L3 having the sequence of SEQ ID NO: 3. In one embodiment the antibody according to the invention contains a variant HVR-L3, where C8 means W, Y, R or S. In certain specified embodiments the variant antibody further comprises HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 4, 5 and 6. In some embodiments HVR-L1 contains SEQ ID NO: 7, 8 or 9. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In one embodiment the antibody with the according to the invention contains HVR-H2, having the sequence of SEQ ID NO: 5. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E2 means F. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E2 denotes V or D. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E6 means G. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E10 means Y. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E12 means A, D or T. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E13 means D, A, or N. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E15 means V. In one embodiment the antibody according to the invention contains a variant HVR-H2, where E17 means G. In some embodiments the specified variant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 3, 4, and 6. In some embodiments HVR-L1 has the sequence of SEQ ID NO: 7, 8 or 9. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/ili. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In one embodiment the antibody according to the invention contains HVR-H3 having the sequence of SEQ ID NO: 6. In one embodiment the antibody according to the invention contains a variant HVR-H3, where F11 denotes Y. In some embodiments, the specified variant antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H3, each of which has the sequence described, respectively, in SEQ ID NO: 1, 2, 3, 4, and 6. In some embodiments HVR-L1 contains SEQ ID NO: 7, 8 or 9. In some embodiments, these antibodies also include a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

In some embodiments of these antibodies EXT is niteline contain a consensus sequence frame area of the human heavy chain subgroup III. In one embodiment of these antibodies consensus sequence frame section contains a substitution at position 71, 73 and/or 78. In some embodiments of these antibodies, position 71 is A, 73 is T and/or at position 78 is A. In one embodiment of these antibodies additionally contain a consensus sequence frame area of the human κI light chain.

Therapeutic drug intended for use in the subject-owner, preferably causes a small immunogenic response or does not cause immunogenic response in the specified owner. In one embodiment of the present invention offers such a tool. For example, in one embodiment of the present invention is proposed humanitariannet antibody that causes and/or supposedly able to cause the subject's response, associated with the formation of human antibodies against antibodies rodents (such as response to mouse or rat antibodies, or human antibodies against human antibodies, at a much lower level than the antibody containing the sequence of SEQ ID NO: 10 and/or 11 (figa and 1B) or SEQ ID NO: 12 and/or 13 (figa and 9B, which shows the rat amino acid sequence Fib504 against mouse antibodies). In another example, the present invention proposed humanitariannet antibody cat what PoE calls and/or supposedly able to provoke a response, associated with the formation of human antibodies against antibodies rodents (such as human antibodies against mouse (HAMA) or rat antibodies, or human antibodies against human antibodies (HAHA).

Variable domain of the heavy and/or light chain gumanitarnogo antibodies according to the invention may contain one or more human and/or human consensus sequence plots, non-hypervariable (e.g., frame). In some embodiments the human and/or human consensus sequence plots, different from the hypervariable contain one or more modifications. In one embodiment the variable domain heavy chain antibodies according to the invention contains the human consensus sequence of frame section, which in one embodiment represents the consensus sequence of frame plot of subgroup III. In one embodiment the antibody according to the invention contains a variant of the consensus sequence of frame plot of subgroup III, the modified at least one amino acid position. For example, in one embodiment variant subgroup III consensus sequence frame section can contain a substitution at one or more of the provisions 71, 73, 7, and/or 94. In one embodiment of this replacement is a R71A, N73T, L78A and/or R94M in any combination.

As is well known in this area and as described in more detail below, the amino acid position/border hypervariable area antibodies can vary, depending on the context and the different options known in the art (as described below). Some provisions in the variable domain can be considered as hybrid hypervariable provisions, which, according to the same criteria are within the hypervariable area, and other criteria - outside the hypervariable area. One or more of these provisions can be found in the extended hypervariable sites (as described below). This invention relates to antibodies containing modifications in these hybrid hypervariable positions. In one embodiment these hybrid hypervariable positions include one or more of the provisions 26-30, 33-35B, 47-49, 49, 57-65, 93, 94 and 102 in the variable domain of the heavy chain. In one embodiment these hybrid hypervariable positions include one or more of the provisions 24-29, 35-36, 46-49, 49, 56, and 97 in the variable domain of the light chain. In one embodiment the antibody according to the invention contains a variant of the human consensus sequence of subgroup to rasnogo plot modified by one or more hybrid hypervariable positions. In one embodiment the antibody according to the invention contains a variable domain of a heavy chain containing a variant of the consensus sequence of the human frame plot of subgroup III, the modified one or more provisions of 28-35, 49, 50, 52a, 53, 54, 58-61, 63, 65, 94 and 102. In one embodiment, the antibody contains replacement T28F, F29I, S30T, S31N, Y32N, A33Y, M34W and S35G. In one embodiment, the antibody contains replacement S49G. In one embodiment, the antibody contains replacement V50F, or V50D, or V50Y. In one embodiment, the antibody contains replacement G53Y. In one embodiment, the antibody contains replacement G54S. In one embodiment, the antibody contains replacement Y58S. In one embodiment, the antibody contains replacement A60N, or A60D, or A60T. In one embodiment, the antibody contains replacement D61P, or D61A, or D61H. In one embodiment, the antibody contains replacement V63L. In one embodiment, the antibody contains replacement G65S. In one embodiment, the antibody contains replacement R94M. In one embodiment, the antibody contains replacement R94A, or R94E, or R94G, or R94Q, or R94S. In one embodiment, the antibody contains replacement G95T. In one embodiment, the antibody contains one or more substitutions at positions 28-35, 49, 50, 52a, 53, 54, 58-61, 63, 65, 94 and 102, in addition, it further comprises one or more substitutions at positions R71A, or N73T, or L78A, or L78F. In one embodiment the AI antibody contains replacement Y102F. As shown in figv, these substitutions are present in the HVR-H1, HVR-H2 and/or HVR-H3 of the heavy chain.

In one embodiment the antibody according to the invention comprises the variable domain of the light chain containing the consensus sequence of the human frame plot of subgroup I, modified by one or more of the provisions of 27, 29-31, 33, 34, 49, 50, 53-55, 91 and 96. In one embodiment, the antibody contains replacement Q27E. In one embodiment, the antibody contains replacement I29V. In one embodiment, the antibody contains replacement S30D. In one embodiment, the antibody contains replacement N31T, or N31S, or N31D. In one embodiment, the antibody contains replacement Y32L. In one embodiment, the antibody contains replacement A34H. In one embodiment, the antibody contains replacement Y49K. In one embodiment, the antibody contains replacement A50Y. In one embodiment, the antibody contains replacement S53Q. In one embodiment, the antibody contains replacement L54S. In one embodiment, the antibody contains replacement E55I or E55V. In one embodiment, the antibody contains replacement Y91G. In one embodiment, the antibody contains replacement W96N or W96L. In one embodiment, the antibody contains replacement A25S. In one embodiment, the antibody contains a substitution at position A25 G, S, T or V. In one embodiment, the antibody contains a modification selected from one or more of the following groups of substitutions. For example, in one embodiment the antibody contains replacement S26 G, I, K, N P, Q or T. In one embodiment, the antibody contains replacement Q27 E, A, D, G, H, I, K, L, N, Q, R or V. In one embodiment, the antibody contains replacement S28 on A, D, G, H, I, K, N, P, R, T, V or Y. In one embodiment, the antibody contains replacement 129 V, A, G, K, L, M, Q or R. In one embodiment, the antibody contains replacement S30 D, A, E, G, H, I, K, L, N, P, S, T or V. In one embodiment, the antibody contains replacement N31 D, T, E, or G. In one embodiment, the antibody contains replacement Y32 L, I or M. In one embodiment, the antibody contains replacement L33 on A, I, M, or V. In one embodiment, the antibody contains replacement A34 H, F, Y or S. In one embodiment, the antibody contains replacement Y49 on K or N. In one embodiment, the antibody contains replacement A50Y. In one embodiment, the antibody contains replacement S53Q. In one embodiment, the antibody contains replacement L54S. In one embodiment, the antibody contains replacement E55 on V, I, or K. In one embodiment, the antibody contains replacement Y91G. In one embodiment, the antibody contains replacement W96 on N, L, W, Y, R, S, A, F, H, I, M, N, R, S, T, V or Y. As shown in figa, these substitutions are present in HVR-L1, HVR-L2 and/or HVR-L3 light chain.

The antibody according to the invention can contain any suitable human or human consensus sequences of frame sections light chain, provided that the antibody has the desirable biological properties (for example, a desirable affinity binding). In one embodiment of antic the lo according to the invention contains at least part of the sequence (or sequence) frame area of the human light chain κ. In one embodiment the antibody according to the invention contains at least a portion of the consensus sequence (or sequence) of the human skeleton plot κ subgroup I.

In one embodiment the antibody according to the invention includes the variable domain of the heavy and/or light chain containing a sequence of frame sections, described in SEQ ID NO: 34-41 and shown in figures 1, 7 and 8, provided that the provisions of 49 light chain and 94 of the heavy chain are extended HVR, and provided that in the above-mentioned position 49 is K, as in the above-mentioned position 94 is preferably, but not necessarily, is M and can be R.

Antagonists according to the invention can be used to modulate one or more aspects beta-associated effects, including, without limitation, in connection with alpha-subunit of integrin, a connection to the subunit of alphae integrin, binding integrin alfaretta with MAdCAM, VCAM-1 or fibronectin binding of integrin alphabet with E-cadherine. These effects can be modulated by any biologically acceptable mechanisms, which include impaired binding of ligand to the subunit beta, or a dimeric integrin alfaretta or alphabet, and/or by interrupting the connection of subunits alpha and beta, in which going on the t inhibition of the formation of dimeric integrin. Accordingly, in one embodiment, the invention provides an antagonistic antibody against beta, which inhibits the binding alpha with beta. In one embodiment, the antagonistic antibody against beta according to the invention disrupts the binding alfaretta with MAdCAM. In one embodiment, the antagonistic antibody against beta according to the invention disrupts the binding alfaretta with VCAM-1. In one embodiment, the antagonistic antibody against beta according to the invention disrupts the binding alfaretta with fibronectin. In one embodiment, the antagonistic antibody against beta according to the invention disrupts the binding beta with algae. In one embodiment, the antagonistic antibody against beta according to the invention disrupts the binding of integrin alphabet with E-cadherine. The violation may be direct or indirect. For example, an antagonistic antibody against beta can contact beta on the phase sequence of the dimerization alfaretta or alphabeta and thereby to inhibit the interaction of integrin subunits and the formation of integrin dimer. In another example, an antagonistic antibody against beta can communicate with the sequence of the ligand-binding domain subunit beta and thereby to inhibit the interaction of the specified linking the house is on its binding partner (such as fibronectin, VCAM and/or MAdCAM for integrin alfaretta; or E-cadherin for integrin alphabeta). In another example, an antagonistic antibody against beta can communicate with the sequence that does not belong to the domain dimerization of the subunits of the integrin or the ligand-binding domain, but where the binding specified antagonistic antibodies against beta leads to impaired ability domain beta to interact with its binding partner (such as a subunit of integrin alpha or algae and/or ligands, such as fibronectin, VCAM, MAdCAM, or E-cadherin). In one embodiment, the antagonistic antibody according to the invention is associated with beta (for example, the extracellular domain) and when this is disrupted dimerization beta with subunit alpha or algae. In one embodiment, the antagonistic antibody according to the invention is associated with beta and the ability beta and/or integrin alfaretta and/or alfabeta associate the corresponding ligand or ligands is reduced. For example, in one embodiment, the invention provides an antagonistic antibody that if the binding molecule beta inhibits dimerization of the specified molecule. In one embodiment, the antagonistic antibody against beta according to the invention specifically binds to the sequence of the ligand-svyazivalsa what about the domain beta. In one embodiment, the antagonistic antibody against beta according to the invention specifically binds to the sequence of the ligand-binding domain beta so that it violates the binding of the ligand (i.e. fibronectin, VCAM and/or MAdCAM) with integrin alfaretta. In one embodiment, the antagonistic antibody against beta according to the invention specifically binds to the sequence of the ligand-binding domain beta so that it violates the binding of the ligand (i.e. E-cadherin) with integrin alphabet.

In one embodiment, the antagonistic antibody according to the invention disrupts the dimerization beta, including heterodimerization (i.e. dimerization beta with a molecule integrin subunit alpha or algae).

In one embodiment, the antagonistic antibody according to the invention binds to an epitope subunit of integrin beta, which contains amino acids 176-237. In another embodiment, the antagonistic antibody according to the invention binds to an epitope integrin beta, which is essentially the same epitope Fib504.64 (ATCC HB-293). The binding epitope analyzed by standard methods, including, without limitation, competitive analysis link.

In one aspect of the present invention proposed an antibody containing one or a combination of two, three, four, five, is whether all sequences of HVR, listed in the table of amino acid substitutions on Fig.

Therapeutic drug intended for use in the subject-owner, preferably causes a small immunogenic response or does not cause immunogenic response in the specified owner. In one embodiment, the invention provides such a means. For example, in one embodiment of the present invention proposed humanitariannet antibody that causes and/or supposedly able to cause the subject's response, associated with the formation of human antibodies against rat antibodies, or human antibodies against mouse antibodies, or human antibodies against human antibodies, at a much lower level than the antibody containing the sequence of SEQ ID NO: 10, 11, 12 and/or SEQ ID NO: 13 (rat antibody against mouse antibodies Fib504 (ATCC HB-293) 1 and 9). In another example, the present invention proposed humanitariannet antibody that causes and/or supposedly able to provoke a response that is associated with the formation of human antibodies against mouse antibodies, human antibodies against rat antibodies, or human antibodies against human antibodies.

Variable domain of the heavy and/or light chain gumanitarnogo antibodies according to the invention may contain one or more human and/or human is such a consensus sequence plots non-hypervariable (e.g., frame). In some embodiments the human and/or human consensus sequence plots, different from the hypervariable contain one or more additional modifications. In one embodiment the variable domain heavy chain antibodies according to the invention contains the human consensus sequence of frame section, which in one embodiment represents the consensus sequence of frame plot of subgroup III. In one embodiment the antibody according to the invention contains a variant of the consensus sequence of frame plot of subgroup III, the modified at least one amino acid position. For example, in one embodiment variant of the consensus sequence of frame plot of subgroup III can contain a substitution at one or more of the provisions 71, 73, 78 and/or 94, although the position 94 is part of the extended hypervariable plot heavy chain H3 according to the invention. In one embodiment of this replacement is a R71A, N73T, L78A and/or R94M in any combination.

The antibody according to the invention can contain any suitable human or human consensus sequences of frame sections light chain provided that antitelomerase desirable biological properties (for example, desirable affinity binding). In one embodiment the antibody according to the invention contains at least part of the sequence (or sequence) frame area of the human light chain κ. In one embodiment the antibody according to the invention contains at least a portion of the consensus sequence (or sequence) of the human skeleton plot κ subgroup I.

Antagonists according to the invention can be used to modulate one or more aspects beta-associated effects. For example, an antagonistic antibody against beta can contact beta on the phase sequence of the dimerization alfaretta or alphabeta and thereby to inhibit the interaction of integrin subunits and the formation of integrin dimer. In another example, an antagonistic antibody against beta can communicate with the sequence of the ligand-binding domain subunit beta and thereby to inhibit the interaction of the specified binding domain with its binding partner (such as fibronectin, VCAM and/or MAdCAM for integrin alfaretta; or E-cadherin for integrin alphabeta). In another example, an antagonistic antibody against beta can communicate with the sequence that does not belong to the domain dimerization of the subunits and tigrina or the ligand-binding domain, but where the binding specified antagonistic antibodies against beta leads to impaired ability domain beta to interact with its binding partner (such as a subunit of integrin alpha or algae, and/or ligands, such as fibronectin, VCAM, MAdCAM, or E-cadherin). In one embodiment, the antagonistic antibody according to the invention is associated with beta (for example, the extracellular domain) and when this is disrupted dimerization beta with subunit alpha or algae. In one embodiment, the antagonistic antibody according to the invention is associated with beta and the ability beta and/or integrin alfaretta and/or alfabeta associate the corresponding ligand or ligands is reduced. For example, in one embodiment of the present invention offers an antagonistic antibody that if the binding molecule beta inhibits dimerization of the specified molecule. In one embodiment, the antagonistic antibody against beta according to the invention specifically binds to the sequence of the ligand-binding domain beta. In one embodiment, the antagonistic antibody against beta according to the invention specifically binds to the sequence of the ligand-binding domain beta so that it violates the binding of the ligand (i.e. fibronectin, VCAM and/or MAdCAM) with integrin alfaretta. In one the embodiment antagonistic antibody against beta according to the invention specifically binds to the sequence of the ligand-binding domain beta so, that it violates the binding of the ligand (i.e., E-cadherin) with integrin alphabet.

In one embodiment, the antagonistic antibody according to the invention disrupts the dimerization beta, including heterodimerization (i.e. dimerization beta with a molecule integrin subunit alpha or algae).

In some cases it is preferable antagonistic antibody against beta, which does not prevent the binding of the ligand (such as fibronectin, VCAM, MAdCAM, or algae) subunit beta, as part of an integrin or integrin alfaretta or alphabet in the form of a dimer. Accordingly, in one embodiment, the invention provides an antibody that does not bind to the binding site of fibronectin, VCAM, MAdCAM, or E-cadherin on beta, and inhibits the interaction of subunit beta and subunit alpha (such as integrin subunit alpha or algae), preventing the formation of biologically active integrin. In one example, an antagonistic antibody according to the invention can be used in combination with one or more other antagonists, where the antagonists directed to other processes and/or functions the way integrin beta. Thus, in one embodiment, the antagonistic antibody against beta according to the invention binds to an epitope on beta different from the epitope bound by another antagonist of integrin beta or integrin alpha/beta (such as the antibody against alfaretta comprising a monoclonal antibody or antibody, such as humanitariannet antibody or monoclonal antibody derived from a mouse antibody and/or having the same or essentially the same characteristics of binding or specificity as the antibody derived from the murine antibodies).

In one embodiment of the present invention proposed an antagonistic antibody against beta that violates multimerization beta-alpha or-algae with the formation of the corresponding integrin, and, in addition, inhibits the binding of the ligand. For example, an antagonistic antibody according to the invention, which inhibits the dimerization beta with integrin subunit alpha or algae, may also have the ability to compete with the ligand for binding to beta or a dimeric integrin (for example, it can inhibit the binding of fibronectin, VCAM, and/or MAdCAM with beta and/or alfaretta; or it can inhibit the binding of E-cadherin with beta or alphabet).

In one embodiment, antagonistic antibodies against beta according to the invention the binding of the antagonist with beta inhibits cell adhesion, activated by binding of the ligand. In another embodiment, the antagonistic antibodies against beta according to the invention the binding of the antagonist with beta in clickinghere recruiting cells to the cells and/or tissues, which is expressed beta-containing integrin.

In one embodiment, the antagonistic antibody against beta according to the invention specifically binds to at least part of the amino acids 176-250 (optional amino acids 176-237) extracellular domain beta (see Tidswell, M. et al. (1997) J. Immunol. 159: 1497-1505) or its variants and reduces or blocks the binding of ligands MAdCAM, VCAM-1, fibronectin and/or E-cadherin. In one embodiment of such blocking binding of the ligand reduces or prevents adhesion of cells expressing the ligand to the cell expressing beta-containing integrin. In one embodiment, the antagonistic antibody according to the invention specifically binds the amino acid sequence beta containing residues 176-237. In one embodiment, the antagonistic antibody according to the invention specifically binds a conformational epitope, formed part of at least one sequence selected from the group consisting of residues 176-237 beta. In one embodiment, the antagonistic antibody according to the invention specifically binds amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% identical or similar AMI is kislotno sequence residues 176-237 or residues l76-250 human beta. In one embodiment, the antagonistic antibody against beta according to the invention binds to the same epitope as the antibody against beta Fib504 obtained using hybridoma ATCC HB-293.

In one aspect of the present invention proposed a composition containing one or more antagonistic antibodies according to the invention and a carrier. In one embodiment the carrier is pharmaceutically acceptable.

In one aspect of the present invention offers a nucleic acid encoding an antagonistic antibody against beta according to the invention.

In one aspect of the present invention proposed the vectors containing the nucleic acid according to the invention.

In one aspect of the present invention proposed cell host containing a nucleic acid or vector according to the invention. The vector may be of any type, for example, it may be a recombinant vector such as an expression vector. As host cells, you can use any of a number of host cells. In one embodiment a host cell is a prokaryotic cell, such as E. coli. In one embodiment a host cell is a eukaryotic cell, such as mammal cells, such as cell Chinese hamster ovary (CHO).

In one aspect, the invention provides FPIC who would obtain antagonist according to the invention. For example, the invention provides a method of obtaining antagonistic antibodies against beta (which, as defined herein, includes a full-sized antibody and its fragments), the said method includes the expression in a suitable cell host recombinant vector according to the invention, encoding the indicated antibody (or fragment), and the allocation of the indicated antibodies.

In one aspect of the present invention proposed a finished product, comprising a container; and a composition contained within the container, where the composition contains one or more antagonistic antibodies against beta according to the invention. In one embodiment, the composition contains a nucleic acid according to the invention. In one embodiment a composition comprising an antagonistic antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment of the finished product according to the invention further comprises instructions for introduction of the composition (e.g., antagonistic antibodies) to a subject.

In one aspect of the present invention proposed a kit comprising a first container containing a composition which includes one or more antagonistic antibodies against beta according to the invention; and second to the container, containing buffer. In one embodiment the buffer is pharmaceutically acceptable. In one embodiment a composition comprising an antagonistic antibody further comprises a carrier, which in some embodiments is pharmaceutically acceptable. In one embodiment the kit further comprises instructions for introduction of the composition (e.g., antagonistic antibodies) to a subject.

Expression beta-integrins and their ligands in a morbid state change. [Expression of MAdCAM-1 ion channel endothelium is increased in areas of inflamed mucosa of patients with inflammatory bowel disease (UC and CD), and the private plate colon of UC and CD patients also have high contents of cells CD3+ and a4b7+ compared with controls IBS (see Souza H., et al., Gut 45:856 (1999)]. It was found that when the liver expression of MAdCAM-1 is associated with inflammation of the portal tract and may play a role in the recruitment of lymphocytes alfaretta+ in the liver during inflammation. (Hillan, K., et al., Liver. 19(6):509-18 (1999)). MAdCAM-1 on cells of the hepatic vessels provides adhesion of lymphocytes a4b7+ patients with IBD and primary sclerosing cholangitis. Adhesion is inhibited by antibodies against MAdCAM-1, against alfaretta or against Alfa. (Grant AJ. et al., Hepatology. 33(5): 1065-72 (2001)). MAdCAM-1, VCAM-1 and E-cadherin expressed on endothelial cells of the hunter and/or on cells of microvessels in the inflamed area of the Central nervous system. Beta-integrins are involved in demyelinizing disease of the Central nervous system (Kanwar et al., J. Neuroimmunology 103, 146 (2000)). Expression alfaretta significantly higher in LPL CD than in control samples and in patients with UC (Oshitani, N. et al., International Journal of Molecule Medicine 12, 715-719 (2003)). IEL in CD patients may be chronically stimulated condition and secretiruetsa from the peripheral system (Meresse, B., et al., Human Immunology, 62, 694-700 (2001)). When liver disease human alphabeta T cells (CD4+ and CD8+) preferentially accumulates in human liver, where E is cadherin is expressed on hepatocytes and epithelial cells of the bile ducts (Shimizu, Y., et al., Journal of Hepatology 39, 918-924 (2003)). In chronic pancreatitis T-cells CD8+CD103+, similar intraepithelial lymphocytes of the gut, infiltrate the pancreas in chronic pancreatitis (Matthias, P., et al., Am J Gastroenterol 93:2141-2147 (1998)). Increasing regulation alfabeta detected in patients suffering from systemic lupus erythematosus with specific inclusion of the epithelium (Pang et al., Arthritis & Rheumatism 41: 1456-1463 (1998)). In sjögren's syndrome T-cells CD8+ alphabeta+ stick to acinose epithelial cells and kill them, Idutywa apoptosis (Kroneld et al., Scand J Rheumatol 27:215-218, 1998). Integrin alfaretta and alfabeta participates in epidermotropism T-cells in skin inflammation and contributes to skin allograft rejection (Sun et al., Transplntation 74, 1202, 2002). Teraki and Shiohara demonstrated preferred expression of integrin aEb7 on T-cells CD8+ in psoriatic epidermis (Teraki and Shiohara, Br. J. Dermatology 147, 1118, 2002). In asthma, COPD and normal subjects T-lymphocytes sputum are activated IEL (CD69+ CD103+) (Leckie et. al., Thorax 58, 23, 2003). CTL CD103+ (aEb7+) accumulate in the epithelium of the graft in clinical rejection of the allograft kidney (Hadley et al., Transplantation 72, 1548, 2001)]. Thus, in one aspect, the invention involves the use of antagonistic antibodies against beta according to the invention for inhibition of the interaction of the integrin beta-ligand, with reduction or relief of symptoms, such as one or more of the above illnesses. In one embodiment the antibody according to the invention is used to produce a medicinal product intended for the treatment and/or prevention of diseases, such as inflammatory disease, including, without limitation, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), inflammatory liver disease, CNS inflammation, chronic pancreatitis, systemic lupus erythematosus, Sjogren syndrome, psoriasis and skin inflammation, asthma, chronic obstructive pulmonary disease (COPD), interstitial lung process, allergies, and commune disease graft rejection, kidney transplant rejection, the reaction of the "graft versus host disease, diabetes and cancer.

In one aspect, the invention proposed the use of a nucleic acid according to the invention for obtaining a medicinal product for the treatment and/or prevention of such diseases, as the immune (such as autoimmune or inflammatory) violation, including, without limitation, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis) and allergic reaction (for example, diseases of the respiratory system, skin, joints, allergic asthma, and diseases of other organs, caused by allergic reaction, mediated beta-containing integrin).

In one aspect, the invention involves the use of expressing the vector according to the invention for obtaining a medicinal product for the treatment and/or prevention of such diseases, as the immune (such as autoimmune or inflammatory) violation, including, without limitation, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis) and allergic reaction (for example, diseases of the respiratory system, skin, joints and other organs, caused by allergic reaction, mediated beta-containing integrin).

In one TSA is regarding subsection of this invention it is proposed to use the host cell according to the invention for obtaining a medicinal product for the treatment and/or prevention of such diseases as an immune (such as autoimmune or inflammatory) violation, including, without limitation, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis) and allergic reaction (for example, diseases of the respiratory system, skin, joints and other organs, caused by allergic reaction, mediated beta-containing integrin).

In one aspect of the present invention proposed the use of the finished product according to the invention for obtaining a medicinal product for the treatment and/or prevention of such diseases, as the immune (such as autoimmune or inflammatory) violation, including, without limitation, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis) and allergic reaction (for example, diseases of the respiratory system, skin, joints and other organs, caused by allergic reaction, mediated beta-containing integrin).

In one aspect of the present invention it is proposed to use set according to the invention for obtaining a medicinal product for the treatment and/or prevention of such diseases, as the immune (such as autoimmune or inflammatory) violation, including, without limitation, inflammatory bowel disease (such as Crohn's disease or ulcerative colitis) and al is ergicheskoe reaction (for example, diseases of the respiratory system, skin, joints and other organs, caused by allergic reaction, mediated beta-containing integrin).

This invention provides methods and compositions used for modulation of painful conditions associated with impaired regulation of intercellular interactions mediated by integrin beta. Integrins beta involved in different biological and physiological processes, such as, for example, inflammatory disorders and allergic reactions. Thus, in one aspect, the invention provides a method comprising introducing to a subject the antibody according to the invention.

In one aspect, the invention provides a method of suppressing inflammation-mediated integrin beta. This method comprises bringing into contact the cell or tissue with an effective amount of the antibody according to the invention, resulting in inhibition of the interaction and binding of lymphocytes or B-cells with a cell expressing the integrin beta.

In one aspect, the invention provides a method of treating a pathological condition associated with dysregulation of binding integrin beta the subject. This method includes the introduction to the subject an effective amount of the antibody according to the invention have beneficial effects on the above-mentioned condition.

In one aspect, the invention provides a method of inhibiting the binding of lymphocytes expressing the integrin ligand beta (for example, cells expressing MAdCAM, VCAM, E-cadherin or fibronectin), with a cell expressing the integrin beta (such as integrin alfaretta or alphabet). This method comprises bringing into contact of the specified cell with the antibody according to the invention and thereby the inhibition or prevention of cell adhesion and decrease the inflammatory response.

In one aspect, the invention provides a method of treatment or prevention of an inflammatory disease associated with increased expression or activity of integrin beta, or increase the interaction between integrin beta on one cell and receptor integrin beta to another cell, the method includes introducing to a subject in need of such treatment, an effective amount of the antibody according to the invention, which provides an effective treatment or prevention of a specified inflammatory diseases. In one embodiment of the aforementioned inflammatory disease is an inflammatory bowel disease (IBD). In another embodiment of the aforementioned inflammatory disease is an allergic reaction.

The methods according to the invention can be used is the substance for influencing any suitable pathological condition for example, cells and/or tissue, in which there is dysregulation of the pathway, mediated by binding of integrin beta. Integrins beta expressed mainly on leukocytes (Tidswell, M. et al. (1997), supra). In one embodiment of the method according to the invention is directed to cells and prevents binding to cells expressing the integrin ligand beta. For example, in accordance with this invention, an antagonistic antibody against beta prevents the binding of intraepithelial lymphocytes expressing E-cadherin, with alphabeta-expressing cell. Antagonistic antibody against beta according to the invention prevents the binding of cells expressing MAdCAM, VCAM-1 or fibronectin, leukocytes expressing alfaretta.

The methods according to the invention can also include other processing stages. For example, in one embodiment the method additionally includes the stage, where the target cell and/or the target tissue (e.g., endothelial cell lining of the intestine) is exposed to antibodies against TNF or low-molecular-weight therapeutic agent, including, without limitation, compounds 5-ASA.

As described in this document, integrins beta mediate important biological processes, dysregulation of which leads to the emergence of a number of Pato is logicheskih States. Accordingly, in one embodiment of the methods according to the invention the cell, which is sent to the method (e.g., endothelial cell)is a cell adhesion to the cell expressing the ligand of integrin beta (where the cell may represent, without limitation, lymphocyte, and the ligand can be a MAdCAM, VCAM or E-cadherin), disturbed, suppressed or prevented as compared with the adhesion in the absence of antagonistic antibodies against beta according to the invention. In one embodiment of the method according to the invention inhibits homing lymphocytes, and the result is inflammation at the site of expression of integrin beta. For example, after contact with the antagonist according to the invention can disrupt the ability of cells to adhere to the cell expressing the ligand of integrin beta.

Brief description of drawings

On figa and 1B comparative analysis of the primary sequences of the variable regions of light and heavy chains for the following areas: human consensus sequences of the light chain subgroup Kappa I (figa, SEQ ID NO: 23), human consensus sequences of heavy chain subgroup III (FIGU, SEQ ID NO: 24), the variable segment the light chain of the rat antibody against mouse antibodies beta (Fib504) (Figa, SEQ ID NO: 10), the variable segment heavy the ETUI rat antibody against mouse antibodies beta (Fib504) (FIGU, SEQ ID NO: 11), as well as options gumanitarnogo antibodies: variable segment light chain gumanitarnogo hu504Kgraft (figa, SEQ ID NO: 25), the variable segment of the heavy chain gumanitarnogo hu504Kgraft (FIGU, SEQ ID NO: 26), option hu504.5 (amino acid changes compared to humanized hu504Kgraft listed on figa (light chain) and figv (heavy chain) for options hu504.5, hu504.16 and hu504.32). Other amino acid substitutions in the HVR-H1 and HVR-H2 hu504Kgraft, leading to the formation of antibodies that bind beta shown in figs.

On figa and 2B shows the full sequence of the human consensus sequences of the light chain subgroup III (figa, SEQ ID NO: 27) and heavy chain (FIGU, SEQ ID NO: 28). HVR underlined.

On figa and 3B shows the full sequence gumanitarnogo 504graft containing hypervariable sections of rat Fib504 (as described herein), grafted on a consensus sequence of human light chain Kappa I (figa, SEQ ID NO: 29) and a consensus sequence of human heavy chain subgroup III (FIGU, SEQ ID NO: 30). HVR underlined.

On figa and 4B shows the full sequence gumanitarnogo 504Kgraft, where in position 49 of the light chain component hu504graft replacement Y49K. The sequence of the light chain hu504Kgraft shown in SEQ ID NO: 31, and the sequence of the heavy C is PI hu504Kgraft shown in SEQ ID NO: 30. HVR underlined.

On figa and 5B shows the full sequence gumanitarnogo hu504K-RFgraft, where in positions 71 and 78 of the heavy chain hu504graft made replacement A71R and A78F compared with the sequence hu504Kgraft. The sequence of the light chain hu504K-RFgraft shown in SEQ ID NO: 31, and the sequence of the heavy chain hu504K-RFgraft shown in SEQ ID NO: 32. HVR underlined.

On figa and 6B shows the full sequence variants hu504.32 containing heavy chain hu504K-RFgraft (SEQ ID NO: 32) and replace T31D and Y32L in the light chain hu504Kgraft (SEQ ID NO: 33). HVR underlined.

On figa-7B and figa-8B are examples of the acceptor human consensus sequences of frame sections used in the implementation of the present invention, the following codes sequences.

The consensus sequence of frame fields variable regions of light chains (VL) (figa,B)

the consensus sequence of frame area of the human VL Kappa subgroup I (SEQ ID NO: 14)

the consensus sequence of frame area of the human VL Kappa subgroup I minus extended HVR-L2 (SEQ ID NO: 15)

the consensus sequence of frame area of the human VL Kappa subgroup II (SEQ ID NO: 16)

the consensus sequence of frame area of the human VL Kappa subgroup III(SEQ ID NO: 17)

the consensus sequence of frame area of the human VL Kappa subgroup IV (SEQ ID NO: 18)

Shaded areas correspond to the light chain HVR (denoted L1, L2 and L3).

The consensus sequence of frame fields variable regions of the heavy chains (VH) (figa,B)

the consensus sequence of frame area of the human VH subgroup I minus Kabat CDRs (SEQ ID NO: 19)

the consensus sequence of frame area of the human VH subgroup I minus extended hypervariable sites (SEQ ID nos: 20-22)

the consensus sequence of frame area of the human VH subgroup II minus Kabat CDRs (SEQ ID NO: 48)

the consensus sequence of frame area of the human VH subgroup II minus extended hypervariable sites (SEQ ID nos: 49-51)

the consensus sequence of frame area of the human VH subgroup III minus Kabat CDRs (SEQ ID NO: 52)

the consensus sequence of frame area of the human VH subgroup III minus extended hypervariable sites (SEQ ID nos: 53-55)

acceptor frame area of the human VH minus Kabat CDRs (SEQ ID NO: 56)

acceptor frame area of the human VH minus extended hypervariable sites (SEQ ID nos: 57-58)

acceptor 2 frame area of the human VH minus Kabat CDRs (SEQ ID NO: 59)

acceptor 2 ka is the red area of the human VH minus extended hypervariable sites (SEQ ID nos: 60-62)

On figa and 9B depicts the amino acid sequence of the variable chains of rat antibodies Fib504 against murine integrin beta obtained using hybridoma ATCC HB-293. HVR underlined. Variable area light chain depicted in figa (SEQ ID NO: 12), and variable plot heavy chain depicted in FIGU (SEQ ID NO: 13).

On figa shows the amino acid position in the heavy chains of different consensus sequences (hu subgroups I-III). The consensus sequence used to obtain antibodies against HER2 Herceptin®, rat Fib504 and frame parts hu504-RL and hu504-RF described in the examples in this document. On FIGU shows a histogram showing the relative binding alfaretta with the antibody hu504graft and antibody hu504Kgraft as a function of modifications of the frame area "RL" or "RF", as described in example 1.

Figa-11C. On figa shows a table that summarizes the changes HVR obtained in the process of affinity maturation using a limited range of amino acid substitutions in the variant hu504.16. The results obtained using the libraries individually modified variant HVR hu504.16 as described in example 2 of this document. Abbreviations of amino acids within the meaning of the amino acids that are most frequently encountered in beta-binding antibodies (EN is the body, selected using phage). On figv and 11C presents the histogram of the results is shown in figa, which indicate the number and type of amino acid substitutions in the variant hu504.16 (light chain, figv; a heavy chain figs), detected using the methods of mutagenesis and selection, described in example 2.

On Fig shows a table in which the results of affinity maturation, obtained using a wide range of possible amino acid substitutions in the variant HVR hu504.32 as described in example 2. In the framework of the indicated amino acids that are most frequently found in antibodies, detected as beta-binding antibodies using methods of mutagenesis and selection, described in example 2.

On figa and 13B depict the sequence HVR rat antibodies Fib504 against mouse antibodies (ATCC-293) and human consensus sequence (left column). Examples of amino acid substitutions found in each position of the HVR (without limitation) using assays described in the examples (amino acid replacement detect using soft amino acid randomization, wide scan of amino acid substitutions and limited scanning amino acid substitutions), shown on the right (modification HVR with the aim of humanizing applicable to variants of the present invention can be found in the application U.S. No. 60/545840,filed February 19, 2004).

On Fig presents a typical graph of activity Fib504 and variant antibodies, binding to MAdCAM, on the concentration of the antibody, which is produced according to the method described in example 3. For all antibodies define values IC50and IC90.

On figa and 15V depicts the amino acid sequence of HVR light and heavy chain antibodies 504.32R against beta, where for six HVR antibodies positions are indicated by the numbering system of Kabat and relative numbering system (A-F). Also indicated amino acid positions 71, 73 and 78 of the site FR3 of the heavy chain. For many of the provisions of the HVR or plot FR3 of the heavy chain specify the amino acid replacement.

On Fig shows histograms showing the relative ability of antibodies 504.32M and 504.32R block homing radiolabelled T-cells in the colon of the mouse with inflammatory bowel disease.

Ways of carrying out the invention

This invention provides methods, compositions, kits and finished products for identification and/or inhibitors of the signaling pathway beta.

These methods, compositions, kits and finished products described in this document.

General procedures

When implementing the present invention, unless otherwise indicated, use traditional methods molecular biologist and (including recombinant techniques), Microbiology, cell biology, biochemistry and immunology, well-known experts in this field. Such methods are described in detail in the literature, for example in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucltotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987 and periodic supplements); "PCR: The Polymerase Chain Reaction", (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

Definitions

The term "subunit beta or β7 subunit" refers to a subunit of human β7 integrin (Erle et al., (1991) J. Biol. Chem. 266: 11009-11016). Subunit beta associated with the integrin subunit alpha, such as human α4 subunit (Kilger and Holzmann (1995) J. Mol. Biol. 73:347-354). Integrin Alfama is expressed on most Mature lymphocytes, and small populations of thymocytes, bone marrow cells and fat cells. (Kilshaw and Murant (1991) Eur. J. Immunol. 21: 2591-2597; Gurish et al., (1992) 149: 1964-1972; and Shaw, S.K. and Brenner, M.B. (1995) Semin. Immunol. 7:335). Subunit beta also associated with suryadinata algae, such as a subunit of algae human integrin (Cepek, K.L, et al. (1993) J. Immunol. 150: 3459). Integrin alphabet is expressed on cells of the intestinal epithelium (ilEL) (Cepek, K.L. (1993), supra). Subunit beta that communicates with a humanized antibody against b is TA according to the invention, may have a natural origin, it can be soluble or localized on the cell surface.

The term "subunit of algae", or "subunit of alphae integrin", or "the αE subunit or subunit αE integrin", or "CD103" refers to an integrin subunit that is associated with integrin beta expressed by intraepithelial lymphocytes, and this integrin alfabeta mediates the binding of iELs with intestinal epithelial cells expressing E-cadherin (Cepek, K.L. et al. (1993) J. Immunol. 150: 3459; Shaw, S.K. and Brenner, M.B. (1995) Semin. Immunol. 7: 335).

The terms "MAdCAM or MAdCAM-1 in the context of the present invention are used interchangeably to refer to a protein molecule cell adhesion - mucosal adressin-1, which is a single-chain polypeptide containing a short cytoplasmic tail, a transmembrane segment and extracellular sequence, consisting of three immunoglobulin-like domains. Were cloned cDNA of murine MAdCAM-1, human MAdCAM-1 and MAdCAM-1 macaques (Briskin, et al, (1993) Nature, 363: 461-464; Shyjan et al., (1996) J. Immunol. 156:2851-2857).

The term "VCAM-1", or "the adhesion molecule vascular cell-1", or "CD106" refers to the ligand alfaretta and 4beta1, which is expressed on activated endothelial cells and plays an important role in the interactions of endothelial cells with leukocyte and, such as binding and movement of leukocytes in inflammation.

The term "E-cadherin" refers to the member of the family catherinew, which is expressed on epithelial cells. E-cadherin is a ligand of integrin alphabet and mediates binding alfabeta expressed on iEL, with the epithelial cells of the intestine, although its role in homing lymphocytes is unclear. Expression of E-cadherin increases under the action of TGF-beta.

The term "fibronectin" refers to fibronectin, which is involved in tissue repair, embryogenesis, blood clotting and migration/adhesion of cells. He serves as a connecting means in the ECM (extracellular matrix) in the form of dimer present in the plasma (plasma fibronectin). The plasma shape is synthesized by hepatocytes, and the ECM form is produced by fibroblasts, chondrocytes, endothelial cells, macrophages, and some epithelial cells. In this context, it interacts with integrin alfaretta and mediates events homing or adhesion of lymphocytes. ECM form of fibronectin plays the role of the main adhesion molecule of the cell, providing the adhesion of cells to collagen or proteoglycan substrates. Fibronectin can also participate in the organization of interaction of cells with the ECM by binding to different components of unclutch the CSOs matrix and membrane-bound fibronectin receptors on cell surfaces. Finally, fibronectin plays an important role in the events associated with cell migration during embryogenesis.

"Gastrointestinal inflammatory diseases are a group of chronic diseases that cause inflammation and/or ulceration in the membrane of the mucosa. These diseases include, for example, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis, indeterminate colitis and infectious colitis), mucositis (e.g., mucositis of the oral cavity, gastrointestinal mucositis, nasal mucositis, proctitis), necrotizing enterocolitis and esophagitis.

The term "inflammatory bowel disease" or "IBD" is used in this description on an equal footing in respect of intestinal diseases that cause inflammation and/or ulceration and include, without limitation, Crohn's disease and ulcerative colitis.

"Crohn's disease (CD)or ulcerative colitis (UC)are chronic inflammatory bowel disease of unknown etiology. Crohn's disease, in contrast to ulcerative colitis, can affect any part of the intestine. The most visible sign of Crohn's disease is granular, reddish-purple edematous thickening of the bowel wall. With the development of inflammation these granulomas frequently lose a clearly defined boundaries and the drain of which are with the surrounding tissue. The predominant clinical signs of this disease are diarrhea and bowel obstruction. Like ulcerative colitis, Crohn's disease may be continuous or recurrent, mild or severe, but unlike ulcerative colitis Crohn's disease is not cured by resection of the affected intestine. For most patients with Crohn's disease require surgery at some parts, but then usually there is a relapse and require continuous medical treatment.

Crohn's disease can affect any part of the digestive tract from the mouth to the anus, although it usually occurs in the ileum-colon, thin or thick-rectal intestine. Histologically, the disease manifests itself in the form of a continuous granulomas, cryptogenic abscesses, fissures and aphthous ulcers. The inflammatory infiltrate is a mixture of lymphocytes (both T and B cells), plasma cells, macrophages and neutrophils. There is a disproportionate increase in IgM - and IgG-secreting plasma cells, macrophages and neutrophils.

For the treatment of moderately active Crohn's disease of the colon using anti-inflammatory drug sulfasalazine and 5-aminosalicylic acid (5-ASA), which are also prescribed to maintain remission of the disease. For the treatment of PE is analnogo diseases especially suitable metronidazole and ciprofloxacin, which on the effectiveness of such sulfasalazine. In more severe cases, effective corticosteroids, which allow to treat active aggravation and even to maintain remission. Azathioprine and 6-mercaptopurine are effective for patients who require regular corticosteroids. These medicines can also be used for long-term prevention. Unfortunately, in some patients the effect occurs with a very high delay (up to six months).

To relieve symptoms in some patients you can also use Antidiarrhoeal funds. Diet therapy or elemental diet can improve the nutritional status of patients and cause symptomatic improvement in acute disease, but they do not lead to long-term clinical remission. For the treatment of secondary over-development of the microflora in the small intestine and pyogenic complications used antibiotics.

"Ulcerative colitis (UC)" affects the small intestine. The disease may be continuous or recurrent, light or heavy. The early pathological change relates to inflammatory infiltration with formation of an abscess at the base of libertyeco crypt. Data fusion stretched and loosened crypt promotes the separation of the overlying mucous membrane from the blood supply, leading to arr is digital ulcers. The symptoms include cramping pain in the lower abdomen, rectal bleeding and frequent, loose bowel movements, consisting mainly of blood, pus and mucus with a small amount of fecal particles. In the case of acute, severe or chronic continuous ulcerative colitis may have a total colectomy.

Clinical signs UC vary greatly, they may develop gradually or abruptly, and may include diarrhea, tanism and recurrent rectal bleeding. When a transient inclusion of the entire colon may develop toxic megacolon threatening condition of life. Not associated with intestinal manifestations include arthritis, septic gangrene, uveitis and nodoso erythema.

For the treatment of UC in mild cases use sulfasalazine and related salicylaldehyde drugs, and in severe cases, corticosteroid funds. Sometimes, especially if the disease is limited to the distal intestine, effectively local introduction of salicylates or corticosteroids, which leads to lower side effects than systemic administration. Sometimes shown supportive therapy, such as the introduction of iron and Antidiarrhoeal funds. In persistent cases, corticosteroid dependence sometimes also prescribe azathioprine, 6-mercapto the purine and methotrexate.

The term "modification" amino acid residue/position in this description refers to the change in the primary amino acid sequence compared to the original amino acid sequence, where the change involves changing these amino acid residues/positions. For example, a typical modifications include the replacement of residue (in position) to another amino acid (e.g., conservative or non-conservative substitution), insert one or more (usually less than 5 or 3) amino acids in the area adjacent to the specified residue/position, and a deletion of the above balances/provisions. The term "replacement of amino acids", or it applies to the replacement of an existing amino acid residue in a known (source) amino acid sequence with other amino acid residue. Typically and preferably, the modification leads to a change in at least one physicomechanical activity of the variant polypeptide compared to a polypeptide containing the source (or "wild-type) amino acid sequence. For example, in the case of antibodies, the variable fisicoquimica activity may represent the affinity of binding ability to bind and/or effect on the molecule-target by binding.

The term "amino acid" in about the Birmingham of the present invention is used in its broadest sense and includes natural Lα-amino acids or their remains. In this description to denote amino acids using a traditional one - or three-letter abbreviations (Lehninger, A.L., Biochemistry, 2d ed., pp.71-92, (Worth Publishers, New York, New York, 1975). This term includes D-amino acids, chemically modified amino acids, such as analogs of amino acids, natural amino acids, which are usually not included in the composition of proteins, such as norleucine, and chemically synthesized compounds, whose properties allow the specialists in this area to characterize them as amino acids. For example, in the determination of amino acids are analogues or mimetics of phenylalanine or Proline, which provide the same conformational restriction of the peptide compounds as natural Phe or Pro. Such analogs and mimetics in this description referred to as "functional equivalents" of amino acids. Other examples of amino acids are listed in the publication Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, Eds., Vol.5, p.341 (Academic Press, Inc., New York, New York, 1983), which is incorporated in this description by reference. Sometimes to denote amino acids using single-letter designations, which can be found in the literature (see, for example, Alberts, B. et al. Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc. 1994, page 57).

"Isolated" antibody is an antibody that was identified and extracted from agoprivate environment, or separated from the components of its natural environment. Unwanted components of its natural environment are substances that interfere with diagnostic or therapeutic application of antibodies, they may include enzymes, hormones and other protein and non-protein soluble substances. In preferred embodiments the antibody is purified (1) to obtain clean, making up more than 95% by weight of antibody that is determined by the method of Lowry, most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a sequencing machine with rotating Cup, or (3) to homogeneity according to the analysis SDS-PAGE in reducing or non conditions with staining of Kumasi blue or, preferably, silver. The selected antibody includes the antibody in situ within recombinant cells, if at least one component of the natural environment antibody is not present. Typically, however, the selected antibody obtained after at least one stage of cleaning.

The term "residue numbering variable domain according to Kabat system" or "the numbering of amino acid positions according to the Kabat system" and its variants refers to the numbering system used for the variable domains of the heavy sludge the light chain in the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system the actual linear amino acid sequence may contain fewer or more amino acids, depending on whether shortening FR or CDR variable domain, or insert in FR or CDR of the variable domain. For example, the variable domain of the heavy chain can include the insertion of one amino acid (residue 52a according to the system of Kabat) after residue 52 H2 and insert multiple residues (e.g. residues 82a, 82b and 82c on the system of Kabat) after residue 82 FR heavy chain. The remains of this antibody can be numbered according to the Kabat system by comparing the homologous sites of the primary sequences of this antibody and the "standard" sequence, numbered according to the Kabat system.

The phrase "essentially similar" or "almost the same" in this description refers to a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody according to the invention and the other associated with a standard antibody/antibody comparison), so that the person skilled in the art may consider the difference between two values little or no biological and/or statistical significance within the context of biological characteristics is determined by the specified knowledge is enemy (for example, the Kd values). The difference between these two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% from the values obtained for standard antibody/antibody comparison.

The term "affinity binding"generally refers to the force of the sum of all non-covalent interactions that occur between the binding site of a molecule (e.g. antibody) and its binding partner (e.g., antigen). Unless otherwise specified, in this specification, the term "affinity binding" refers to the native affinity of the binding, which reflects the interaction between members of a binding pair (e.g., antibody and antigen) 1:1. The affinity of a molecule X for its partner Y can be estimated using the dissociation constants (Kd). Affinity can be measured by traditional methods known in this field, including those described in this document. Antibodies with low affinity is usually associated antigen slowly, and the resulting complex has a tendency to easily dissociate, whereas antibodies with high affinity is usually associated antigen faster, and the resulting complex has a tendency to stay longer associated with the condition. In this area, the number of known ways to measure the affinity of binding, each of which can be used for the purposes of the present invention. Specific illustrative embodiment described below.

In one embodiment of the "Kd" or "Kd value" according to this invention is measured by analysis of binding antigen, radiolabelled (RIA)performed with the use of Fab-version of the target antibody and its antigen as described in the following analysis, where the affinity of binding of the Fab to the antigen measured in solution by balancing Fab with a minimal concentration (125I)-labeled antigen in the presence of a series tetraoxa solutions its antigen, then associated antigen capture using a tablet coated with antibodies against Fab (Chen, et al., (1999) J. Mol Biol 293:865-881). To set conditions analysis microtiter plates (Dynex) are covered during the night solution (5 μg/ml) detecting antibodies against Fab (Cappel Labs) in 50 mm sodium carbonate solution (pH 9,6) and then blocked with 2% solution (wt./about.) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In readsorbing tablet (Nunc #269620) mix 100 PM or 26 PM [125I]-antigen and serial dilution of the target Fab (for example, by the method used for analysis of antibodies is Rotel VEGF, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). Then the target Fab incubated over night; however, the incubation can be continued for a longer period of time (for example, 65 hours)to ensure achievement of equilibrium. Then the mixture is transferred into a tablet to capture and incubated at room temperature (for example, within one hour). Then the solution is removed and the plate washed eight times with 0.1% solution of tween-20 in PBS. After drying, the tablets add scintillator in the amount of 150 μl/well (MicroScint-20; Packard), and consider the tablets on the gamma counter Topcount (Packard) for ten minutes. In the analysis of competitive binding for each Fab is chosen such concentrations that provide the binding, not exceeding 20% of the maximum binding. In accordance with another embodiment the Kd or Kd value is measured by means of surface plasmon resonance using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) and CM5 chips with immobilized antigen at ~10 response units (RU) and 25°C. Briefly, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm sodium acetate solution, pH of 4.8, to 5 μg/ml (~0.2 μm) and then injected him with what speed of flow of 5 μl/minute to achieve approximately 10 response units (RU) of the associated protein. To block unreacted groups, after entering the antigen is injected 1M ethanolamine. To measure the kinetic parameters enter double serial dilutions of Fab (0.78 nm to 500 nm) in PBS containing 0.05% tween-20 (PBST) at 25°C with a flow rate of approximately 25 μl/min Rate of Association (kon) and dissociation rate (koff) calculate, using a simple model linking the Langmuir one-to-one (software for data processing BIAcore, version 3.2) by simultaneous fitting sensogram Association and dissociation. The equilibrium dissociation constant calculated as the ratio of koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol 293: 865-881. However, if the on-rate, determined by the above method, surface plasmon resonance, greater than 106M-1S-1then this speed may be determined by the method of quenching the fluorescence, which allows us to measure the increase or decrease in the intensity of fluorescence emission (wavelength excitation 295 nm; the wavelength of emission of 340 nm, a bandwidth of 16 nm) at 25°C and concentration of antibodies against the antigen 20 nm (form Fab) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen determined with a spectrometer, such as a spectrophotometer with a stopped flow (Aviv Instruments) or a spectrophotometer SLM-Aminco with the series 8000 (ThermoSpectronic) with a cuvette, equipped with a stir bar.

"on-rate"or "rate of Association"or "Association rate"or "kon"in accordance with this invention can be determined using the above method, surface plasmon resonance using a BlAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) and CM5 chips with immobilized antigen at ~10 response units (RU) and 25°C. Briefly, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm sodium acetate solution, pH of 4.8, to 5 μg/ml (~0.2 μm) and then injected with a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of the associated protein. To block unreacted groups, after entering the antigen is injected 1M ethanolamine. To measure the kinetic parameters enter double serial dilutions of Fab (0.78 nm to 500 nm) in PBS containing 0.05% tween-20 (PBST) at 25°C with a flow rate of approximately 25 μl/min Rate of Association (kon) and dissociation rate (koff) calculate, using a simple model linking the Langmuir one-to-one (software for data processing BIAcore, version 3.2) by simultaneous fitting sensogram Association and d is Sociali. The equilibrium dissociation constant calculated as the ratio of koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol 293: 865-881. However, if the on-rate, determined by the above method, surface plasmon resonance, greater than 106M-1S-1then this speed is preferably determined by the method of quenching the fluorescence, which allows us to measure the increase or decrease in the intensity of fluorescence emission (wavelength excitation 295 nm; the wavelength of emission of 340 nm, a bandwidth of 16 nm) at 25°C and concentration of antibodies against the antigen 20 nm (form Fab) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen determined with a spectrometer, such as a spectrophotometer with a stopped flow (Aviv Instruments) or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a cuvette equipped with a stirrer. "Kd" or "Kd value" according to this invention is measured by analysis of binding antigen, radiolabelled (RIA)performed with the use of Fab-version of the target antibody and its antigen as described in the following analysis, where the affinity of binding of the Fab to the antigen measured in solution by balancing Fab with a minimal concentration (125I)-labeled antigen in the presence of a series tetraoxa solutions its antigen, then include the hydrated antigen capture using tablet covered with antibodies against Fab (Chen, et al., (1999) J. Mol Biol 293:865-881). To set the conditions of analysis, microtiter plates (Dynex) are covered during the night solution (5 μg/ml) detecting antibodies against Fab (Cappel Labs) in 50 mm sodium carbonate solution (pH 9,6) and then blocked with 2% solution (wt./about.) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In readsorbing tablet (Nunc #269620) mix 100 PM or 26 PM [125I]-antigen and serial dilution of the target Fab (for example, by the method used for analysis of antibodies against VEGF, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). Then the target Fab incubated over night; however, the incubation can be continued for a longer period of time (for example, 65 hours)to ensure achievement of equilibrium. Then the mixture is transferred into a tablet to capture and incubated at room temperature for one hour. Then the solution is removed and the plate washed eight times with 0.1% solution of tween-20 in PBS. After drying, the tablets add scintillator in the amount of 150 μl/well (MicroScint-20; Packard), and consider the tablets on the gamma counter Topcount (Packard) for ten minutes. In the analysis of competitive binding for each Fab is chosen such concentrations that provide the binding, not exceeding 20% of max is th link. In accordance with another embodiment the Kd or Kd value is measured by means of surface plasmon resonance using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) and CM5 chips with immobilized antigen at ~10 response units (RU) and 25°C. Briefly, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm sodium acetate solution, pH of 4.8, to 5 μg/ml (~0.2 μm) and then injected with a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of the associated protein. To block unreacted groups, after entering the antigen is injected 1M ethanolamine. To measure the kinetic parameters enter double serial dilutions of Fab (0.78 nm to 500 nm) in PBS containing 0.05% tween-20 (PBST)at 25°C with a flow rate of approximately 25 μl/min Rate of Association (kon) and dissociation rate (koff) calculate, using a simple model linking the Langmuir one-to-one (software for data processing BIAcore, version 3.2) by simultaneous fitting sensogram Association and dissociation. The equilibrium dissociation constant calculated as the ratio of koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol 93: 865-881. However, if the on-rate, determined by the above method, surface plasmon resonance, greater than 106M-1S-1then this speed may be determined by the method of quenching the fluorescence, which allows us to measure the increase or decrease in the intensity of fluorescence emission (wavelength excitation 295 nm; the wavelength of emission of 340 nm, a bandwidth of 16 nm) at 25°C and concentration of antibodies against the antigen 20 nm (form Fab) in PBS, pH of 7.2, in the presence of increasing concentrations of antigen determined with a spectrometer, such as a spectrophotometer with a stopped flow (Aviv Instruments) or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a cuvette equipped with a stirrer.

In one embodiment of the "on-rate"or "rate of Association"or "Association rate"or "kon"in accordance with this invention can be determined using the above method, surface plasmon resonance using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) and CM5 chips with immobilized antigen at ~10 response units (RU) and 25°C. Briefly, biosensor chips with carboxyethylgermanium dextran (CM5, BIAcore Inc.) activate the hydrochloride of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mm p is the target sodium acetate, pH of 4.8, to 5 μg/ml (~0.2 μm) and then injected with a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of the associated protein. To block unreacted groups, after entering the antigen is injected 1M ethanolamine. To measure the kinetic parameters enter double serial dilutions of Fab (0.78 nm to 500 nm) in PBS containing 0.05% tween-20 (PBST) at 25°C with a flow rate of approximately 25 μl/min Rate of Association (kon) and dissociation rate (koff) calculate, using a simple model linking the Langmuir one-to-one (software for data processing BIAcore, version 3.2) by simultaneous fitting sensogram Association and dissociation. The equilibrium dissociation constant calculated as the ratio of koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol 293: 865-881. However, if the on-rate, determined by the above method, surface plasmon resonance, greater than 106M-1S-1then this speed is preferably determined by the method of quenching the fluorescence, which allows us to measure the increase or decrease in the intensity of fluorescence emission (wavelength excitation 295 nm; the wavelength of emission of 340 nm, a bandwidth of 16 nm) at 25°C and concentration of antibodies against the antigen 20 nm (form Fab) in PBS, pH of 7.2, in the presence of increasing to the of ncentrate antigen, measured by a spectrometer, such as a spectrophotometer with a stopped flow (Aviv Instruments) or a spectrophotometer SLM-Aminco 8000 series (ThermoSpectronic) with a cuvette equipped with a stirrer.

The phrase "substantially reduced" or "essentially different" in this description refers to a sufficiently high degree of difference between two numeric values (generally one associated with an antibody according to the invention and the other associated with a standard antibody/antibody comparison), so that the person skilled in the art may consider the difference between the two values is statistically significant in the context of biological characteristics is determined by the specified values (e.g., Kd values, the HAMA response). The difference between these two values is preferably more than about 10%, preferably more than about 20%, preferably more than about 30%, preferably more than about 40%, preferably more than about 50% of the values obtained for standard antibody/antibody comparison.

"Percent identity (%) amino acid sequence" as applied to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in the sequence candidate, identical amino acid residues in identificar the bathroom peptide or polypeptide sequence, by sequence alignment and, if necessary, the introduction of gaps to achieve the maximum percent sequence identity, and conservative substitutions are not considered a part of the identity sequence. The alignment of the sequence to determine percent identity of amino acid sequences can be accomplished in various ways known in this field, for example, using widely available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Specialists in this field can determine the settings that are appropriate for carrying out the alignment, including any algorithms needed to achieve maximal alignment over the entire length of the compared sequences. However, for the purposes of this invention the values of the % identity of amino acid sequences obtained using the computer program to compare sequences ALIGN-2, wherein source code of the program ALIGN-2 is shown below in table A. Computer program for comparing sequences ALIGN-2 developed by Genentech, Inc., and the code below in table A, registered with the accompanying documentation in the office for the protection of the copyright of the USA, Washington D.C., 20559, number TXU510087. The program ALIGN-2 comes Genentech, Inc., South San Francisco, California, or it can with kompilirovat from the source code, following on Fig. The program ALIGN-2 can be compiled for use on the UNIX operating system, preferably digital UNIX V4.0D. All parameters of the comparison of sequences is determined by the program ALIGN-2 and do not change.

If the ALIGN-2 is used to compare the amino acid sequence % identity to a certain amino acid sequence and A certain amino acid sequence B, or compared with some amino acid sequence B (or in other words that some amino acid sequence contains A certain % amino acid sequence identity compared to a certain amino acid sequence B, or in relation to certain amino acid sequence (B), calculated as follows:

100 × X/Y,

where X means the number of amino acid residues of the sequence And that the program to compare sequences ALIGN-2 identifies it as identical to the corresponding residues of the sequence B, and where Y represents the total number of amino acid residues of the sequence B. note that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % identity of amino acid sequence A with respect to B is not equal to % identichnost and amino acid sequence of B relative to A.

Unless otherwise noted, all values % identity of amino acid sequences used in this description, get in the way described in the previous paragraph using the computer program ALIGN-2.

Table A

The term "vector" in this description refers to a nucleic acid molecule that can carry associated with another nucleic acid. One type of vector is a "plasmid", predstavlyaemoi circular double-stranded loop DNA you can ligitamate other segments of DNA. Another type of vector is a phage vector. Another type of vector is a viral vector, where other segments of DNA can be ligitamate in the viral genome. Certain vectors are capable of Autonomous replication in a cell host, in which they are introduced (e.g., bacterial vectors containing bacterial origin replication, and epilimnia vectors mammals). Other vectors (e.g., napisanie vectors mammals) after introduction into the cell of the host can be integrated into the genome of that cell and replicated along with the genome of the host. Moreover, certain vectors are capable of driving the expression of genes with which they are functionally linked. Such vectors in this description referred to as "recombinant expression vectors" (or simply "recombinant vectors"). As a rule, as vectors of expression in the methods of recombinant DNA often use plasmids. In the present description, the terms "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used type of the vector.

The terms "polynucleotide" or "nucleic acid"as used herein interchangeably, refer to a polymeric nucleotides of any length, and include DNA and RNA. Nucleotides can before the presentation a deoxyribonucleotides, the ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into the polymer using a DNA or RNA polymerase or by the reaction of synthesis. Polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogues. If the nucleotide modification patterns takes place, it can be entered before or after Assembly of the polymer. In the nucleotide sequence can be included dinucleotide components. Polynucleotide can be further modified after synthesis, for example, by conjugation with a label. Other types of modifications include, for example, "kierowanie, the replacement of one or more natural nucleotide analogs, mezhnukleotidnyh modifications such as, for example, modification, education-related communications that are not carrier charge (for example, methylphosphonate, phosphotriester, phosphoamide, carbamates and other), and communications, carrier charge (for example, phosphorothioate, phosphorodithioate and others), modification, containing one or more additional fragments, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine and other), modification, contain intercalating agents (e.g., acridine, psoralen, and others), modification, containing chelating agents (e.g., metals, radioactive meta the crystals, boron, oxidative metals, and others), modification, containing alkylating agents, modification, containing modified linkages (e.g., alpha anomeric nucleic acids, and others), as well as unmodified forms of polynucleotides. In addition, all the hydroxyl groups, generally present in the sugars may be replaced, for example, phosphonate groups, phosphate groups, they can also be protected by the standard protective groups, or activated to education links with other nucleotides, or they can participate in conjugation with solid or semi-solid media. 5'- and 3'-OH end groups can be phosphorylated or they can be substituted amines or organic kapinowski fragments containing from 1 to 20 carbon atoms. Other hydroxyl group can also be derivateservlet obtaining standard protective groups. Polynucleotides can also contain analogous forms Robotnik or deoxyribose sugars, which are widely known in the field and include, for example, 2'-O-methyl, 2'-O-allyl, 2'-fluoro - or 2'-azido-ribose, carbocyclic analogues of sugars, alpha-anomeric sugars, epimeria sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugar, sedoheptulose, acyclic analogs and basicosta nucleoside analogues, such as METI ribose. One or more fosfolipidnyh relations can be replaced by alternative linking groups. These alternative linking groups include, without limitation, the embodiment in which the phosphate is replaced by P(O)S("tiat"), P(S)S("ditial"), "(O)NR.sub.2("amidate"), P(O)R, P(O)OR', CO or CH.sub.2("Formatul"), where each R or R' independently denotes H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether linkage (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldi. Not all communication polynucleotide must be identical. The foregoing applies to all polynucleotides mentioned in this document, including RNA and DNA.

The term "oligonucleotide" in this description generally refers to short, usually single-stranded and synthetic polynucleotides, which are often, but not necessarily, have a length of less than about 200 nucleotides. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. All of the above in respect of polynucleotides equally and fully applicable to oligonucleotides.

The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, the floor is specific antibodies (e.g., bespecifically antibodies, if they possess the desired biological activity), these terms may also include certain antibody fragments (as described in detail in this document). The antibody can be human, humanized and/or affinity-Mature.

"Antibody fragments" contain only a portion of an intact antibody, which preferably retains at least one function, preferably greater part of the functions or all functions of this part, when it is part of the intact antibody. In one embodiment the antibody fragment contains antigennegative plot intact antibodies and, therefore, retains the ability to bind antigen. In another embodiment the antibody fragment, for example, which contains a plot Fc, retains at least one biological functions normally performed by the plot Fc when it is part of intact antibodies, such as FcRn binding, modulation of the half-life of antibodies, ADCC function and binding of complement. In one embodiment, the fragment of the antibody is a monovalent antibody, which has essentially the same half-life in vivo, as the intact antibody. For example, such an antibody fragment may contain antigennegative shoulder, coupled with the Fc sequence that is capable of Prodavat is this fragment stability in vivo.

The term "monoclonal antibody" in the present description refers to an antibody obtained from an almost homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies have a high specificity against a single antigen. In addition, unlike drugs polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant.

The monoclonal antibodies herein separately include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, whereas the other part of the chain (chain) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, this term also includes fragments of such antibodies, if they retain the desired biological activity (U.S. patent No. 4816567; and Morrison et al, Proc. Natl. Acad. Sci USA 81: 6851-6855 (1984)).

Humanized" forms of inhuman (for example, murine) antibodies are chimeric antibodies that contain minimal sequence nonhuman immunoglobulin. Mainly, humanized antibodies are human immunoglobulins (recipient antibody), where the remains of the hypervariable part of the recipient to replace the remnants of the hypervariable area species other than human (donor antibody)such as mouse, rat, rabbit or non-human Primate, which has the desired specificity, affinity and antigennegative capacity. In some cases the remains of the skeleton of the plot (FR) of a human immunoglobulin are replaced by corresponding nonhuman remains. Furthermore, humanized antibodies may contain residues that are not present in the recipient antibody or in the donor antibody. Such modifications carried out to further improve the properties of the antibodies. As a rule, humanitariannet antibody contains almost all of at least one, typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to areas nonhuman immunoglobulin and all or substantially all of the FR have the sequence of a human immunoglobulin. Humanitariannet antibody may also optionally include at measures is part of the constant part of the immunoglobulin (Fc), usually human immunoglobulin. More details can be found in Jones et al., Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2: 593-596 (1992). Cm. the following review articles and the links: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op.Biotech. 5: 428-433 (1994).

The term "antigen" refers to an identified antigen which selectively binds the antibody. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other natural or synthetic compound. Preferably, the target antigen is a polypeptide. "Acceptor human skeleton plot"used in the present invention is a frame area containing the amino acid sequence of frame section VL or VH obtained from the frame area of the human immunoglobulin or human consensus wireframe plot. Acceptor human skeleton plot, "derived from" a frame area of the human immunoglobulin or from a consensus of the human frame section can have the same amino acid sequence as these areas, or it may contain changes to the original amino acid sequence. If changes and the initial amino acids, preferably there are not more than 5, preferably 4 or less, or 3 or less, change the source of amino acids. If changes to the original amino acid present in VH, preferably these changes are only three, two or one of the provisions 71H, 73H and 78H; for example, amino acid residues in these positions can represent 71A, 73T and/or 78A. In one embodiment the sequence of the acceptor human frame section VL identical sequence of frame section VL of a human immunoglobulin or human consensus sequence frame section.

"Human consensus wireframe plot" is a framed site, which contains amino acid residues most frequently in the set of sequences of frame sections VL or VH of a human immunoglobulin. Typically, the set of sequences of the VL or VH of a human immunoglobulin belongs to the subgroup of sequences of the variable domain. Typically, the subgroup of sequences is a subgroup as described in Kabat et al. In one embodiment, in the case of VL, a subgroup is a subgroup Kappa I described in Kabat et al. In one embodiment, in the case of VH, the subgroup is a subgroup III, described in Kabat et al.

"The consensus is the first frame section VL subgroup I includes the consensus sequence, derived from the amino acid sequence of the variable light chain Kappa subgroup I, Kabat et al. In one embodiment of the consensus amino acid sequence of frame section VL subgroup I contains at least partially or completely each of the following sequences:

DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 34)-L1-WYQQKPGKAPKLLI (SEQ ID NO: 35)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 36)-L3-FGQGTKVEIKR (SEQ ID NO: 37).

"Consensus wireframe plot VH subgroup III contains a consensus sequence derived from the amino acid sequences of variable regions of heavy chain subgroup III, Kabat et al. In one embodiment the amino acid consensus sequence of frame section VH subgroup III contains at least partially or completely each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 38)-H1-WVRQAPGKGLEWV (SEQ ID NO: 39)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA (SEQ ID NO: 40)-H3-WGQGTLVTVSS (SEQ ID NO: 41).

"Unmodified human skeleton plot" is a human skeleton plot, which has the same amino acid sequence as acceptor human skeleton plot, which, for example, does not contain substitutions of the human amino acids at inhuman.

The term "modified hypervariable area" in this description refers to the hypervariable segment containing one Il is a few (for example, from one to about 16) amino acid substitutions.

The term "unmodified hypervariable area" in this description refers to the hypervariable segment containing the same amino acid sequence as nonhuman antibody from which it is derived, i.e. this sequence does not contain amino acid substitutions.

The term "hypervariable area", "HVR" or "HV"in this description refers to the sections of the variable domain of the antibody sequences which are hypervariable and/or form determined by the structure of the loop. Usually antibodies contain six hypervariable sites; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). The concept of the number of boundaries hypervariable sites used and included in the scope of this description. The most widely used hypervariable sites Kabat (CDR)based on the variability of the sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The location of the structural loops described by Chothia (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). Hypervariable sites AbM represent something of a cross between Kabat CDR structural loops (Chothia and used in the program for simulation of Oxford Molecular's AbM. "Contact" hypervariable areas determined on the basis of a comprehensive analysis of available crystal with whom ructur. The remains of these hypervariable sections below.

Table 1
LoopKabatAbMChothiaContact
L1L24-L34L24-L34L26-L32L30-L36
L2L50-L56L50-L56L50-L52L46-L55
L3L89-L97L89-L97L91-L96L89-L96
H1
Numbering according to Kabat
N-NWN-NWN-NN-NW
H1 numbering
By Chothia
N-NN-NN-NN-N
H2Uw50-NUw50-N N-NN-N
H3N-NN-NN-NN-N

Hypervariable sites may include the following "extended hypervariable sites": 24-36 or 24-34 (L1), 46-56 or 49-56, or 50-56, or 52-56 (L2), and 89-97 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2)and 93-102, 94-102 or 95-102 (H3) in the VH. The remains of the variable domain are numbered according to the Kabat system (Kabat et al., above) for each of the above definitions.

In this description remains "frame area" or "FR" are variable domain residues other than residues from a hypervariable site.

"Human antibody" is an antibody that has an amino acid sequence corresponding to the sequence of the antibody produced by a human and/or obtained using the methods for obtaining human antibodies described in this document. This definition of human antibodies is not included humanitariannet antibody containing inhuman antigennegative remains.

The term "affinity maturation" of an antibody refers to one or more changes in one or more CDRs that result in increased affinity of the antibody for antigen, compared to the original ant the body, which does not contain such a change. Preferred affinity-Mature antibodies will have nanomolar or even picomolar affinity for the target antigen. Affine-Mature antibodies produced using known in the field of methods. Marks et al. Bio/Technology 10: 779-783 (1992) describes affinity maturation in the reshuffling of the VH and VL domains. Nonspecific mutagenesis of residues of CDR and/or frame of the plot is described in: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

"Blocking" antibody or an "antagonist" antibody is an antibody that inhibits or reduces biological activity associated antigen them. Preferred blocking or antagonistic antibodies essentially or completely inhibit the biological activity of the antigen.

The term "agonist antibody" is used in this description refers to an antibody that mimics at least one of the functional activity of the target polypeptide.

"Violation" is any condition, improving as a result of treatment with the use of the substance/molecule or method according to the invention. This term includes chronic and acute disorders or diseases including those pathological conditions which provoc the shape the development of the mammal in question of the violation. Non-limiting examples of disorders that can be treated using the methods according to the invention include malignant and benign tumors; malignant diseases other than leukemia, and tumors of the lymph nodes; neuronal, glial, astatically disorders, diseases of the hypothalamus and other glands of internal secretion, macrophagal, epithelial, stromal and blastocladia disorders; and inflammatory, immunological and other related angiogenesis disorders.

The term "disease associated with the immune system" refers to the condition in which a component of the immune system of a mammal causes, mediates or otherwise contributes to a painful condition of the mammal. This term also includes diseases in which stimulation or enactment of the immune response has a beneficial effect on the development of the disease. In the scope of this term are mediated by the immune system inflammatory diseases, not mediated by the immune system inflammatory diseases, infectious diseases, immunodeficiency diseases, neoplasia, and other

Examples associated with the immune system and inflammatory diseases, some of which are mediated by immune or T-cells that can be treated with the method is in accordance with the invention, include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathy, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, mediated by the immune system thrombocytopenia), thyroiditis (diffuse toxic goiter, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, mediated by the immune system kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the Central and peripheral nervous systems such as multiple sclerosis, idiopathic demyelinizing polyneuropathy or Guillain-Barre syndrome and chronic inflammatory demyelinizing polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other nagapattanam viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease (ulcerative colitis: Crohn's disease), glute is sensitive enteropathy, and Whipple's disease, autoimmune or mediated by the immune system skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, hypersensitivity to food and urticaria, immunologic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitive pneumonitis, diseases associated with transplantation, including graft rejection and graft-versus-host. Infectious diseases, including viral diseases such as AIDS (HIV), hepatitis A, B, C, D and E, herpes and other infections, bacterial infections, fungal infections, protozoal infections and parasitic infections.

The term "autoimmune disease" or "autoimmune disease" herein used interchangeably and refer to a non-malignant disease or a disorder, directed against the subject's own tissue. Described in this document autoimmune diseases do not include malignant or cancerous disease or condition, in particular they do not include B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia and chronic leukemia of myeloblasts. the reamers autoimmune diseases or disorders include, without limitation, inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (e.g. atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including the syndrome of respiratory disorders in adults; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; insufficient adhesion of leukocytes; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes mellitus (e.g., diabetes mellitus type I or insulin-dependent diabetes), multiple sclerosis, Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; syndrome Serena; juvenile diabetes; and immune responses associated with acute delayed-type hypersensitivity mediated by cytokines and T-lymphocytes typically present in tuberculosis, sarcoidosis, polymyositis, Wegener and vasculitis; pernicious anemia (Addison disease); diseases related to diapedesis cells; inflammatory disorders of the Central nervous system (CNS); the syndrome lesions multiple organs;hemolytic anemia (including, without limitation, cryoglobulinemia or positive anemia of Coombs); myasthenia gravis; diseases mediated by a complex of antigen-antibody; disease directed against the glomerular basement membrane; antiphospholipid syndrome; allergic neuritis; graves ' disease; myasthenic syndrome Lambert-Eton; pemphigidae bladderwort; pemphigus; autoimmune polyendocrine syndromes; disease Reiter; the syndrome of limited mobility; disease Bechet; giant cell arteritis diagnostics; nephritis mediated by immune complexes; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and other

The term "gastrointestinal inflammatory disease" refers to a group of chronic disorders that cause inflammation and/or ulceration of the mucous membrane. As such, the term includes inflammatory bowel disease (such as Crohn's disease, ulcerative colitis, colitis of unknown origin and infectious colitis), mucositis (e.g., mucositis of the oral cavity mucositis of the gastrointestinal tract, nasal mucositis, proctitis), necrotizing enterocolitis and esophagitis.

The term "cell-proliferative violation" and "proliferative violation" otnostisya to violations associated with some abnormalities in cell proliferation. In tromboplastin cell-proliferative infringement is cancer.

The term "tumor" in this description refers to any growth or proliferation of neoplastic cells, both malignant and benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancer", "cell-proliferative violation", "proliferative violation" and "tumor" in this description are not considered mutually exclusive.

The terms "cancer" and "cancerous" refer to the physiological condition of the mammal or describe the physiological condition of a mammal, characterized by unregulated growth/proliferation of cells. Examples of cancers include, without limitation, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, cancer of the abdominal cavity, liver cell cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, cancer of the colon and rectum, carcinoma of the endometrium or uterine carcinoma, salivary glands, kidney cancer, liver cancer, prostate cancer, cancer of the female external genitals, thyroid cancer, liver carcinoma and various types of head and neck cancer.

Dysregulation of angiogenesis can lead to various disorders that can be treated using the compositions and methods according to the invention. These violations include both non-neoplastic and neoplastic condition. Neoplastic condition include, without limitation, the above-described diseases. Non-neoplastic condition include, without limitation, such conditions as undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathy, including retinopathy of premature development of retrolental fibroplasia, - neovascular glaucoma, age-related macular degeneration, diabetic edema yellow spots, revascularization of the cornea, revascularization of the graft corneal graft rejection retinal revascularization of the retina/choroid, revascularization angle (reddening of the iris), an eye disease associated with revascularization, vascular restenosis, arteriovenous defect (AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasia (including diffuse toxic goiter), transplantation of the cornea and other tissues, chronic inflammation, lung inflammation, acute damage to egka/acute respiratory distress syndrome, sepsis, primary pulmonary hypertension, malignant pulmonary effusions, cerebral edema (e.g., associated with acute stroke/closed skull trauma/trauma), synovial inflammation, pannus formation in RA, references for additional information myositis, hypertrophic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovarian syndrome, endometriosis, liquid-diseases 3rd range (pancreatitis syndrome lifestealing space, burns, bowel disease), fibromyoma of the uterus, premature labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), rejection of renal allograft inflammatory disease intestines, nephrotic syndrome, undesired or aberrant growth of tissue mass (noncancerous), hemarthrosis, hypertrophic scars, slow hair growth, syndrome Osler-Weber, retrolental fibroplasia pyogenic granuloma, scleroderma, trachoma, vascular adhesion, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.

In this description, the term "treatment" refers to clinical intervention aimed at changing the natural process in the subject or in the cell to be treated, and the treatment can be performed either for prophylaxis or during clinical pathology. Desirable effects Les the program include preventing occurrence or recurrence of the disease, improving symptoms, reducing direct or indirect pathological consequences of the disease, prevention of metastasis, reducing the rate of disease development, improvement or temporary relief of painful conditions, and remission or improved prognosis. In some embodiments the antibodies according to the invention is used to slow down the progression of disease or impairment.

The term "effective amount" refers to an amount effective to achieve the desired therapeutic or prophylactic result within the required time period and with use of necessary metering mode.

The term "therapeutically effective amount" of a substance/molecule, agonist or antagonist according to the invention can vary depending on factors such as the disease state, age, sex and weight of the subject, as well as the ability of a substance/molecule, agonist or antagonist, to cause the subject of the desired effect. Therapeutically effective amount is the amount at which therapeutically beneficial effects caused by the substance/molecule, agonist or antagonist, prevail over toxic or harmful effects. Since a prophylactic dose is administered to the subject prior to the onset of the disease or early ill the of prophylactically effective amount often but not always, below therapeutically effective amount.

The term "cytotoxic agent" in this description refers to a substance that suppresses or stops functioning cells and/or destroys cells. Understood that this term includes radioactive isotopes (e.g., At211I131I125, Y90That Re186That Re188Sm153Bi212, P32and radioactive isotopes of Lu), chemotherapeutically substances, such as methotrexate, adriamicin, Vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleotidase enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants, as well as various antitumor and anticancer means disclosed below. Other cytotoxic tools described below. Means, destroys tumor cells, causing the destruction of tumor cells.

"Chemotherapeutic agent" is a compound used to treat cancer. Examples of chemotherapeutic agents include alkylating tools, such as thiotepa and cyclophosphamide CYTOXAN®; the alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodepa, Carbogen, matureup and uredepa; ethylenimines and methylmelamine, including altretamin, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and triethylenemelamine; acetogenins (especially bullatacin, bullatacin); Delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulin acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcystein, scopoletin and 9-aminocamptothecin); bryostatin; callistemon; CC-1065 (including its synthetic analogues of adozelesin, carzelesin and bizelesin); podophyllotoxin; podofillina acid; teniposide; cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, halophosphate, estramustine, ifosfamide, mechlorethamine, hydrochloride oxide mechlorethamine, melphalan, novemberin, finestein, prednimustine, trofosfamide, braziliera; nitrosoanatabine, such as carmustine, chlorozotocin, fotemustine, lomustin, nimustine and ranimustine; antibiotics such as enediyne antibiotics (for example, calicheamicin, osobennostyami gamma and calicheamicin omega (see, for example, Agnew, Chem Int. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin a; spiramycin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores) aclacinomycin, actinomycin, autralian, azaserine, bleomycin, actinomycin, carubicin, karminomitsin, casinopolis, chromomycin, dactinomycin, daunorubicin, demoralizing, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morphosyntactical, cyanomethaemoglobin, 2-pyrrolidinecarbonyl and desoxidation), epirubicin, zorubicin, idarubitsin, marsellaise, mitomycin, such as mitomycin C, mycofenolate acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, gentamicin, radiobeacon, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); analogues Polevoy acid, such as deeperin, methotrexate, peripherin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, timipre, tioguanin; pyrimidine analogues such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antihormones of the adrenal glands, such as aminoglutaric the ID, mitotane, trilostane; compensators Polevoy acid, such as prolinnova acid; Eagleton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; astroball; bisantrene; edatrexate; defaming; demecolcine; diazinon; alternity; slipline acetate; epothilone; etoposide; gallium nitrate; hydroxyurea; lentinan; londini; maytansinoid, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitrean; pentostatin; penomet; pirarubicin; losoxantrone; 2-acylhydrazides; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tinoisamoa acid; creation; 2,2',2"-trihlortrietilamin; trichothecenes (especially toxin T-2, verrucarin a, roridin A and unguided); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; Galitsin; arabinoside ("Ara-C"); thiotepa; taxoid, for example, paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, N.J.), not containing cremophor composition of paclitaxel from the nanoparticles based on albumin ABRAXANE™ (American Pharmaceutical Partners, Schaumberg, Illinois), docetaxel TAXOTERE® (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR(R)); 6-tioguanin; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxal the sinkers; leucovorin; vinorelbine (NAVELBINE®); Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; deformational (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of the above compounds; and combinations of two or more of the above compounds, such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and lacavalier.

This definition also includes antihormone tools that regulate, reduce, reduce, block, or inhibit the effects of hormones that can stimulate tumor growth, these funds are often used for systemic injections or across the body. They can imagine themselves hormones. Examples include antiestrogens and selective modulators of estrogen receptors (SERM), including, for example, tamoxifen (including NOLVADEX tamoxifen®), raloxifene EVISTA®, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; antiprogesterone; step-down regulators of estrogen receptor (ERD); a means of suppressing or extinguishing the functioning of the ovaries, such as agonists releasing factor is luteinizing hormone (LHRH), such as LUPRON® and leuprolide acetate ELIGARD®, goserelin acetate, buserelin acetate and triptorelin; other anti-androgens such as flutamide, nilutamide and bikalutamid; and aromatase inhibitors that inhibit the enzyme aromatase, which inhibits estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate MEGASE®, exemestane AROMASIN®, formestane, fadrozole, vorozole RIVISOR®, letrozole, FEMARA® and anastrozole ARIMIDEX®. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate DIDROCAL®, NE-58095, zoledronicaa acid/zoledronate ZOMETA®, alendronate FOSAMAX®, AREDIA pamidronate®, tiludronate SKELID® or risedronate ACTONEL®; and troxacitabine (casinoby similar 1,3-dioxolane nucleoside); antisense oligonucleotides, particularly the nucleosides that inhibit expression of genes in signaling pathways involved in abberant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras and the receptor for epidermal growth factor (EGF-R); vaccines such as THERATOPE vaccine® and vaccines gene therapy, for example a vaccine ALLOVECTIN®vaccine, LEUVECTIN® vaccine VAXID®; topoisomerase inhibitor 1 LURTOTECAN®; ABARELI® rmRH; lapatinib ditosylate (double low molecular weight inhibitor of the tyrosine kinase ErbB-2 and EGFR, also known as GW572016); and pharmaceutically priemel the appropriate salt, acid or derivatives of all the above compounds.

The term "agent, inhibiting the growth" in this description refers to compounds or compositions that inhibit the growth of cells, which does not depend on beta activation, either in vitro or in vivo. Thus, by means of inhibiting the growth of, can be a substance that is able to significantly reduce the percentage beta-dependent cells in S-phase. Examples of means, inhibiting growth, include means blocking the development of the cell cycle (period other than S phase), such as tools to interrupt execution of phase G1 and M. Classical blockers M-phase include the Vinca alkaloids (vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Means terminating the G1 phase and, as a side effect, the S phase, include, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p.13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew is who, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel activate the Assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

"Doxorubicin" is an anthracycline antibiotic. Full chemical name of doxorubicin is (8S-CIS)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetic)-1-methoxy-5,12-naphthacenedione.

Compounds that can be used in combination therapy with an antagonistic antibody against beta, according to the invention include antibodies (including, without limitation, other antagonistic antibodies against beta (Fib 21, 22, 27, 30 (Tidswell, M. (1997), supra) or humanized derivatives), antibodies against alpha (such as ANTEGEN®), antibodies against TNF (REMICADE®)) or non-protein compounds, including, without limitation, compounds 5-ASA ASACOL®, PENTASA™, ROWASA™, COLAZAL™, and other compounds, such as purinethol, and steroids, such as prednisone. In one embodiment, the invention encompasses a method of treating a patient, such as a person, an antagonistic antibody against beta according to the invention alone or in combination with a second compound, which is used for is ecene inflammation. In one embodiment the second compound selected from the group consisting of Fib 21, 22, 27, 30, or humanized derivatives, antibodies against alpha, ANTEGEN®, antibodies against TNF, REMICADE®, compounds 5-ASA, ASACOL®, PENTASA™, ROWASA™, COLAZAL™, purinethol, steroids and prednisone. In one embodiment according to the invention the introduction of antagonistic antibodies against beta according to the invention allows to significantly reduce the dose of the second compound. In one embodiment of the specified reduction of the dose of the second compound is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. In one embodiment according to the invention the combination of the antibodies according to the invention with a low dose of the second connection facilitates the patient's symptoms are almost the same or higher level as the introduction of one of the second connection.

Obtaining variant antibodies, causing a reduced response HAMA or not the caller of this response

The reduction or elimination of HAMA response (formation of human antibodies against mouse (also true of human antibodies against rat or human antibodies against human)) is an important aspect of clinical development of suitable therapeutic agents. See, for example, Khaxzeli et al., J. Natl. Cancer Inst. (1988), 80: 937; Jaffers et al., Transplantation (1986), 41: 572; Shawler et al., J. Immunol. (1985), 135: 1530; Sears et al., J. Biol. Response Mod. (1984), 3: 138; Miller et al., Blood (1983), 62: 988; Hakimi et al., J. Immunol. (1991), 147: 1352; Reichmann et al., Nature (1988), 332: 323; Junghans et al., Cancer Res. (1990), 50: 1495. As indicated in the present description, the invention provides antibodies, humanized to reduce or eliminate the HAMA response. Using known in the field of methods (some of them described below) can also be obtained variants of these antibodies.

For example, as described herein, the amino acid sequence of the antibody may serve as the original (parent) sequence for derivatization frame section and/or a hypervariable sequence (hypervariable sequences). The selected sequence of frame section to which is attached the original hypervariable sequence, herein referred to as the acceptor human skeleton plot. Although you can use the acceptor human frame sections from a human immunoglobulin or derived from a human immunoglobulin (areas VL and/or VH immunoglobulin), preferably using the acceptor human frame sections from the human consensus sequence frame of the site or obtained from this sequence is, since it has been shown that such frame sections cause minimal immune response or not cause an immune response in humans.

If the acceptor sequence derived from a human immunoglobulin, you do not have to use the human sequence frame area, which is chosen on the basis of homology with the donor sequence frame section by a comparison of the primary sequences of the donor frame section and different human frame sections from the collection of the human sequences of frame sections with the choice of the sequence of frame section having a maximum homology with respect to the acceptor sequence.

In one embodiment of the use of human consensus framework regions or derived from a consensus sequences of frame sections VH subgroup III and/or VL Kappa subgroup I.

Thus, the acceptor human skeleton plot VH may contain one, two, three or all of the following frame sections:

FR1 contains EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 38),

FR2 contains WVRQAPGKGLEWV (SEQ ID NO: 39),

FR3 contains RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYCA (SEQ ID NO: 42), where X1 denotes A or R, X2 means T or N, and X3 means A, L or F,

FR4 contains WGQGTLVTVSS (SEQ ID NO: 41).

Examples to sensonic frame sections VH include:

the consensus sequence of frame area of the human VH subgroup I minus Kabat CDRs (SEQ ID NO: 19);

the consensus sequence of frame area of the human VH subgroup I minus extended hypervariable sites (SEQ ID nos: 20-22);

the consensus sequence of frame area of the human VH subgroup II minus Kabat CDRs (SEQ ID NO: 48);

the consensus sequence of frame area of the human VH subgroup II minus extended hypervariable sites (SEQ ID nos: 49-51);

the consensus sequence of frame area of the human VH subgroup III minus Kabat CDRs (SEQ ID NO: 52);

the consensus sequence of frame area of the human VH subgroup III minus extended hypervariable sites (SEQ ID nos: 53-55);

acceptor frame area of the human VH minus Kabat CDRs (SEQ ID NO: 56);

acceptor frame area of the human VH minus extended hypervariable sites (SEQ ID nos: 57-58);

acceptor 2 frame area of the human VH minus Kabat CDRs (SEQ ID NO: 59) or

acceptor 2 frame area of the human VH minus extended hypervariable sites (SEQ ID nos: 60-62).

In one embodiment of the acceptor human skeleton plot VH contains one, two, three or all of the following frame sections:

FR1 contains EVQLVESGGGLVQPGGSLRLSCAAS (SE ID NO: 38),

FR2 contains WVRQAPGKGLEWV (SEQ ID NO: 39),

FR3 contains RFTISADTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO:

43), RFTISRDTSKNTAYLQMNSLRAEDTAVYYCA (SEQ ID NO: 44),

RFTISRDTSKNTFYLQMNSLRAEDTAVYYCA (SEQ ID NO: 45),

RFTISADTSKNTFYLQMNSLRAEDTAVYYCA (SEQ ID NO: 46),

FR4 contains WGQGTLVTVSS (SEQ ID NO: 41).

Acceptor human skeleton plot VL contains one, two, three or all of the following frame sections:

FR1 contains DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 34),

FR2 contains WYQQKPGKAPKLLI (SEQ ID NO: 35),

FR3 contains GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 36),

FR4 contains FGQGTKVEIKR (SEQ ID NO: 37).

Examples of consensual frame sections VL include:

the consensus frame area of the human VL Kappa subgroup I (SEQ ID NO: 14);

the consensus frame area of the human VL Kappa subgroup I (advanced HVR-L2 (SEQ ID NO: 15);

the consensus frame area of the human VL Kappa subgroup II (SEQ ID NO: 16);

the consensus frame area of the human VL Kappa subgroup III (SEQ ID NO: 17) or

the consensus frame area of the human VL Kappa subgroup IV (SEQ ID NO: 18).

Although the acceptor sequence may be identical to a sequence selected human skeleton plot, obtained either from a human immunoglobulin or human consensus skeleton of the plot, in the present invention, it is assumed that in the acceptor sequence may already was present is W ith amino acid substitutions compared to the sequence of human immunoglobulin or sequence, human consensus wireframe plot. Data already existing replacement preferably present in a minimum amount; typically the difference from the sequence of human immunoglobulin or sequence, human consensus frame area is four amino acids, three amino acids, two amino acids, or one amino acid.

The remains of the hypervariable area nonhuman antibodies injected into the acceptor human frame areas VL and/or VH. For example, you can enter the residues corresponding to residues of Kabat CDR, the remnants of the hypervariable loops Chothia, the remnants of the Abm and/or contact residues. Optional enter the following residues expansion hypervariable plot: 24-34 (L1), 49-56 (L2) and 89-97 (L3), 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3).

Although this document describes the "introduction" hypervariable residues of the plot, it should be understood that it can be made in various ways, for example you can get a nucleic acid encoding a target amino acid sequence by mutation of the nucleic acid coding sequence of the murine variable domain, so that the residues encoded by this nucleic acid skeleton of the plot has changed on the remnants of the acceptor human frame the site, or by mutation of the nucleic acid that encodes posledovatelnostyakh variable domain, so that remains of the hypervariable domain changed to inhuman remnants, or by synthesis of nucleic acid that encodes a target sequence, and others

In the example in this description and the examples options with grafted hypervariable area obtained by mutagenesis of the nucleic acid that encodes a human acceptor sequence, Councel method using a separate oligonucleotide for each hypervariable segment. Kunkel et al, Methods Enzymol. 154: 367-382 (1987). To adjust and tweak the interaction hypervariable site-specific antigen, in the frame area and/or a hypervariable area of traditional methods, enter the appropriate replacement.

Method of phage (fahmideh) display (also referred to in some aspects of this description of phage display) can be used as a quick and convenient method of obtaining and screening of libraries of different potential variant antibodies obtained by the randomization sequence. However, experts in this area and other known methods of obtaining and screening of modified antibodies.

Method of phage (fahmideh) display is an effective tool for obtaining and selection of new proteins that bind to the ligand, such as an antigen. Application of methods of phage (fahmideh) dis is Leah allows you to make large libraries of protein variants, which you can easily sort the sequences with high affinity associated with the molecule-target. Nucleic acids encoding variant polypeptides, usually hybridized with a nucleic acid sequence that encodes a viral envelope protein, such as protein gene III or gene VIII protein. Developed monovalent system fahmideh displays, where the nucleic acid sequence that encodes a protein or polypeptide, hybridized with a nucleic acid sequence that encodes a protein fragment of the gene III (Bass, S., Proteins, 8: 309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3: 205 (1991)). In monovalent system fahmideh display hybrid gene is expressed at a low level, while the gene III wild-type, too, is expressed with the protein synthesis, therefore, remains infective ability. Methods of obtaining peptide libraries and screening these libraries are disclosed in many patents (for example, U.S. patent No. 5723286, U.S. patent No. 5432018, U.S. patent No. 5580717, U.S. patent No. 5427908 and U.S. patent No. 5498530).

Libraries of antibodies or antigenspecific polypeptides receive a variety of ways, including changing one gene by inserting a random DNA sequence or by cloning of a family of related genes. Methods for producing antibodies or antigenspecific fragments and the use of phage (fahmideh) displays described in U.S. patent No. 5750373, 5733743, 5837242, 5969108, 6172197, 5580717 and 5658727. Then the library are screened for the expression of antibodies or antigenspecific proteins with desirable characteristics.

Methods of replacement of certain amino acids through changes nucleic matrix is well known in this field and some of them are described in this document. For example, the remains of a hypervariable area can be replaced by Councel method. See, for example, Kunkel et al., Methods Enzymol. 154: 367-382 (1987).

The sequence of the oligonucleotide contains one or more sets of desirable codons encoding the variable remains hypervariable segment. The set of codons represents the number of different sequences of nucleotide triplets used for encoding the desired variant amino acids. Sets of codons can be represented by characters that mean individual nucleotides or equimolar mixture of nucleotides, as shown below in accordance with the system of IUB.

Reduction system IUB

G - Guanine

A - Adenine

T - Thymine

C - Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S (C or G)

W (A or T)

H (A or C or T)

B - (C or G or T)

V - A, or C, or G)

D - (A, or G, or T)H

N (A or C or G or T)

For example, in the set of codons DVK D may indicate the nucleotides A or G or T; V can mean A, or G, or C; and K m which may mean G or T. This set of codons may contain 18 different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Sets of oligonucleotides or primers can be synthesized by standard methods. For example, by the method of solid-phase synthesis is possible to synthesize a set of oligonucleotides, which includes a sequence that contains all possible for a given set of codons combination of nucleotide triplets, and which encodes a desired set of amino acids. In this area a well-known method for synthesis of oligonucleotides using the specified "degeneracy" of the nucleotides at specific positions. These sets of oligonucleotides containing specific sets of codons, can be synthesized using a commercial nucleic acid synthesizers (supplied, for example, Applied Biosystems, Foster City, CA) or they can be obtained from commercial suppliers (e.g., from Life Technologies, Rockville, MD). Therefore, the set of oligonucleotides synthesized with a specific set of codons, usually contains a set of oligonucleotides with different sequences, and the differences are determined by the set of codons in the full sequence. In accordance with this invention, the oligonucleotides have a sequence that is capable of gibridizatsiya with nucleic matrix of variabel the second domain, and, in addition, in order to clone may contain areas enzymatic restriction.

In the same way nucleotide sequence encoding variant amino acids, can be obtained by mediated oligonucleotide mutagenesis. This method is well known in this field and described by Zoller et al. Nucleic Acids Res. 10: 6487-6504 (1987). Briefly, the nucleotide sequences encoding variant amino acids, obtained by hybridizing set of oligonucleotides encoding the desired set of codons, DNA matrix, where the matrix is a single-stranded plasmid containing the nucleotide sequence of the matrix variable segment. After hybridization using a DNA polymerase synthesize a full-sized second complementary chain matrix, which will contain the oligonucleotide primer, and will contain a set of codons caused by the set of oligonucleotides.

As a rule, use oligonucleotides containing at least 25 nucleotides in length. Optimal oligonucleotide contains from 12 to 15 nucleotides that is fully complementary to the matrix on either side of the nucleotide (nucleotides)encoding (encoding) mutation (mutation). This ensures that the oligonucleotide will be appropriately gibridizatsiya with the single-stranded molecularity DNA. Oligonucleotides can be easily synthesized using known in the field of methods, such as described by Crea et al., Proc. Natl. Acad. Sci. USA, 75: 5765 (1978).

The DNA matrix is obtained using vectors that receive any of bacteriophagy vectors M13 (you can use commercially available vectors M13mp18 and M13mp19), any of the vectors that contain the origin of replication of single-stranded phage as described by Viera et al., Meth. Enzymol., 153: 3 (1987). Thus, DNA is subject to mutagenesis, it is possible in the paste in one of the data vectors to obtain single-stranded matrix. Obtaining single-stranded matrix described in sections 4.21-4.41 above publication Sambrook et al.

To change the natural DNA sequence, oligonucleotide hybridized stranded matrix in appropriate hybridization conditions. Then add DNA polimerizuet enzyme, usually DNA polymerase T7 or piece maple DNA polymerase I, and synthesize the complementary chain of the matrix, using the oligonucleotide as a seed for the synthesis. So get heteroduplex the DNA molecule, one strand which encodes the mutant form of the gene 1, and the other strand (the original matrix) encodes natural, unaltered sequence of the gene 1. Then a suitable cell host, typically prokaryotic, such as E. coli JM101, transform received the Oh heteroduplexes molecule. After growing the cells are placed on an agarose Cup and subjected to screening using oligonucleotide primers labeled with radioactive 32-phosphate, in the presence of bacterial colonies that contain the mutated DNA.

The above method can be modified to obtain homoduplex molecule plasmids, both chains which contain the mutation(s). Modify the method as follows: single-stranded oligonucleotide is annealed to single-stranded matrix, as described above. A mixture of three deoxyribonucleotides, desoxyephedrine (dATP), deoxyribofuranosyl (dGTP) and desoxyepothilone (dTT), together with a modified timezonebias called dCTP-(aS) (which can be obtained from Amersham). This mixture was added to the complex matrix-oligonucleotide. After adding to the mixture of DNA polymerase produces DNA strand that is identical to the matrix, except for the mutant bases. In addition, the new DNA strand contains dCTP-(aS) instead of dCTP, which protects it from cleavage by the endonuclease. After cutting the matrix circuit of the double-stranded heteroduplex appropriate restriction enzyme chain matrix can be split by ExoIII nuclease or other appropriate nuclease after the section that contains the site(s)subject to mutagenesis. Then the reaction stopped by and get a molecule which is only partially single-stranded. Then in the presence of all four deoxyribonucleotides, ATP and DNA ligase using DNA polymerase get a complete double-stranded homoduplex DNA. Received homoduplex molecule can be used to transform a suitable host cell.

As indicated previously, the sequence of the set of oligonucleotides of sufficient length to gibridizatsiya with nucleotide matrix and may also, but need not, contain the restriction sites. The DNA matrix is obtained using vectors that receive any of bacteriophagy vectors M13 (you can use commercially available vectors M13mp18 and M13mp19), any of the vectors that contain the origin of replication of single-stranded phage as described by Viera et al., Meth. Enzymol., 153: 3 (1987). Thus, DNA is subject to mutagenesis, can be inserted into one of the data vectors to obtain single-stranded matrix. Obtaining single-stranded matrix described in sections 4.21-4.41 above publication Sambrook et al.

In accordance with another way the library can be obtained through the use of sets of oligonucleotides above and below in the course of reading, and each kit contains a set of oligonucleotides with different sequences, which are determined by the sets of codons in the sequence of the oligonucleotide. Sets of oligonucleotides above and the who in the course of reading along with the sequence of the matrix nucleic acid, encodes a variable domain, used in polymerase chain reaction with obtaining libraries of PCR products. The PCR products can be called "nucleotide cassette, as they can be hybridizat, using known methods of molecular biology with other related or unrelated nucleotide sequences, for example, protein-coding viral envelope and dimerizes domains.

The sequence of PCR primers comprises one or more sets of desirable codons in solvent-accessible and highly varying the provisions of the hypervariable segment. As described above, the set of codons is a set of sequences of different nucleotide triplets encoding the desired variant amino acids.

Antibodies selected using the appropriate stages of the screening/selection and, therefore, meets the desired criteria, you can select and clone using standard recombinant methods.

The vectors, cells of the host and recombinant methods

To obtain the antibody according to the invention using recombinant methods, allocate encoding its nucleic acid to be inserted into a vector capable of replication, with the aim of further cloning (amplification of the DNA) or expression. DNA encoding the antibody can Le is to be extracted and sequenced using conventional methods (for example, through the use of oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are commercially available. The choice of vector, in particular, depends on the host cell. Generally, the preferred cells of the host can be either prokaryotic or eukaryotic cells (typically, mammalian cells).

Obtaining antibodies using prokaryotic host cells

Vector design

Polynucleotide sequences encoding polypeptide components of the antibodies according to the invention, can be obtained using standard recombinant methods. The desired polynucleotide sequence can be distinguished from cells that produce the antibody, such as hybridoma cells, and then sequenced. Alternatively, polynucleotide can be synthesized using nucleotide synthesizer or PCR. Obtained sequences encoding the polypeptides are inserted into the recombinant vector capable of replication and expression of heterologous polynucleotides in prokaryotic hosts. For the purposes of the present invention can use various commercially available and known in the field vectors. The selection of the appropriate vector depends mainly on RA is a measure of nucleic acids, inserted into the vector, and from a specific host cell transformed by the vector. Each vector contains various components for performing its function (amplification or expression of heterologous polynucleotide or both), and compatibility with a particular cell of the host in which it is located. Vector components generally include, without limitation, the origin of replication, a gene marker selection, the promoter, the binding site of the ribosome (RBS), signal sequence, the insertion of a heterologous nucleic acid and the sequence termination of transcription.

Generally, plasmid vectors containing replicon and control sequences derived from a species compatible with the host-cell, can be used to transform these cells. Typically, the vector contains an origin of replication and a marker sequence that helps to hold the phenotypic selection of the transformed cells. For example, E. coli is typically transformed pBR322, a plasmid derived from the species E. coli. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), which allows for easy identification of transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or after appropriate modification could the t to contain promoters, which can be used by the microorganism for the expression of endogenous proteins. Examples of derivatives of pBR322, used for the expression of specific antibodies, are described in detail in Carter et al., U.S. patent No. 5648237.

In addition, phage vectors containing replicon and control sequences that are compatible with the microorganism host, can be used to transform the data owners. For example, on the basis of the bacteriophage, such as λGEM.TM.-11, can be obtained recombinant vector used for transformation of such host cells as E. coli LE392.

The expression vector according to the invention may contain more than two pairs of the promoter-cistron encoding each polypeptide components. The promoter is a noncoding regulatory sequence above (5') cistron and modulating its expression. Prokaryotic promoters are usually divided into two classes, induced and constitutive. Inducible promoter is a promoter that initiates an increase in the transcription level of cistron under its control in response to a state change of culture, such as changing the number of nutrients or temperature changes.

There are a large number of promoters recognized by a number of potential host cells. The selected promoter can functionally St. the show with DNA cistron, encoding the light or heavy chain by cutting out the promoter of the DNA source using a restriction enzyme and paste the selected sequence of the promoter in the vector according to the invention. For direct amplification and/or expression of a target gene can be used as native promoters, and many heterologous promoters. In some embodiments using heterologous promoters, because they usually provide a higher level of transcription and higher output expressed by the target gene compared to the native promoter of the target polypeptide.

Promoters suitable for use in prokaryotic hosts include the PhoA promoter, the promoter system β-galactosi and lactose promoter system tryptophan (trp), and hybrid promoters such as the tac promoter or the trc. However, you can use other promoters capable of functioning in bacteria (such as other known bacterial or phage promoters). The nucleotide sequences of these promoters published that helps experienced professionals functionally connect them with cisternae coding target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269)using linkers or adapters providing the necessary restriction sites.

In one aspect according to the SNO invention each cistron in the recombinant vector contains a component signal secretion sequence, which controls the movement expressed polypeptide through the membrane. Typically, the signal sequence may be a component of the vector, or it may constitute a part of the DNA that encodes a target polypeptide, which has been inserted into the vector. Signal sequence, ispolzuya in order according to the invention should be recognized by the host-cell and subjected to processing (i.e. to split the signal peptidases) in this cell. In the case of a prokaryotic host cells that do not recognize the sequence of amplified relative to the heterologous polypeptide, and do not perform their processing, this signal sequence is substituted for prokaryotic signal sequence selected, for example, from the group consisting of the leader sequence of alkaline phosphatase, penitsillinazy, Ipp or thermostable enterotoxin II (STII), LamB, PhoE, Pe1B, OmpA and MBP. In one embodiment according to the invention the signal sequence used in both cistronic expression systems represent the STII signal sequences or their variants.

In another aspect of the production of immunoglobulins in accordance with this invention can occur in the cytoplasm of the host cell, and therefore, the presence of a signal sequence in which each cistron is not required. In this case expressed light and heavy chains of immunoglobulins and their installation and Assembly with the formation in the cytoplasm of functional immunoglobulins. In the cytoplasm of some strains of hosts (for example, strains trxB' E.coli) there are conditions that promote the formation of disulfide bonds, which ensure the correct Assembly and laying expressed protein subunits. Proba and Pluckthun Gene, 159: 203 (1995).

The present invention provides an expression system in which the quantitative ratio of the expressed polypeptide components can be modulated to maximize the yield of secreted and correctly assembled antibodies according to the invention. Such a modulation is carried out at least partially by simultaneous modulation of the intensity of broadcast polypeptide components.

One method of modulating the intensity of the broadcast described in Simmons et al., U.S. patent No. 5840523. In this method, use the options of the site of translation initiation (TIR) within cistron. For a given TIR you can create a number of variants with amino acid or nucleotide sequences which provide different intensity of the broadcast, and then choose the value of this factor corresponding to the desired level of expression of a particular circuit. Options TIR can be obtained by the method of traditional mutage is ESA change the codon resulting in change of amino acid sequence, although the nucleotide sequence of the preferred "silent" changes. Changes in TIR may include, for example, quantitative or spatial variations in the sequence of the Shine-Dalgarno, along with changes in the signal sequence. One way of obtaining mutant signal sequence is to obtain Bank codons" in the beginning of the coding sequence, which does not change the amino acid sequence of the signal sequence (i.e. the changes are "silent"). This is done by changing the third nucleotide position in each codon; in addition, some amino acids such as leucine, serine and arginine, have several first and second positions, which can create complications when receiving the Bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzyniol. 4: 151-158.

Preferably, get a set of vectors with a range of TIR-intensity for each cistron included in the vectors. This limited set allows us to compare the levels of expression of each chain, and the yield of the target product antibodies at different combinations of TIR-intensity. TIR-intensity can be determined by quantitative measurement of the expression level of the reporter is wow gene, as described in Simmons et al., U.S. patent No. 5840523. Depending on the results of the comparison of the intensity of the broadcast, select the desired specific TIR, which enter into the composition of the vector expression constructs according to the invention.

Prokaryotic cells are the owners that are suitable for expression of the antibodies according to the invention, include Archaebacteria and Eubacteria, such as gram-negative or gram-positive organisms. Examples of usable bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P.aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment of use gram-negative cells. In one embodiment according to the invention as hosts use E.coli cells. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol.2 (Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC No. 27325) and its derivatives, including strain 33D3 containing genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR(U.S. patent No. 5639635). You can also use other strains and derivatives thereof, such as E. coli 294 (ATCC 31446), E. coli B, E. coliλ1776 (ATCC 31537), and E. coli RV308 (ATCC 31608). These examples are illustrative rather than limiting. Methods of obtaining derivatives of any of the above strains that have specific genotypes know what TNA in this area and described, for example, Bass et al., Proteins, 8: 309-314 (1990). As a rule, it is necessary to choose the appropriate bacteria with the capacity replicon to play in the bacterial cell. For example, varieties of E.coli, Serratia, or Salmonella can be successfully used as hosts, if replicon uses well known plasmids such as pBR322, pBR325, pACYC177, or pKN410. Usually cells are the masters secrete minimal amounts of proteolytic enzymes in the composition of the culture medium, it is desirable to introduce additional protease inhibitors.

Antibodies

Cell owners transform the above expression vectors and cultured in conventional nutrient medium, modified in such a way as to ensure inducing promoters, selecting transformed cells, or amplifying the genes encoding the target sequence.

Transformation is the introduction of DNA into a prokaryotic host so that the DNA was capable of replication either in the form of an extrachromosomal element or integrated into the chromosome. The transformation is carried out using standard methods appropriate for the selected host cell. Bacterial cells with a significant barrier cell membrane is usually treated with calcium in the form of calcium chloride. In another method of transformation used is jut glycol/DMSO. In addition, for transformation using electroporation method.

Prokaryotic cells used to obtain the polypeptides according to the invention, is grown in an environment known in the field and suitable for culturing a selected host cells. Examples of suitable media include broth, Luria (LB)containing the necessary nutritional supplements. In some embodiments, the medium also contains a tool that facilitates the selection, selected depending on the design of the expression vector, the tool provides a selective growth of prokaryotic cells containing the expression vector. For example, the medium used for the cultivation of cells expressing the gene of resistance to ampicillin, add ampicillin.

Any necessary additives, in addition to carbon and nitrogen sources, inorganic phosphate, can also be included in the composition of the medium in suitable concentrations individually or in a mixture with other additives or environment, such as a complex nitrogen source. The culture medium may optionally contain one or more reducing agents selected from the group consisting of glutathione, cysteine, applied, thioglycolate, dithioerythritol and dithiothreitol.

Prokaryotic cells are the owners of cultivated at a suitable temperature. For example, temperature, preference is sustained fashion for the growth of E. coli varies approximately from 20°C to 39°C, more preferably from about 25°C to 37°C, even more preferably it is approximately 30°C. the pH of the medium may have any value in the range from about 5 to 9 and depends mainly on the type of the host body. In the case of E.coli pH preferably is in the range of approximately from 6.8 to 7.4, and more preferably it is equal to approximately 7,0.

If the expression vector according to the invention using inducible promoter, expression of the protein induce under conditions suitable for activation of the promoter. In one aspect according to the invention for regulating transcription of the polypeptide using the PhoA promoter. Accordingly, in order to induce the transcription of transformed cell hosts are cultivated in limited phosphate environment. Preferably, as limited by phosphate environment use environment C.R.A.P (see, for example, Simmons et al., J. Immunol. Methods (2002), 263: 133-147). As is well known in this field, you can use a number of other inducing factors, depending on the applied vector design.

In one embodiment of the expressed polypeptides of the present invention are secreted into periplasm cells-owners and retrieved from the database. Extraction of protein typically involves the destruction of the microorganism, usually with the help of such with osobov, as osmotic shock, sonication or lysis. After the destruction of the cell fragments or whole cells can be removed by centrifugation or filtration. Proteins can be further cleaned, for example by chromatography on affinity resin. Alternatively, proteins can be transported into the culture medium and to extract from it. Cells can be removed from the culture, and the culture supernatant filtered and concentrated, followed by purification of the obtained proteins. Expressed polypeptides can be isolated and identified using well known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting.

In one aspect according to the invention the antibody receive in large quantities by using a fermentation process. To obtain recombinant proteins can be used various methods of large-scale periodic fermentation feed. The capacity of the large-scale fermentation is at least 1000 liters, preferably from about 1000 to 100,000 liters. In such fermenters for the distribution of oxygen and nutrients, especially glucose (the predominant source of carbon/energy), using a paddle stirrer. Small-scale fermentation is usually carried out in a fermenter, volume vestimenti which does not exceed approx the tion of 100 liters and can vary from about 1 liter to 100 liters.

In the fermentation process the induction of expression of the protein is usually started after the cell in suitable conditions, achieved the desired density, for example corresponding to the value of OD550approximately 180-220, in this state, the cells are in an early phase of a stationary population. As is well known in this field and described above, depending on the design of the vector, you can use different inducing factors. Before induction, the cells can be grown in a shorter period of time. Usually cells induce for approximately 12-50 hours, although you can use a longer or shorter induction.

Fermentation condition can be modified to improve the yield and quality of the polypeptides according to the invention. For example, to improve the accuracy of Assembly and installation of secreted polypeptides antibodies, it is possible to carry out simultaneous transformation of prokaryotic host cells other vectors providing for the overexpression of proteins, chaperones, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (peptideprophet-CIS, TRANS-isomerase with chaperone activity). It was shown that protein chaperones facilitate proper Assembly and increase the solubility of heterologous proteins produced in bacterial cells-hosts. Chen et al. (199) J. Bio. Chem. 274:19601-19605; Georgiou et al., U.S. patent No. 6083715; Georgiou et al., U.S. patent No. 6027888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Environ. 39:199-210.

To minimize proteolysis of heterologous proteins (particularly sensitive to proteolysis), for the purposes of the present invention can be used strains-owners with a deficit of proteolytic enzymes. For example, strains of host cells can be modified by mutations (mutations) in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and their combinations. Some deficient in protease strains of E.coli are commercially available and are described, for example, in Joly et al. (1998), above; Georgiou et al., U.S. patent No. 5264365; Georgiou et al., U.S. patent No. 5508192; Hara et al., Microbial Drug Resistance, 2:pp.63-72 (1996).

In one embodiment the expression system according to the invention as host cells using E. coli strains with a deficit of proteolytic enzymes, and transformed by the plasmid providing the overexpression of one or more chaperone proteins.

Purification of antibodies

In one embodiment the protein-antibody obtained by the method according to the invention, additionally cleaned with obtaining almost homogeneous preparations that can be used in other analyses and for other purposes. Cleaning the proteins can be performed by standard methods, known in this field. The following procedures are examples of suitable methods for cleaning up: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, HPLC on reversed phase chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, precipitation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.

In one aspect immunoaffinity purification of full-size antibodies according to the invention using protein A immobilized on a solid phase. Protein A is A protein of the cell wall of Staphylococcus aureas size of 41 kDa, which with high affinity binds to Fc-plot antibodies. Lindmark et al. (1983) J. Immunol. Meth. 62: 1-13. Solid phase on which immobilized A protein, preferably represents a column with the surface of glass or silicon oxide, more preferably a glass column with a defined pore size or a column of silicic acid. In some applications, to prevent nonspecific binding of impurities column cover such a reagent as glycerin.

At the first stage of purification of the product obtained by culturing cells according to the above method, applied to a solid phase with immobilized protein A, thus there is specific binding of the decree of the nogo antibodies with protein A. Then the solid phase is washed to remove nonspecific related impurities. Finally, the specified antibody extracted from the solid phase by elution.

Obtaining antibodies using eukaryotic host cells

Vector components generally include, without limitation, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and the sequence termination of transcription.

(i) a Signal sequence

The vectors used in eukaryotic cells-the owners may also contain an element that encodes a signal sequence or other polypeptide containing a specific cleavage site at the N-end described the Mature protein or polypeptide. Preferably chosen such heterologous signal sequence that can be recognized by the host-cell and subjected to processing (i.e. to split the signal peptidases) in a cage. In the case of expression in mammalian cells can be used commercially available signal sequences of mammalian and viral secretory leader sequence, such as the signal sequence of the gD of herpes simplex virus.

DNA encoding such ucast the predecessor, are ligated in the same reading frame with a DNA that encodes the antibody.

(ii) the origin of replication

As a rule, the origin of replication is not required for expression vectors mammals. For example, the origin of replication of SV40 often used only because it contains the early promoter.

(iii) a Gene which facilitates the selection

The expression vectors and cloning may contain a gene selection, also called marker selection. Typically, genes breeding encode proteins that (a) give resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, if necessary, or (c) supply critical nutrients that are missing in a complex environment.

In one method of selection use the tool, which stops the growth of the host cell. Cells transformed with a heterologous gene produce a protein which imparts stability to the specified tool, and, therefore, survive in the conditions of selection. In such dominant selection as specified tools are used, for example, neomycin, mycophenolate acid and hygromycin.

Other examples of suitable selection markers are compounds that facilitate the identification of cells containing the nucleic acid encoding the antibody, such as DHFR, Tim is dikinase, metallothionein-I and-II, preferably genes metallothionein primates, adelaideans, interdiscursivity and other

For example, cells transformed with the gene for DHFR selection, identified initially by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR as a suitable host cell using the cell line of Chinese hamster ovary (CHO) with a deficit of DHFR activity (e.g., ATCC CRL-9096).

Alternatively, cell owners (especially wild-type hosts that contain endogenous DHFR) transformed or simultaneously transform sequences encoding antibodies, protein DHFR wild-type and other markers of breeding wild type, such as an aminoglycoside-3'-phosphotransferase (APH)can be selected in the process of cell growth in medium containing the agent of selection, such as aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. Cm. U.S. patent No. 4965199.

(iv) a Promoter

The expression vectors and cloning usually contain a promoter that is recognized by the body-master and functionally linked to a nucleic acid that encodes a polypeptide antibodies. Promoter sequences of eukaryotes known. Almost all the genes of eukaryotes contain AT-rich area, the location is hydrated approximately 25-30 bases above the site of transcription initiation. Many genes 70-80 bases above the start of transcription found another sequence, plot CNCAAT, where N can mean any nucleotide. At the 3'end of most eukaryotic genes is the sequence AATAAA, which can serve as a signal adding tail poly-A 3'-end of the coding sequence. All these sequences are appropriately inserted into eukaryotic expression vectors.

Transcription of the polypeptide antibodies by a vector matrix in the cells of the host mammal is regulated, for example, by promoters obtained from the genomes of these viruses as polyoma, avian pox, adenovirus (such as adenovirus 2), human papilloma virus of cattle, sarcoma virus of birds, cytomegalovirus, a retrovirus, hepatitis B virus and simian vacuolating virus 40 (SV40), from heterologous mammalian promoters, e.g. the actin promoter or promoter of the immunoglobulin from the promoters of heat shock proteins, provided such promoters are compatible with the systems of host cells.

Early and late promoters of SV40 virus are usually obtained in the form of fragments of SV40 restriction, which also contain the origin of replication of SV40 virus. The immediate early promoter of human cytomegalovirus are usually obtained in the form of a restriction fragment HindIII E. Sist the mA in the expression of DNA-hosts-mammals using as a vector of human papilloma virus in cattle are described in U.S. patent No. 4419446. Modification of this system is described in U.S. patent No. 4601978. Expression of cDNA for human β-interferon in mouse cells under the control of the promoter timedancing of herpes simplex virus described in Reyes et al., Nature 297: 598-601 (1982). Alternatively, as a promoter, you can use the long terminal repeat of rous sarcoma virus.

(v) Enhancer element

Transcription of DNA encoding the polypeptide antibodies according to the invention, in higher eukaryotes is often increased by the insertion in the vector enhancer sequence. Currently, there are many enhancer sequences from mammalian genes (globin genes, elastase, albumin, α-fetoprotein, and insulin). However, the most common use of enhancers from viruses of eukaryotic cells. Examples include the SV40 enhancer on the late side of the origin of replication (BP 100-270), the enhancer early promoter of cytomegalovirus enhancer of virus polyoma on the late side of the origin of replication and the enhancers of adenoviruses. The enhancers that enhance the activation of eukaryotic promoters are also described in Yaniv, Nature 297: 17-18 (1982). In the vector enhancer can be linked to a sequence that encodes a polypeptide antibody to 5'- or 3'-position, but preferably it is placed with the 5'side of the promoter.

(vi) the Site of transcription termination

Also what about the, the expression vectors used in eukaryotic cells-the owners, usually contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are usually found in the 5'-sometimes in the 3', untranslated parts of eukaryotic or viral DNA or cDNA. These sites contain nucleotide segments transcribed as fragments polyadenylation of the untranslated region of the mRNA that encodes the antibody. As the site of termination of transcription can be used, for example, the site of polyadenylation bovine growth hormone. Cm. WO94/11026 and described expression vector.

(vii) Selection and transformation of host cells

Cell-hosts suitable for cloning or expression of the DNA in the vectors described in this document include described here, cells of higher eukaryotes, including cell-vertebrate hosts. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of cell lines mammals used as hosts, are the cell line of monkey kidney CV1 transformed by SV40 (COS-7, ATCC CRL 1651); cell line of human embryo kidney (293 or 293 cells subcloned in suspension culture, Graham et at., J. Gen Virol. 36:59 (1977)); the kidney cells baby hamster (BHK, ATCC CCL 10); cells aicn the ka Chinese hamster ADHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); the kidney cells of monkeys (CV1 ATCC CCL 70); the kidney cells of the African green monkey (VERO-76, ATCC CRL-1587); carcinoma cells human cervical (HELA, ATCC CCL 2); cells of the dog kidney (MDCK, ATCC CCL 34); liver cells of cattle, rats (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); the cells of the human liver (Hep G2, HB 8065); tumor cells of mouse mammary cancer (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY. Acad. Sci 383:44-68 (1982)); the cells, MRC 5; FS4 cells and cell line human hepatoma (Hep G2).

Cell owners transform the above expression vectors or cloning, ensuring production of antibodies, and is cultivated in a suitable nutrient medium, which change so as to ensure the induction of promoters, selection of transformants, or amplifying the genes encoding the target sequence.

(viii) Culturing host cells

Cell owners used to obtain the antibodies according to the invention, can be grown in different environments. For culturing the host cells can be used commercially available medium such as Ham''s F10 (Sigma-Aldrich, St. Louis, MO, USA), minimum maintenance medium ((MEM), (Sigma), RPMI-1640 (Sigma) and modified by way of Dulbecco Wednesday Needle ((DMEM), Sigma). In addition, as a nutrient medium for cell-hose which you can use environment, described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem 102:255 (1980), U.S. patents№№4767704; 4657866; 4927762; 4560655; or 5122469; WO 90/03430; WO 87/00195; or in U.S. patent Re. 30985. In any of these environments, you can optionally add hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as a drug GENTAMYCIN™), trace elements (inorganic compounds, the final concentrations are usually found in the micromolar range), and glucose or an equivalent energy source. In the composition of the medium may include any other necessary additives in appropriate concentrations, known to specialists in this field. The cultivation is carried out in conditions (temperature, pH and the like), previously used to host cells selected for expression, and known to ordinary specialist in the field.

(ix) Purification of antibodies

The antibody produced using recombinant methods, can be found inside cells or immediately secretariats on Wednesday. If the antibody is inside the cell, in the first stage removes debris cells, or cells of the owners, or fragments resulting from lysis, for example, by centrifugation or ultra is istratii. If the antibody is secreted into the medium, supernatant such expression systems generally first concentrated using a commercially available filter for concentrating proteins, such as filter for ultrafiltration Amicon or Millipore Pellicon. At all previous stages can be used protease inhibitors such as PMSF to inhibit proteolysis and antibiotics to prevent the growth of contaminating organisms.

Obtained from cells drug antibodies can be cleaned, for example, methods hydroxiapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and the preferred method of cleaning is affinity chromatography. The possibility of using protein A as an affinity ligand depends on the species and isotype Fc domain of immunoglobulin present in the antibody. Protein A can be used for purification of antibodies on the basis of human heavy chain γ1, γ2, or γ4 (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). In the case of mouse isotypes and for human γ3 chain, it is recommended to use protein G (Guss et at., EMBO J. 5:15671575 (1986)). Most often, the affinity ligand is attached to the agarose, but you can use other carriers. Mechanically stable media such as glass with a certain pore size or poly(Stradivari)benzene, provide higher flow rates and shorter time check the tion process, than agarose. If the antibody contains a CH3 domain, for cleaning, you can use the resin Bakerbond ABX™ (J. T. Baker, Phillipsburg, NJ). Depending on recoverable antibodies can use other methods of protein purification such as fractionation on ionoobmennoi column, ethanol precipitation, HPLC on reversed phase chromatography on silica, chromatography on SEPHAROSE™containing heparin chromatography on anyone - or cation-exchange resin (such as a column with poliasparaginovaya acid), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate.

After the preliminary stage (stages) purification of a mixture containing the target antibody and impurities, can be subjected to hydrophobic chromatography at low pH using a buffer for elution with pH in the range of from about 2.5 to 4.5 and preferably low salt concentrations (e.g., salt concentration may range from 0 to 0,25M).

Analyses of activity

Physical/chemical properties and biological function of the antibody of the present invention can be analyzed by various methods known in this field.

After cleaning, the immunoglobulins can be characterized using a number of assays, including, without limitation, N-terminal sequencing, amino acid analysis, sedentarius helpanimals you ecoeffectiveness liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain cleavage.

In some embodiments according to the invention analyze the biological activity of antibodies, obtained as described in this document method. In some embodiments analyze antigennegative activity of the immunoglobulins of the present invention. Applicable for the present invention and known in the field of methods of analysis of the binding of the antigen include, without limitation, all direct or competitive binding assays using techniques such as Western blotting, radioimmunoassay analysis, ELISA (ELISA analysis), immunological sandwich-analysis, immunoprecipitation, fluorescent immunological assays and immunological assays using protein A. the Example of analysis of binding antigen below in the "Examples"section.

In one embodiment of the present invention provides an altered antibody, with some but not all effector functions, which makes it a desirable candidate for many applications in which it is set to the half-life of antibodies in vivo, and some effector functions (such as the binding of complement and ADCC) are unnecessary or harmful. In some embodiments measure the Fc-activity received immuno is lobeline, to ensure that these are desirable properties. Decrease/decrease in the activity of CDC and/or ADCC can be determined using assays of cytotoxicity in vitro and/or in vivo. For example, using the assays of the binding of the Fc receptor (FcR) can confirm that the antibody has lost the ability to bind FcγR (and, therefore, in all probability, have lost the ADCC activity), but retained the ability to bind FcRn. Basic cells mediating ADCC, NK cells, Express FcγRIII only, whereas monocytes Express FcγRI, FcγRII and FcγRIII. The FcR expression on hematopoietic cells is shown in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). The example in vitro analysis of ADCC activity of the target molecule described in U.S. patent No. 5500362 or 5821337. Used in such analyses effector cells include mononuclear cells of peripheral blood (PBMC) and natural killer cells (NK). Alternative or additionally, ADCC activity of the target molecule can be determined in vivo, for example, in animal models, such as described in Clynes et al. PNAS (USA) 95:652-656 (1998). By analyzing the binding of C1q can also confirm that the antibody is unable to bind C1q and hence lost the CDC activity. Activation of complement can be estimated, for example, by analyzing the CDC, for example, by the method described in Gazzano-three-bet et al., J. Immunol. Methods 202:163 (1996). Analysis swazilan what I FcRn and determine values clearance/half-life in vivo can be performed using known in the field of methods, for example, as described in the "Examples"section.

Humanized antibodies

The present invention encompasses humanized antibodies. In this area known different ways of humanizing the inhuman antibodies. For example, in humanitariannet antibody can enter one or more amino acid residues of the non-human sequence. Data inhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization mainly carried out according to the method of winter and co-authors (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by replacing sequences of the hypervariable portion at the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. patent No. 4816567), in which the fragment is substantially less than an intact human variable domain has been replaced by the corresponding sequence differs from the human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable residues of the plot and possibly some FR residues are replaced by residues from analogous sites of the antibodies of the rodent.

The choice of a human being is variable domains as easy, and the heavy chain used to obtain gumanitarnogo antibodies is very important to reduce antigenicity. Using the so-called "best match", the sequence of the variable domain of the antibody rodent subjected to screening against a library of known sequences of human variable domain. Then the human sequence that best matches the rodent, used as human frame section gumanitarnogo antibody (Sims et al. (1993) J. Immunol 151:2296; Chothia et al. (1987) J. Mol Biol. 196:901. In another method uses a particular frame plot derived from a consensus sequence of all human antibodies containing light or heavy chain specific subgroups. The same frame section can be used to obtain different humanized antibodies (Carter et al (1992) Proc. Natl. Acad. Sci USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623.

It is also important that humanitariannet antibody retained high affinity for the antigen and other desirable biological properties. To achieve this objective, in accordance with one method, humanized antibodies are obtained by analysis of the original sequences and various conceptual humanized products using three-dimensional models of iskhodnoye humanized sequences. Three-dimensional models of immunoglobulins are widely available and well-known experts in this field. Available computer programs which illustrate and display probable three-dimensional conformation of a particular candidate sequences of immunoglobulins. Image data allow an analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., to identify residues that influence the ability of the candidate immunoglobulin to bind antigen. Using this method, you can select and combine the remains of FR from the recipient and import sequences that provide antibodies with desired characteristics, such as increased affinity for the target antigen (antigens target). Typically, the remains of a hypervariable site directly and strongly affect the binding to the antigen.

Variants of antibodies

In one aspect, the invention provides a fragment of an antibody containing modifications in the edge region of the Fc polypeptides, including the site of the Fc, where modifications to facilitate and/or provide heterodimerization. These modifications include the creation of a protrusion on the first Fc polypeptide and the cavity of the second Fc polypeptide, and the protrusion can be positioned in the cavity so that when iniziare the camping complex formation of the first and second Fc polypeptides. Methods of obtaining antibodies with these modifications known in this field, for example, their description can be found in U.S. patent No. 5731168.

In some embodiments discusses the modification of the amino acid sequence described in this document antibodies. For example, sometimes it is desirable to increase the affinity of binding and/or improve other biological properties of the antibody. Variants of the amino acid sequence of the antibody produced by introducing appropriate nucleotide changes into a nucleic acid encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequences of the antibodies. For the final design, you can use any combination of deletions, insertions and substitutions, provided that the final construct possesses the desired characteristics. Amino acid changes can be introduced in the amino acid sequence of the antibody at the time of receiving the order.

The method of identification is preferred for mutagenesis of residues or regions of the antibody referred to as "scanning mutagenesis to alanine", its description can be found in Cunningham and Wells (1989) Science, 244:1081-1085. In this way identify a residue or group of target residues (for example, charged OST the key, such as arg, asp, his, lys, and glu) and replaced them by a neutral or negatively charged amino acid (most preferably alanine or polyalanine), which affects the interaction of amino acids with antigen. Then amino acid position having a functional sensitivity to the substitutions will improve by introducing additional or different options in these areas. Thus, while the site for introducing the variations of amino acid sequences previously identified, the nature of the mutation per se need not be defined in advance. For example, for the analysis of mutations at the specified position spend ala scanning or random mutagenesis at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

Insertions of amino acid sequences include amino - and/or carboxykinase insertion of one residue, or a polypeptide with a length of several hundred or more residues, as well as insertion of one or more amino acid residues within the sequence. Examples of terminal insertions include an antibody with an N-terminal methioninol residue or the antibody conjugated with the cytotoxic polypeptide. Other insertional variants of the antibody molecules include hybrid antibodies to the N - or C-Termini with the enzyme (e.g. the R, ADEPT) or a polypeptide which increases the half-life of antibodies in the serum.

Another type of variant is the variant with amino acid substitution. This option includes the antibody molecule in which one amino acid residue is replaced by another. Lots of interest from the point of view of the substitutional mutagenesis include the hypervariable sites, but also discusses the changes in FR. Conservative substitutions are shown in table 2 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then you can enter more significant substitutions listed in table 2 under the heading "standard replacement" or described in more detail below with reference to classes of amino acids, with subsequent screening of antibodies.

Table 2
The original balanceGeneric replacementPreferred replacement
Ala(A)Val; Leu; IleVal
Arg(R)Lys; Gln; AsnLys
Asn(N)Gln; His; Ap; Lys; ArgGln
Asp(D)Glu; AsnGlu
Cys(C)Ser; AlaSer
Gln(Q)Asn; GluAsn
Glu(E)Asp; GlnAsp
Gly(G)AlaAla
His(H)Asn; Gln; Lys; ArgArg
Ile(I)Leu; Val; Met; Ala;
Phe; Norleucine
Leu
Leu(L)Norleucine; Ile; Val;
Met; Ala; Phe
Ile
Lys(K)Arg; Gln; AsnArg
Met(M)Leu; Phe; IleLeu
Phe(F)Trp; Leu; Val; Ile; Ala; TyrTyr
Pro(P)AlaAla
Ser(S)ThrThr
Thr(T)Val; SerSer
Trp(W)Tyr; PheTyr
Tyr(Y)Trp; Phe; Thr; SerPhe
Val(V)Ile; Leu; Met; Phe; Ala; NorleucineLeu

Significant changes in the biological properties of the antibodies reach by selecting substitutions that have a significant impact on (a) the structure of the polypeptide skeleton in the replacement of, for example, have folded or helical conformation, (b) the charge or hydrophobicity of the molecule at a given site, or (c) the volume of the side chain. Depending on the properties of the side chains of amino acids can be divided into the following classes (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A)Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);

(2) uncharged polar: Gly (G), Ser (S)Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

(3) acidic: Asp (D)Glu (E);

(4) basic: Lys (K), Arg (R), His(H).

Alternatively, depending on the General properties of the side chains of natural residues can be divided into the following classes:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) atelinae hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions include the substitution of the residue of one of these classes for the remainder of the other class. Such parts can also be carried out in the areas of conservative substitutions or, more preferably, in the remaining (non-conservative) sites.

One type of substitution variant involves substituting one or more residues of the hypervariable area of the original antibody (for example, gumanitarnogo or human antibody). Typically, the received option(s)selected for further improvement, has improved biological properties compared to the original antibody, from which he received. The traditional way of obtaining such substitution variants include affinity maturation using phage display. In short, several provisions of the hypervariable segment (for example, 6-7 provisions) is subjected to mutagenesis with obtaining all possible amino acid substitutions at each position. The resulting antibodies are exposed from filamentous phage particles as hybrids with the gene III product M13 contained in each particle. Then use the methods described here to analyze the biological will stifle activity (for example, the affinity of binding of the options presented in phage display. To identify the position of the hypervariable area, which will be modified, using alanine-scanning mutagenesis identify the remains of the hypervariable area, with a significant impact on binding to the antigen. Alternative or additionally, the contact point of the antibody with the antigen can be identified by analyzing the crystal structure of the complex of antigen-antibody. Parts of such contact points, as well as adjacent areas are candidates for replacement by using the methods described in this document. The collection of obtained variants is subjected to screening as described herein, the method and antibodies, which are the results of one or more appropriate tests have better properties chosen for further development.

Molecules of nucleic acids encoding amino acid sequence variants of antibodies, receive a variety of ways known in this field. These methods include, without limitation, the selection is from a natural source (in the case of a natural amino acid sequence variants) or the receipt by oligonucleotide-mediated (or site-specific) mutagenesis, PCR mutagenesis, and cassette m is Magenta obtained earlier variant or variant version of the antibody.

Variant plot Fc can also be obtained by modifying one or more amino acids in the plot Fc. Variant plot Fc may contain a sequence of the human plot Fc (for example, section Fc of human IgG1, IgG2, IgG3, or IgG4)that is modified (for example, through the introduction of replace) one or more amino acid positions, including the position of the hinge cysteine.

In accordance with this description and principles adopted in this area, it is believed that in some embodiments the antibody used in the methods according to the invention may contain one or more changes in comparison with the analogous wild-type, for example, in the plot of Fc. However, these antibodies retain almost all of the properties required for therapeutic applications present their counterpart wild-type. For example, it is assumed that the plot of Fc can be made that lead to change (i.e. increase or decrease) of the C1q binding and/or complementability cytotoxicity (CDC), for example as described in WO 99/51642. Other examples of plot Fc described in Duncan & Winter Nature 322:738-40 (1988); U.S. patent No. 5648260; U.S. patent No. 5624821 and WO 94/29351.

Immunoconjugate

This invention also relates to immunoconjugates or conjugates of the antibody-drug (ADC), which pre whom are conjugate antibody with a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments), or a radioactive isotope (i.e radioactive conjugate).

The use of conjugates of the antibody-drug for local delivery of cytotoxic or cytostatic funds, i.e. funds that destroy tumor cells or inhibit the growth of these cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. patent No. 4975278) theoretically allows the directed delivery of medicinal fragment to tumors and accumulate inside cells, in cases where systemic introduction of such drug in unconjugated form can have an unacceptable toxic effects on normal cells along with cancer cells to be destroyed (Baldwin et al., (1986) Lancet pp.(Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibody '84: Biological And Clinical Applications, A. Pinchera et al. (ed.s), pp.475-506). Thus the maximum efficiency with minimal toxicity. In these methods are used both polyclonal and monoclonal antibodies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Used to indicated the data how drugs include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986) supra). Toxins used in the conjugates of the antibody-toxin include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins, such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoid (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623) and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Toxins can exert cytotoxic and cytostatic effect through mechanisms including tubulin binding, DNA binding or inhibition of topoisomerase. Some cytotoxic funds may lose activity or become less active after conjugation with major antibodies or protein ligands of receptors.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is a conjugate of the antibody-radioactive isotope, consisting of murine monoclonal antibodies IgG1 Kappa directed against the CD20 antigen found on the surface of normal and malignant B-lymphocytes, and radioactive isotope111In or90Y connected via a chelate forming linker of the thiourea (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Cli. Oncol. 20(15):3262-69). Although the activity of ZEVALIN directed against B-cell non-Hodgkin lymphoma (NHL), its introduction in most patients causes severe and prolonged cytopenia. MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), conjugate antibody-drug consisting of antibodies huCD33 connected with calicheamicin, approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future(2000) 25(7):686; U.S. patents№№4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001). Cantuzumab mertansine (Immunogen, Inc.), conjugate antibody-drug consisting of huC242 antibody linked via the disulfide linker SPP to the fragment maytansinoids medicines, DM1, has entered phase II trials of drugs against cancerous diseases associated with expression of CanAg, such as colon cancer, pancreas, stomach and other organs. MLN-2704 (Millennium Pharm., BZL Biologies, Immunogen Inc.), conjugate antibody-drug consisting of monoclonal antibodies against specific membrane antigen prostate (PSMA), connected with a fragment of maytansinoids medicines, DM1, is being developed as a potential tool for the treatment of prostate tumors. Auristatin peptides, auristatin E (AE), monomethylaniline E (MMAE), synthetic analogs of dolastatin conjugated with hemery and monoclonal antibodies cBR96 (specific to Lewis Y antigen, present in carcinomas) and cAC10 (specific to CD30 antigen, present in malignant blood diseases) (Doronina et al. (2003) Nature Biotechnology 21(7):778-784; and Francisco, et al. (2003) Blood 102, 1458-1465)are under therapeutic development. Other compounds used as conjugated cytotoxic funds include, without limitation, auristatin E (AE), MMAF (auristatin E (MMAE), containing a phenylalanine at the C-end of the molecule) and AEVB (auristatin E valerianaceae, acid-sensitive linker at the C-end AE). The linkers used in the conjugates to connect medicines with the antibody include, without limitation, MC (maleimidomethyl), Val Cit (valine-citrulline, dipeptide site cleaved by the protease linker), citrulline (2-amino-5-breedopedia acid), PAB (t-aminobenzimidazole, "self-destructing" the fragment linker), Me (N-methyl-valine-citrulline, where the linker peptide bond has been modified to prevent its cleavage by cathepsin B), MC(PEG)6-OH (maleimidomethyl-glycol, attached to cysteine residues of the antibody), SPP (N-Succinimidyl 4-(2-pyridylthio)pentanoate) and SMCC (N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). These and other suitable to the use of conjugates of drugs and methods of their obtaining a revelation is s, for example, in Doronina, S.O. et al., Nature Biotechnology 21:778-794 (2003), included in this description by reference in its entirety. Particularly preferred linker molecules include, for example, N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (see, for example, Carlsson et al., Biochem. J., 173, 723-737 (1978)), N-Succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, for example, U.S. patent No. 4563304), N-Succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (see, for example, CAS registration number 341498-08-6), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (see, for example, Yoshitake et al., Eur. J. Biochem., 101, 395-399 (1979)) and N-Succinimidyl 4-methyl-4-[2-(5-nitropyridine)dithio]pentanoate (SNMP) (see, for example, U.S. patent No. 4563304).

Chemotherapeutic agents used for such immunoconjugates described above. Suitable for use with enzymatic active toxins and fragments thereof include A-chain of diphtheria toxin, neisvaziuosiu active fragments of diphtheria toxin A-chain, exotoxin a (from Pseudomonas aeruginosa), A-chain of ricin, A-circuit abrina And-chain medecine, alpha sarcin, proteins Aleurites fordii proteins of diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor MOMORDICA CHARANTIA, Curtin, krotin, inhibitor sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. To obtain conjugates of antibodies containing radioactive isotopes, you can use a number of the of radionuclides. Examples of such radionuclides include212Bi131I131In90Y and186Re. Conjugates of the antibody and cytotoxic receive funds using a variety of bifunctional reagents for crosslinking proteins such as N-Succinimidyl-3-(2-[pyridyldithio]propionate (SPDP)), aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCl), activated esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido-compounds (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained according to the method described in Vitetta et al, Science, 238: 1098 (1987). Labelled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is an example of a chelating means used to obtain the conjugates of the radionuclide to the antibody. Cm. WO 94/11026.

This paper also discusses the conjugates of antibodies with one or more small molecule toxins, such as calicheamicin, maytansinoid, trichothecin and CC1065, and with proizvodnymi these toxins have a toxic actinistia.

M is stanzin and maytansinoid

In one embodiment the antibody (full-length or fragments) according to the invention kongugiruut with one or more maytansinoid molecules.

Maytansinoid are inhibitors of mitosis, which act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. patent No. 3896111). Then it was discovered that certain microbes also produce maytansinoid, such as maytansine and C-3 esters maytansine (U.S. patent No. 4151042). Synthetic maytansines and its derivatives and analogues are disclosed, for example, in U.S. patents№№4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663 and 4371533, the description of which is specifically incorporated herein by reference.

Conjugates maytansinoid-antibody

To improve therapeutic performance of maytansine and maytansinoids, kongugiruut with antibodies that specifically bind with the antigens of tumor cells. Immunoconjugate containing maytansinoid, and their therapeutic use are disclosed, for example, in U.S. patent No. 5208020, 5416064 and in the European patent EP 0425235 B1, the description of which is specifically incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) describe immunoconjugate, with whom containing a series of maytansinoid DM1, coupled with a monoclonal antibody C242 directed against cancer of the colon and rectum of humans. This conjugate is highly toxic to the cell culture of cancer of the colon and rectum of humans and demonstrates antitumor activity in the analysis of tumor growth in vivo. Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which maytansinoid kongugiruut via a disulfide linker to the murine antibody A7 binding to an antigen cell line of cancer of the colon and rectum of a person, or to another murine monoclonal antibody TA.1 that binds to the oncogene HER-2/neu. The cytotoxicity of the conjugate TA.1-maytansinoid analyze the in vitro cell line breast cancer human SK-BR-3, which Express 3×105surface antigens HER-2 on the cell. The level of cytotoxicity of the conjugate containing the drug reaches the level of cytotoxicity of free maytansinoid funds, and this level can be increased by increasing the number of maytansinoid molecules per molecule of antibody. Conjugate A7-maytansinoid has low systemic cytotoxicity in mice.

Conjugates of the antibody-maytansinoid (immunoconjugate)

Conjugates of the antibody-maytansinoid obtained by chemical attach antibodies to the molecule maytansinoid the ez significant reduction of the biological activity of the antibodies or molecules maytansinoid. It is shown that the conjugate containing an average of 3-4 molecules maytansinoid molecule antibodies, effectively increases the cytotoxicity of the target cells and not have a negative impact on the function and solubility of antibodies, although presumably even one molecule of the toxin to the antibody enhances cytotoxicity naked antibodies. Maytansinoid well known in this field, they can be synthesized using well known methods or to isolate from natural sources. Suitable maytansinoid disclosed, for example, in U.S. patent No. 5208020 and in other patents and non-patent publications cited above. Preferred maytansinoids are maytansines and analogues maytansine modified aromatic cycle or other provisions of molecules maytansine, such as various esters maytansine.

In this area there are many linker groups used to obtain conjugates antibody-maytansinoid, which includes, for example, disclosed in U.S. patent No. 5208020 or in patent EP 0425235 B1 and Chari et al., Cancer Research 52: 127-131 (1992). Linker groups include disulfide groups, thioester groups, kislotolabilen group, photolabile group, sensitive to peptidase group or sensitive esterase group, as described in the above patents, and preference is sustained fashion disulfide and thioester groups.

Conjugates of the antibody and maytansinoid can be obtained using a variety of bifunctional reagents for cross-linking proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCl), activated esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-azido-compounds (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis-page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). Especially preferred condensing agents include N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 [1978]) and N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), ensuring the formation of disulfide bonds.

The linker can be attached to the molecule maytansinoid at different positions, depending on the type of communication. For example, the ester bond can be obtained by reacting with a hydroxyl group using traditional methods of condensation. This reaction can be carried out according to the position C-3, containing a hydroxyl group at position C-14, modified hydroximate the Ohm, position C-15, modified hydroxyl group, and at position C-20) containing a hydroxyl group. In the preferred embodiment, the bond formation occurs at position C-3 maytansine or similar maytansine.

Calicheamicin

Other interest immunoconjugate contain antibody conjugated with one or more molecules calicheamicin. Antibiotics calicheamicin family can cause rupture of double-stranded DNA at concentrations close to picomolar. Obtaining conjugates calicheamicin family described in U.S. patents 5712374, 5714586, 5739116, 5767285, 5770701, 5770710, 5773001, 5877296 (they are all owned by American Cyanamid). Usable structural analogues calicheamicin include, without limitation, γ1Iα2Iα3IN-acetyl-γ1I, PSAG and Θ1I(Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998) and the aforementioned U.S. patents, owned by American Cyanamid). Other antitumor agent, which can be konjugierte with the antibody, is QFA, representing antifolate. And calicheamicin, and QFA act inside the cell and hardly penetrate the cytoplasmic membrane. Therefore, the penetration of these funds into the cells in the indirect antibody and is ternalization greatly enhances their cytotoxic effects.

Other cytotoxic funds

Other antitumor agents that can be konjugierte with the antibody according to the invention include BCNU, streptozocin, vincristine and 5-fluorouracil, the family of the tools, collectively known as the complex LL-E33288 and described in U.S. patent 5053394, 5770710 and espiramicina (U.S. patent 5877296).

You can also use enzymatically active toxins and fragments thereof that include A-chain of diphtheria toxin, neisvaziuosiu fragments of diphtheria toxin A-chain, exotoxin a (from Pseudomonas aeruginosa), A-chain of ricin, A-circuit abrina And-chain medecine, alpha sarcin, proteins Aleurites fordii proteins of diantin, proteins, Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor MOMORDICA CHARANTIA, Curtin, krotin, inhibitor sapaonaria officinalis, gelonin, mitogillin, restrictocin, vanomycin, inomycin and tricothecene. See, for example, WO 93/21232, published October 28, 1993

The present invention also includes immunoconjugate formed by the antibody and the connection with nucleotidase activity (e.g., a ribonuclease or a DNA endonuclease such as desoksiribonukleaza; DNA-ASE).

Selective tumor destruction can be achieved using antibodies containing highly radioactive atom. To obtain radioactive conjugates antibodies can be used a number of radioactive isotopes. Examples of what aka isotopes include At 211I131I125, Y90That Re186That Re188Sm153Bi212, P32, Pb212and radioactive isotopes of Lu. If the conjugate is used for detection, it may contain radioactive atoms used for scintigraphic studies, for example tc99mor I123or spin the label used for imaging method for nuclear magnetic resonance (NMR) (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Radioactive or other label you can enter in the composition of the conjugate using known methods. For example, the peptide can be obtained by biosynthesis or it can be synthesized by chemical amino acid synthesis using suitable amino acid precursors, for example, fluorine-19 instead of hydrogen. Labels such as tc99mor I123That Re186That Re188and In111can be attached to the peptide via a cysteine residue. Yttrium-90 can be attached via a lysine residue. Iodine-123 can be entered using the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Other methods are described in detail in "Monoclonal Antibody in Immunoscintigraphy" (Chatal, CRC Press 1989).

Conjugates of the antibody with a cytotoxic agent can be obtained by a series of bifunctional reage the tov for fusion proteins, such as N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, aminothiols (IT), bifunctional derivatives of imidapril (such as dimethylacetamide HCl), activated esters (such as disuccinimidyl), aldehydes (such as glutaraldehyde), bis-azido-compounds (such as bis(p-azidobenzoyl)hexanediamine), derivatives of bis -, page (such as bis-(p-disoriented)Ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-debtor-2,4-dinitrobenzene). For example, rezinovy immunotoxin can be obtained according to the method described in Vitetta et al, Science, 238: 1098 (1987). Labelled with carbon-14 1-isothiocyanatobenzene-3-metallienjalostuksessa acid (MX-DTPA) is an example of a chelating means used to obtain the conjugates of the radionuclide to the antibody. Cm. WO94/11026. The linker can be a "biodegradable linker"facilitating release of cytotoxic funds in the cell. For example, you can use acid-sensitive linker sensitive peptidase linker, photolabile linker, dimethyl linker or dyslipidaemias linker (Chari et al., Cancer Research 52: 127-131 (1992); U.S. patent No. 5208020).

Compounds according to the invention particularly include, without limitation, the ADC obtained is by using cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, fairs are forthcoming-Siab, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-fairs are forthcoming-Siab, sulfo-SMCC, sulfo-SMPB, and SVSB (Succinimidyl-(4-vinylsulfonic)benzoate)which are commercially available (they can be obtained, for example, from Pierce Biotechnology, Inc., Rockford, IL., U.S.A). Cm. pages 467-498, 2003-2004 Applications Handbook and Catalog.

Obtaining conjugates antibody-drug

In the conjugates of the antibody-drug (ADC) according to the invention, the antibody (Ab) is connected to one or more fragments of a drug (D), for example from about 1-20 fragments drug molecule antibody, through a linker (L). The ADC of formula I can be obtained in different ways using the reactions of organic chemistry, conditions, and reagents known to specialists in this field, which include (1) the interaction of a nucleophilic group of an antibody with a bivalent linker reagent with obtaining Ab-L, via the formation of covalent bonds with subsequent interaction with a fragment of drug D and (2) the interaction between the nucleophilic group of a fragment of a medicinal product with a bivalent linker reagent with obtaining D-L through the formation of covalent bonds with subsequent interaction with the nucleophilic group of an antibody.

Ab-(L-D)p
I

Nucleophilic groups on the antibody include, without limitation, (i) N-terminal amino group, (ii) amino side chains, for example, lysine, (iii) tirinya groups of the side chains, for example, cysteine, and (iv) a hydroxyl group or amino sugars, if the antibody is glycosylated. Amino group, tirinya groups and hydroxyl groups are nucleophilic and can interact with the formation of covalent bonds with electrophilic groups on linker fragments and linker reagents including : (i) activated esters such as NHS esters, esters, HOBt, halogenfree and acid halides; (ii) alkyl and benzyl halides such as halogenated; (iii) aldehydes, ketones, carboxylic groups and maleimide group. Some contain antibodies capable of restoring disulfide cross-links, i.e. cysteine bridges. Antibodies capable of interacting with linker reagents, can be obtained by processing the regenerating agent such as DTT (dithiothreitol). In this case, each cysteine bridge theoretically forms two reactive tylnej of the nucleophile. Other nucleophilic groups can be introduced in antibodies through interaction of the lysine residues with 2-aminothiophenol (reagent Troth) with PR the rotation of the amino group in Tilney.

Conjugates of the antibody-drug according to the invention can also be obtained by introducing into the molecule antibodies electrophilic fragments that can interact with nucleophilic substituents on the linker reagent or drug. Sugar glycosylated antibodies can oxidize, for example, controllable periodic destruction oxidizing reagents with obtaining aldehyde or ketone groups which can interact with the amino group of the linker reagents or fragments medicines. Received aminogroup Chippewa base can form a stable connection or they can be recovered, for example, barginregister.com the reagents to form stable amine linkages. In one embodiment of the interaction of carbohydrate fragment of a glycosylated antibody with galactosialidosis or metaperiodate sodium can lead to the formation of carbonyl (aldehyde and ketone) groups in the protein molecule that can interact with appropriate groups on the drug (Hermanson, Bioconiugate Techniques). In another embodiment of proteins containing N-terminal residues of serine or threonine can interact with metaperiodate sodium with the formation of aldehyde instead of the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). This aldehyde can in order to imagestate with a nucleophile fragment of a drug or linker.

Similarly, the nucleophilic group on medicinal fragment include, without limitation, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazinecarboxamide and arylhydrazines group capable of interacting with the formation of covalent bonds with electrophilic groups on linker fragments and linker reagents including : (i) activated esters such as NHS esters, esters, HOBt, halogenfree and acid halides; (ii) alkyl and benzylchloride, such as halogenated; (iii) aldehydes, ketones, carboxylic groups and maleimide group.

Alternatively, a hybrid protein containing the antibody and cytotoxic agent, can be obtained, for example, recombinant methods, or by peptide synthesis. The DNA molecule may contain relevant areas encoding the two parts of the conjugate, which are adjacent to each other or separated by plot, encoding the linker peptide which does not have an adverse effect on the desired properties of the conjugate.

In another embodiment, the antibody can be konjugierte with the "receptor" (such as streptavidin) for pre-marking of the tumor, where the conjugate of the antibody-receptor is administered to the patient, then the unbound conjugate is removed from the bloodstream by using outputs of the reagent, after chowdah "ligand" (e.g., avidin)that is conjugated with a cytotoxic agent (e.g., radioactive nucleotide).

Derived antibodies

Antibodies of the present invention also can be modified by introducing an additional non-protein fragments, known in this area and easily accessible. Preferably, the fragments suitable for derivatization of the antibody, are water-soluble polymers. Non-limiting examples of water soluble polymers include, without limitation, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, a copolymer of ethylene/maleic anhydride, polyaminoamide (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, homopolymers of propylene, copolymers of propylene oxide/ethylene oxide, polyoxyethylene polyols (e.g. glycerol), polyvinyl alcohol and mixtures thereof. Propionaldehyde of polyethylene glycol may have an advantage when used in production due to the stability in the water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if you attached more than the same polymer, they may have the same or different molecules. Typically, the number and/or type of polymers used for derivatization determined depending on such factors as, among others, specific properties or functions of antibodies, as well as whether derived antibody used to treat certain conditions, and others

The pharmaceutical composition

Therapeutic compositions containing the antibody according to the invention, receive for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions, and also in the form of lyophilized or other dried compositions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the used doses and concentrations, and include buffers, for example, containing phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyltrimethylammonium chloride; hexammine chloride; benzalkonium chloride, benzene chloride; phenol, butyl or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; the cycle is hexanol; 3-pentanol and m-cresol); low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The composition described herein can also contain several active compounds, if necessary at a particular diagnosis, preferably such compounds have synergistic activity and does not adversely effect each other. Such molecules are present in the composition in amounts effective for their intended use.

The active ingredients can also be enclosed in a microcapsule obtained, for example, using the methods koatservatsii or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin microcapsule and poly(methyl methacrylate) microcapsule, sootvetstvenno is, you can also use colloidal systems drug delivery (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsion. Such methods are described in Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Composition used for administration in vivo, must be sterile. Sterilization can be performed by filtering through a membrane for sterilization by filtration.

You can get drugs with a slow release. Suitable examples of drugs with a slow release include semi-permeable matrices of solid gidrofobnykh polymers containing the immunoglobulin according to the invention, the data matrices are in the form of particles of a certain shape, e.g. films, or microcapsules. Examples of matrices that provide slow release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol)), polylactide (U.S. patent No. 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem the ethylene vinyl acetate, degradiruete copolymers of lactic and glycolic acids, such as the LUPRON DEPOT™ (suitable for introduction microspheres consisting of a copolymer of lactic and glycolic acids and leuprolide), and poly-D-(-)-3-hydroxybutiric acid. Although polymers such as ethylenevinyl the Etat and a copolymer of lactic and glycolic acids, may release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. If enclosed in a capsule antibodies remain in the body for a long time, they can denaturing or aggregate as a result of exposure to moisture at 37°C, which leads to loss of biological activity and possibly to changes in immunogenicity. To stabilize antibodies can develop a rational strategy based on the mechanism of the process. For example, if aggregation proceeds through the formation of intermolecular S-S in ridiculing interactions, stabilization can be performed by modifying sulfhydryl residues, lyophilization from acidic solutions, controlling moisture content, using the respective additives, and developing specific compositions based on polymer matrices.

Application

The antibody according to the invention can be used, for example, in the methods of treatment in vitro, ex vivo and in vivo. Antibodies according to the invention can be used as antagonists for the partial or complete blockade of specific activity of the antigen in vitro, ex vivo and/or in vivo. Moreover, at least some of the antibodies according to the invention are capable of neutralizing the activity of the antigen from other species. Antibodies according to the obreteniyu can also be used for the inhibition of the specific activity of the antigen, for example, in a cell culture containing the antigen, or in humans or other mammals (such as chimpanzee, baboon, marmoset, cynomolgus monkeys and macaque-rhesus, pig or mouse)containing the antigen with which the antibody according to the invention gives a cross-reaction. In one embodiment the antibody according to the invention can be used for inhibiting the activity of antigen by bringing into contact antibodies to the antigen so that was the inhibition activity of the antigen. Preferably the antigen is a molecule of the human protein.

In one embodiment the antibody according to the invention can be used in a method of inhibiting antigen in the subject, stradayuschego from a disease in which activity of the antigen is harmful, this method includes the introduction of the subject antibodies according to the invention for inhibiting the activity of the antigen. Preferably the antigen is a molecule of the human protein, and the subject is a human. Alternatively, the subject can be a mammal expressing an antigen associated antibody according to the invention. In addition, the subject may be a mammal containing an exogenous antigen (for example, by injection of the antigen or by expression of the transgene, codereuse the antigen). The antibody according to the invention it is possible to enter a person for therapeutic purposes. In addition, the antibody according to the invention it is possible to enter non-human mammal expressing an antigen to which the immunoglobulin gives a cross-reaction (e.g., a Primate, pig or mouse) for treatment, or to obtain an animal model of human disease. In the latter case, the animal model can be used to evaluate therapeutic efficacy of antibodies according to the invention (for example, to determine doses and period of injection drugs). Blocking antibodies according to the invention, which can be used for therapeutic purposes include, without limitation, antibodies against HER2 antibodies against VEGF, antibodies against IgE, antibodies against CD11 antibodies against interferon and antibodies against tissue factor. Antibodies according to the invention can be used for treating, inhibiting, slowing down the development of, prevent/delay recurrence, improvement or prevention of diseases, disorders or conditions associated with abnormal expression and/or activity of one or more antigen molecules, which include, without limitation, malignant and benign tumors; placemates and lymphoid malignant disease is; neural, glial, astatically disorders, diseases of the hypothalamus and other glands of internal secretion, macrophagal, epithelial, stromal and blastocladia disorders; and inflammatory, associated with angiogenesis and immune disorders.

In one aspect of the blocking antibody according to the invention is specific to the ligand-antigen and inhibits the activity of an antigen by blocking the interaction of ligand-receptor or inhibit this interaction, which involves ligand-antigen, and as a result of inhibition of the corresponding signaling pathways and other molecular or cellular events. This invention also provides a receptor-specific antibodies, which may not prevent the binding of ligand and inhibit activation of the receptor and thereby inhibit any responses that are typically initiated as a result of binding of the ligand. The invention also encompasses antibodies that bind preferentially or exclusively with complexes of the ligand-receptor. The antibody according to the invention can also act as an agonist of the receptor-specific antigen, and, as such, it can promote, strengthen and activate all functions that can be called ligand-mediated activation of the receptor, or part of these functions.

In the treatment of the antibody according to the invention can be used alone or in combination with other compositions. For example, the antibody according to the invention can be entered together with another antibody, chemotherapeutic agent (means) (including mixtures of chemotherapeutic agents), other cytotoxic agent (means), antiangiogenic agent (means), cytokines and/or growth inhibitors. If the antibody according to the invention inhibits tumor growth, it is sometimes desirable to combine it with one or more other terapeutiche the Kimi means, also any abscopal tumor growth. For example, in the treatment of the antibody according to the invention can be combined with anti-VEGF antibody (e.g., AVASTIN) and/or antibodies against ErbB (for example, an antibody against HER2 HERCEPTIN®), for example, when treating any of the diseases described in this document, including cancer of the colon and rectum, metastatic breast cancer and kidney cancer. Alternative or additionally, the patient may receive combined radiation therapy (e.g., external irradiation or treatment with the use of tools, radiolabelled, such as an antibody). Such of the above combined treatment methods include introduction of joint (where two or more funds are part of the same or different compositions) and a separate introduction, in which the antibody according to the invention is administered before and/or after administration of one or more auxiliary medicines.

The antibody according to the invention (and supporting drug) is administered using any suitable means, including parenteral, subcutaneous, intraperitoneal, intra-lungs and intranasal introduction, and if you want local treatment, ingestion of affected tissues. Parenteral administration includes intramuscular, intravenous, intraarterial, NutriBar the tire or subcutaneous administration. In addition, the antibody can be administered by pulse infusion, particularly with lower doses of antibody. The dose can be accomplished in any suitable way, for example by injection, such as intravenous or subcutaneous injection, and this method depends partly on whether the introduction of short or long.

The composition of the antibodies according to the invention receives, divided into doses and administered using methods that are widely used in medical practice. This should take into account such factors as the particular disease to be treated, the type of mammal to be treated, the clinical condition of the individual patient, the cause of the disease, the plot, which is delivered to the means, route of administration, mode of administration and other factors known to experts in the field of medicine. In the composition containing the antibody, you can optionally enter one or more of the funds currently used for the prevention or treatment discussed the disease, although it is not necessary. The effective amount of such auxiliary means depends on the number of antibodies according to the invention, present in the composition, the type of disease or treatment method, as well as other factors listed above. These devices usually injected using the above methods and in the above doses or doses components from 1 to 99% from previously used.

In the case of prevention or treatment of disease, the appropriate dosage of the antibody according to the invention (using antibodies alone or in combination with other agents, such as chemotherapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, from enter whether the antibody in the preventive or therapeutic purposes, previous treatment, the patient's medical history and response to the antibody, as well as the personal opinion of the attending physician. The antibody can enter the patient once or over several courses. Depending on the type and severity of the disease as a suitable initial dose to the patient can be entered from about 1 μg/kg to 15 mg/kg (for example, 0.1 to 10 mg/kg) antibodies, for example, by one or more separate injections or by continuous infusion. As a rule, the daily dose varies in the range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. In the case of multiple injections over several days or longer depending on the condition of the treatment continued until the desired suppression of the observed symptoms. The typical dose of antibody is usually in the range from about 0.05 mg/kg to 10 mg/khetani way the patient can enter a single or multiple doses, constituting approximately 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination). Such doses can be entered periodically, for example every week or every three weeks (e.g. such that the patient receives from about two to twenty, for example about six doses of the antibody). First, you can enter a higher loading dose and then one or more lower doses. A typical dosing regimen includes the introduction of the initial loading dose, a component of approximately 4 mg/kg, and then weekly introduction maintenance dose of the antibody component of approximately 2 mg/kg, However, you can use other metering modes. The results of this method of treatment can be monitored using traditional methods and analysis.

The finished product

In another aspect, this invention features a finished product containing the substance used for treatment, prevention and/or diagnosis of the above violations. The finished product includes a container and a label or an insert inside the packaging or attached to the container. Suitable containers include, for example, bottles, vials, syringes and other Containers can be obtained from different materials, such as glass or plastic. The container has a comp the stand, which by itself or after a merger with another composition is effective for treating, preventing and/or diagnosing the condition, and may have a hole to sterile injection (for example, the container may be a bag of intravenous solution or bottle with a lid that can be punctured by a needle for subcutaneous injection). At least one active agent in the composition is an antibody according to the invention. On the label or an insert in the packaging indicates that the composition is intended for treatment of a particular condition, such as cancer. In addition, the finished product may include (a) a first container containing the composition, where the composition comprises the antibody according to the invention; and (b) a second container containing the composition, where the composition contains another cytotoxic agent. The finished product in this embodiment of the present invention may further comprise an insert in the packaging, which indicated that the first and second compositions of the antibodies can be used for treating a particular condition, such as cancer. Alternative or additionally, the finished product may additionally include a second (or third) container containing a pharmaceutically Bramley buffer, such as bacteriostatic water for injection (BWFI), sabaf is provided a phosphate saline solution, the ringer's solution and dextrose. It can optionally contain other substances which are desirable from the point of view of the supplier and the consumer, including other buffers, diluents, filters, needles and syringes.

Below are examples of the methods and compositions according to the invention. It should be understood that the above General description can be made of various other embodiments.

EXAMPLES

In the examples described getting gumanitarnogo antibodies against beta from rat antibody against mouse antibodies, which binds to the subunit beta integrin alfaretta.

Example 1:

Humanization antagonistic antibodies against beta

Materials and methods

Residues are numbered according to the Kabat system (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Use one-letter symbols of amino acids. The degeneracy of the DNA mean using the IUB code (N = A/C/G/T, D = A/G/T, V = A/C/G, B= C/G/T, H= A/C/T, K = G/T, M = A/C, R = A/G, S=G/C, W= A/T, Y = C/T).

Direct grafting hypervariable sites in the acceptor human consensus framework section

In this work, the use fahmida pVO350-2b, which represents a monovalent display-vector Fab-g3 containing 2 open reading frames under control of the phoA promoter, practically the same as described in Lee et al., J. Mol. Biol. (2004), 340(5): 1073-93. The first open reading frame includes a signal sequence stII connected with domains VL and CH1 acceptor light chain, and the second includes the signal sequence stII connected with domains VH and CH1 acceptor heavy chain, and then protestirovanny form minor protein ragovoy shell P3 (Lowman, H. et al. (1990) Biochemistry 30:10832).

The primary sequence of domains VL and VH rat Fib504 (antibody FIB504.64 obtained using hybridoma ATCC HB-293, American type culture collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA) compared with the sequences of human consensus domain Kappa I (huKI) and human consensus VH domain subgroup III (huIII). Grafting hypervariable sites (HVR) use the following frame areas: HuKI used for wireframe plot variable domain of the light chain (see figa and 7). For wireframe plot variable domain of the heavy chain acceptor skeleton plot VH, you can use a modified human consensus VH domain subgroup III (humIII), which differs from the sequence humIII 3: R71A, N73T and L78A (see Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992)) (see figv). Upon receipt of the antibodies according to the invention the insert 504K-RF also get modified the human con is encynova domain VH subgroup III by carrying out the following amino acid substitutions: A71R and A78F.

Hypervariable sections of rat antibodies Fib504 (obtained using hybridoma ATCC HB-293) using genetic engineering techniques is inserted into the acceptor human consensus wireframe plot VH subgroup III with getting directly inoculated plot HVR (Fib504graft) (see figv). In the VL domain of human consensus acceptor huKI instill plots rat Fib504, containing the following provisions: 24-34 (L1), 50-56 (L2) and 89-97 (L3) (Figa)in the VH domain of the inoculation sites containing provisions 26-35 (H1), 49-65 (H2) and 94-102 (H3) (pigv). In addition, construct a second insert HVR, Fib504Kgraft, which also include the HVR, position 49 VL, in accordance with the broader definition of L2 (see MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)). The analysis of the crystal structure of the complex of the antibody with the antigen MacCallum et al. found that the position 49 of the light chain and the provisions of 49 and 94 of the heavy chain are part of the contact section of the antigen and, therefore, these provisions are included in the definition of the HVR-L2, HVR-H2 and HVR-H3 gumanitarnogo antibodies against beta described in this document.

Variants containing directly inoculated plots, obtained by mutagenesis of Kunkele (Kunkel et al. (1987), above) using a separate oligonucleotide for each hypervariable segment. The desired clones determined by the method of DNA sequencing.

Soft R is homesale hypervariable sites

Is the process "soft randomization (see application U.S. No. 60/545840) refers to the procedure mutagenesis of variance (biased mutagenesis) in the selected protein sequence, such as the hypervariable area of the antibodies. This method allows you to keep the deviation (bias) in sequences of mouse, rat, or other source hypervariable sites in the introduction of approximately 10-50 percent of mutations at each selected position. This method allows to increase the productivity of screening libraries, and to avoid changing the antigenic epitope recognized by the antibody. In accordance with this method soft randomization, change the sequence of each hypervariable site using a strategy that allows you to save deviations with respect to the sequence of murine hypervariable segment. To use this method for the synthesis of poisoned oligonucleotides, first described by Gallop et al., J. Med. Chem. 37: 1233-1251 (1994). However, you can use other methods to keep the deviation in relation to the remains inhuman hypervariable sites, such as misdirected PCR, DNA shuffling and other

If, in accordance with conventions used in this description by way of mutation is subjected to a certain position hypervariable area, the codon encoding the amino acid wild-type, poison mixture (for example, a mixture of 70-10-10-10) nucleotides and the result of mutation in 50 percent of cases in each selected position of the hypervariable segment. For this codon encoding subjected to amino acid mutagenesis of the wild type of the hypervariable area, synthesized using a mixture of low-level impurities of the other three nucleotides, such as a mixture of nucleotides 70-10-10-10. Thus, for example, in the case of soft randomization PRO (CCG), first synthesized position is a mixture of 70% C and 10% of each of the G, T and A; the second position is a mixture of 70% C and 10% each of A, G and T; and the third position is a mixture of 70% G and 10% each of A, C and T. it Should be understood that the deviation can increase or decrease depending on the codon synthesized in this situation, the number of codons encoding a particular amino acid, and the degree poisoning of the oligonucleotide nucleotide composition of the synthetic mixture.

The resulting soft randomization oligonucleotides can be inserted after a mouse, rat or other source sequence hypervariable area, these oligonucleotides are surrounded by such plots, obtained by direct insertion of hypervariable sites. Optional two amino acid position at the beginning of H2 and H3 of the VH domain may have the limited variability: codon RGC can be used for coding in position 49 A, G, S or T and codon AKG can be used to encode position 94 M or R.

Obtaining phage libraries

Pools of randomized oligonucleotides were designed for each hypervariable area, phosphorylate separately in six reaction mixtures with a volume of 20 μl, each containing 660 ng of oligonucleotide, 50 mm Tris, pH 7.5, 10 mm MgCl2, 1 mm ATP, 20 mm DTT, and 5 units of polynucleotide kinase for 1 h at 37°C. Then six phosphorylated oligonucleotide pools combined with 20 μg of the matrix of Kunkele in 500 μl of a solution containing 50 mm Tris, pH 7.5, 10 mm MgCl2when the ratio of the oligonucleotide to the matrix 3:1. The mixture is subjected to annealing at 90°C for 4 min, at 50°C for 5 min and then cooled on ice. To prevent excessive denaturation of annealed DNA, excessive neotony oligonucleotide is removed, using a set of PCR-purification QIAQUICK™ (Qiagen kit 28106, Qiagen, Valencia, CA) and modified method of cleaning. To 500 ál of the annealed mixture, add 150 ál of buffer PB (Qiagen, and the mixture is partitioned between 2 columns with silicon oxide. Each column was washed with 750 ál of buffer PE, Qiagen, dried method extraterritorially (extra spin) and then elute 110 μl mixture containing 10 mm Tris, 1 mm EDTA, pH 8. After that, annealed and purified matrix (220 μl) complement by adding 1 μl 100 mm ATP, 10 μl of 25 mm dNTP (25 mm each of dAP, dCTP, dGTP and dTTP), 15 μl of 100 mm DTT, 25 μl buffer 10× TM (0.5 M Tris, pH 7.5, 0.1 M MgCl2), 2400 units of T4 ligase and 30 units of polymerase for 3 h at room temperature.

Augmented product is analyzed on Tris-acetate-EDTA/agarose gels (Sidhu et al:, Methods in Enzymology 328:333-363 (2000)). Usually there are three bands: the lower band corresponds properly supplemented and legirovannom product, average bar corresponds added, but delegirovano product, and the upper band corresponds to the offset circuit. The upper band corresponds to the product resulting from adverse activity characteristic of the T7 polymerase, education is hard to avoid (Lechner et al., J. Biol. Chem. 258: 11174-11184 (1983)); however, this band is transformed into a 30-fold less efficiently than the lower band, and usually makes a low contribution in the resulting library. The product corresponding to the average band, formed in result of the lack of 5'-phosphate, required for the final ligation reaction; this product transformnode effectively and gives essentially the sequence of the wild type.

Then the augmented product is distilled and the method of electroporation give SS320 cells grown in the presence of helper phage M13/KO7, as described by Sidhu et al, Methods in Enzymology 328: 333-363 (2000). The size of the libraries varies in the range 1-2×109depending on the clone. To evaluate the quality is biblioteki, spend sequencing of random clones from the original libraries.

Selection of phage

A full-sized human integrin alfaretta Express in 293 cells (Graham et al., J. Gen Virol. 36: 59 (1977)), purified by the method of affinity chromatography using Fib504 and used as a target for selection of phage. In order to carry out the immobilization integrin on microtiter tablets MaxiSorp ™ (Nalge Nunc, Rochester, NY)add 100 μl of the solution of human integrin alfaretta with a concentration of 5 μg/ml in a mixture containing 150 mm NaCl, 50 mm Tris, pH 7.5, 2 mm CaCl2, 2 mm MgCl2and 2 mm MnCl2(TBSM), overnight at 4°C. the Wells are blocked for 1 h with TBSM, containing 1% BSA. In the first round of selection using 8 wells, coated with the target; for a successful breeding cycles use the wells, once covered by the target. Phage harvested from the culture supernatant and suspended in TBSM, containing 1% BSA and 0.05% TWEEN™ 20 (TBSMBT). After incubation with holes for 2 h, the unbound phage are removed by copious rinsing TBS containing 0.05% TWEEN 20 (TBST). Bound phage elute by incubation of the wells with 100 mm HCl for 30 min Phage amplified using Top10 cells and helper phage M13/KO7, and grown overnight at 37°C in 2YT, in the presence of 50 μg/ml of carbenicillin. To assess the enrichment, the titers of phage, lirovannomu with holes on rytych target compared with the titers of phage recovered from holes not covered by the target. After four cycles of selection analyze a sequence of randomly selected clones.

Getting Fab and determination of affinity

To Express the Fab to measure the affinity, the stop codon is introduced into the vector phage display between sequence that encodes a heavy chain, and g3. The resulting clones are used for transformation of E.coli cells 34B8 grown in an environment AP5 at 30°C (Presta, L. et al., Cancer Res. 57: 4593-4599 (1997)). Cells are harvested by centrifugation, suspended in a mixture containing 10 mm Tris, 1 mm EDTA, pH 8, and destroy with the help of microfluidizer. Fab purified by the method of affinity chromatography using protein G.

Affinity is determined by means of surface plasmon resonance using a BIAcore™-3000 (Biacore, Piscataway, NJ). Options Fab gumanitarnogo Fib504 immobilized in 10 mm acetate buffer, pH 4.5 (in the range of from 250 to 1500 response units (RU)) on the sensor chip CM5, and introduce a 2-fold dilution of human integrin alfaretta (from 1.5 to 770 nm) in TBSM containing 2% n-octylglucoside. Each sample analyzed, using a 5-minute interval of the Association and 5-60-minute interval dissociation. After each input chip regenerate using three 1-minute introductions 8 M urea. Answer by linking adjusted by subtracting the EN of the blank flow cells. For analysis of the kinetics using the model of Langmuir 1:1 with simultaneous fit of konand koff.

Results and discussion

Humanization of rat Fib504

Used for humanization of the human acceptor frame portion which is received on the base frame section used for HERCEPTIN®, consists of a consensus human VL domain subgroup Kappa I (huKI) and variants of the consensus human domain VH subgroup III (humIII). The specified variant of the VH domain contains 3 replacement compared with the human consensus sequence: R71A, N73T and L78A. Sequence domains VL and VH rat Fib504 compared with the sequences of the domains of human chains subgroup Kappa I and subgroup III; each hypervariable segment (HVR) identify and teach to the human acceptor frame section with the receiving insert HVR (504graft), which is detected in the phage display Fab (figa and 1B).

The analysis of available crystal structures of complexes of antibody with antigen MacCallum et al. (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)) the proposed definition HVR-based variable domain residues that are frequently in contact with antigens. Thus, the provisions 49G 94M and the heavy chain comprises inserting HVR Fib504 (pigv). In addition, receive a second insert HVR, Fib504Kgraft, which contains provisions is their 49K light chain, because this provision is also included in the contact definition HVR-L2, and may participate in the establishment of contact with the antigen (Figa). If the insert Fib504 or Fib504K fixed on phage display and analyze binding to the immobilized alfaretta, binding does not occur.

Libraries receive using inserts HVR in Fib504 and Fib504-K, where all areas of HVR were simultaneously subjected to soft randomization. Each library inserts HVR sort against immobilized alfaretta within 4 cycles of selection. Enrichment is not observed, and the clones selected for analysis of DNA sequences contain only random changes in the sequence directed on the 6 areas of HVR.

Two other sequences of frame sections VH, "RL" and "RF"is used as acceptor frame sections contain substitutions at positions 71 and 78. The amino acid at position 71 is substituted for arginine, as in the human consensus sequence of subgroup III, and the amino acid at position 78 is replaced by leucine, as in the human consensus sequence of subgroup III (acceptor skeleton plot "RL"), or phenylalanine, as in the human consensus sequence of subgroup II and in the sequence of frame section VH rat Fib504 (acceptor skeleton plot "RF") (figa). If the insert Fib504 and the and Fib504K acceptor in wireframe plot "RL" (Fib504-RL and Fib504K-RL) or "RF" (Fib504-RF and Fib504K-RF) is fixed on phage display and analyze binding to the immobilized alfaretta, specific binding of phage observed only in the case of insertion Fib504K using wireframe plot "RF" (pigv). Moderate binding phage insert Fib504-RF compared to other inserts, not containing Y49K (light chain) and L78F (heavy chain), indicates the importance of these provisions when selecting the acceptor frame section.

Libraries receive the above-described method using the strategy of soft randomization simultaneously on all 6 HVR for inserts Fib504K-RL and Fib504K-RF, and then sorted against immobilized alfaretta within 4 cycles of selection, as described above. Enrichment is observed only for the library, based on the insertion Fib504K-RF. The clones obtained after 4 cycles of selection library Fib504K-RF are selected for sequence analysis, which detects amino acid changes in HVR-L1. Most of the clones contains replacement Y32L; in addition, the amino acid at position 31 is often replaced with D, S, P or N (figs). In addition to the original insert, Fib504K-RF, 3 clone, Express and purify in the form of Fab proteins that are analyzed using Biacore as described above. Clones hu504-5, hu504-16 and hu504-32 (variants of SEQ ID NO: 1 containing replacement T31S and Y32L (option hu504.5), Y32L (option hu504.16) or T31D and Y32L (option hu504.32); see figs) much better link alfaretta than inserting Fib504K-RF, and the possession is t the same or a higher affinity to Alfama, as chimeric Fab Fib504. The results of the Biacore analysis are shown below in table 3 and show that the resulting selection of the variants disclosed in the present description, the HVR and/or frame sections antagonistic antibodies against alfaretta have a higher affinity than the original antibody. In table 3 the results show that humanitarianly option 504.32 demonstrates the highest affinity compared to the original mouse antibody, as associated with alfaretta 3 times stronger.

Table 3
Fab
(Analysis BIAcore™)
Affinity for alfaretta
(nm)
Fib50411
Option 504.59
Option 504.1623
Option 504.323

In table 3 the results also show that the reconstruction of the HVR-L1 may lead to the restoration of high affinity for binding to antigen. In particular, among the different clones most common mutation Y32L. Other changes in position 31, as well as several other substitutions in the sequence is the HVR-H1 are valid and may provide additional improvement. The results show that there are many sequence changes that result in improved affinity site Fib504, grafted on the human frame section, and provide affinity, which is identical to the original affinity of rat antibodies or greater.

Thus, on the basis of the insert 6 HVR rat Fib504 in the human acceptor framework to receive the extension HVR-L2, with the inclusion of provisions 49 (lysine), expansion HVR-H2, with the inclusion of provisions 49 (glycine) and the expansion of the HVR-94 with the inclusion of a provision 94 (methionine), and amino acid changes at position 32 VHR-L1 (where L, or I is replaced by Y) and, optionally, at position 31 VHR-L1 (where T is substituted, for example, D or S). Acceptable amino acid substitutions in the frame area carry out the provisions of 71 (A71R) and 78 (L78F) of the VH domain. Such amino acid substitutions produce fully human antibodies, variants hu504.32, for example, with a 3-fold increase in the affinity of binding to the integrin alfaretta. In addition, it was shown that the resulting selection of the humanized antibodies of the present invention have a biological activity at least comparable to the biological activity of the original rat antibodies Fib504 (see example 3 of this specification).

Example 2

Other humanized variants HVR Fib504

Amino is islote sequence HVR gumanitarnogo option Fib504.32 additionally modify with obtaining other options having antagonistic activity against the integrin subunit beta and/or integrins containing subunit beta.

Getting library broad amino acid scan

A library for scanning the positions of selected HVR other amino acid residues, which can lead to beta-binding variants variant hu504.32, obtained using 3 oligonucleotides: 504-L1, designed to achieve a soft randomization fragment HVR-L1 deviation with respect to the sequence HVR-L1 hu504.32 (i.e. sequence ASESVDDLLH (SEQ ID NO: 47, the relative position of the A2-A11) was gently is randomized by the above method); and an HVR-L3 504-N96 and HVR-H3 504-M94, which are obtained by the introduction of NNS in position 96 light chain HVR-L3 and in position 94 of the heavy chain HVR-H3, thus ensuring the presence of all 20 amino acids in these positions. Using these three oligonucleotides receive a library of broad amino acid scan according to the above method, using a matrix that contains three stop codon in the light chain (positions 31 and 32 in HVR-L1 and position 96 in HVR-L3), and 1 stop codon in the heavy chain (position 94 in HVR-H3).

Broad amino acid scan hu504-32

To more fully explore the preferred sequence, razresheniem HVR-L1, as well as to improve the stability 504-32, the authors present invention has created a phage library, which (a) contains gently randomized HVR-L1 504-32 in the area in which the changes occur (i.e. ASESVDDLLH (SEQ ID NO: 47, the relative position of the A2-A11) in the process of humanization (figs)), and (b) ensures the presence of all possible amino acids at position N96 HVR-L3 and in position M94 HVR-H3. After 4 cycles of selection against immobilized full-sized human integrin alfaretta, as described above, 96 random clones selected for sequence analysis. The frequency of occurrence of amino acids at each position in the library broad amino acid scan allows us to conclude that the sequence of HVR-L1, which is present in hu504-32, and methionine at position 94 of the heavy chain are optimal for high affinity binding (Fig). The most preferred amino acids are defined through selection of options, starting with version 504.32 (Fig) are highlighted in yellow. On the contrary, although at position 96 light chain hu504-32 is asparagine, the high occurrence of leucine in this position suggests that the mutation N96L could further improve the affinity of humanized Fib504 variants to Alfama and, in addition, it can also fix any problems associated with the possibility on the amidation under this provision. On Fig provides information about the number of amino acid substitutions, which, in all probability, are acceptable for most positions, without causing reduction of binding affinity. For example, to prevent oxidation of M94 in HVR-H3, glutamine or arginine, apparently, must be replaced.

Obtaining libraries of limited amino acid scan

Get six libraries limited amino acid scan using six different matrices of Kunkele, each of which contains one of the stop codon in one of the six HVR. Each library is obtained using one of the oligonucleotide encoding one HVR, and with the use of codons that are listed on figa (column "codon"), resulting in change of amino acid residues, followed by testing for binding to beta or alfaretta. The same methods are used to modify amino acid residues of antibodies against beta and analysis of their binding to integrins alphabet.

Limited amino acid scan hu504-32

Limited amino acid scan hu504-16 take in order to further increase the similarity of hu504-16 and human consensus sequences of light and heavy chains, and to identify using this method the minimum elements of a sequence of rat Fib504 required for binding. On ucaut six libraries, targeting these provisions in each HVR, which differ among hu504-16 and human consensus sequences of the light chain Kappa or I heavy chain subgroup III (figa and 1B); these provisions libraries can be rat or human amino acid (figa). To ensure that the coding of both amino acids in the process of oligonucleotide synthesis and mutagenesis in some cases, introducing other amino acids (see "encoded amino acids", figa). Selection of libraries of limited amino acid scan is conducted against immobilized full-sized human integrin alfaretta, as described above, and after 3 cycles of approximately 32 random clones from each library is sequenced. The frequency of occurrence of each amino acid at each position is shown in figv and 11C.

Like a broad amino acid scan, limited amino acid scan also gives information about the changes that are acceptable for many of the provisions gumanitarnogo Fib504. However, in contrast to the broad amino acid scan, variability, valid for each randomizearray position, with limited amino acid scan is reduced to a pair of amino acids. Thus, the absence of any of the observed substitutions in a given situation does not mean that it is hydrated residue cannot be changed, and the high frequency of occurrence of any particular amino acid in a given position does not indicate that this amino acid is necessarily the best solution for obtaining high affinity.

Some regulations (regulation 27, 29, 30, 53, 54 light chain and 50, 54, 58, 60, 61 and 65 of the heavy chain) human consensus amino acid is present often enough and this suggests that reverse mutation to the human consensus amino acid does not lead to a strong change of binding to human alfaretta. Actually in position 54 light chain (HVR-L2) human consensus amino acid is present more often than amino acid from rat Fib504, and therefore this change in 504-32 results in usable beta-binding antibodies.

In addition, some provisions of the obtained library more likely to be amino acids that are not present neither in the human consensus sequence, nor in rat Fib504, these amino acids provide potential replacements, which may increase the affinity of humanized Fib504 variants. These substitutions include, without limitation, D30A and I55V in the light chain and Y50F in the heavy chain. The results of the analysis 2 of these libraries show that many of the provisions of the HVR other possible amino acid substitutions cover what anoniem comparable biological activity.

Data on the observed amino acid changes are shown on figures 13 and 15. The figure 15 shows the different amino acids used in each of the provisions CDR variant antibodies according to the invention, are numbered using the Kabat system or relative numbering system. Each additional antibody specific to the cases depicted in figures 13 and 15, is an embodiment according to the invention.

Example 3

Analyses of cell adhesion

The ability of some humanized Fib504 variants according to the invention to bind ligands expressed on the cell surface, tested by analysis of cell adhesion. Linking alfaretta with another integrin beta, alphabeta, analyze the ability of humanized variants to inhibit the binding of integrin to its natural receptor. Similarly analyze the binding of humanized Fib504 variants only subunit beta expressed on the cell surface. Methods and results are described below.

Production of IgG

Humanized IgG variants Fib 504 transtorno Express in 293 cells (Graham et al. (1977) supra), using separate vectors for light and heavy chains. Vectors design by sublimirovanny variable domains of the light or heavy chain in a suitable vector, the expression for each of the light and heavy chains. The supernatant 1.1 l of cell culture CHO containing humanitarianly Fib504 variant, filtered through a filter with pore diameter of 0.45 μm on a new column HiTrap Protein A HP column (Amersham/Pharmacia) in a volume of 1 ml, equilibrated with buffer A (10 mm Tris, pH 7.5, 150 mm NaCl). The samples applied with a speed of 0.8 ml/min, over night at 4°C. Then each column is washed and balance 30 ml of buffer A. elute Antibody at room temperature by chromatography on FPLC (liquid chromatograph quick resolution) (Amersham/Pharmacia) using a linear gradient of buffer B (100 mm glycine, pH 3.0) from 0 to 100%for 14 min at 1 ml/min. and the Obtained fractions of 1 ml immediately neutralized by adding 75 ál of 1 M Tris, pH 8. Eluruumis protein detected by absorption at 280 nm, the peak fractions are combined and absoluut in PBS for disposable columns for gel filtration with PD10 Sephadex G-25 (Amersham/Pharmacia). Protein detected by optical density at 280 nm and peak fractions pooled. The antibody solution in PBS, filtered through a filter with pore size of 0.22 μm and stored at 4°C. the concentration of the obtained purified antibodies was determined by amino acid analysis as the average of two separate measurements.

Tagging BCECF

In each of the analyses presented in this example 3, the cells have been labelled using the following method. All cells used for analysis the hell is esii, mark 10 μm solution acetoxymethyl ester of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in medium RPMI 1640 containing 10% FBS, in the case of RPMI8866 cells and 38C13 cells, transfected subunit beta (cells BEA), and the mixture F-12:DMEM (50:50)containing 10% FBS, in the case of 293 cells, transfected alfabeta (cells alfabeta-293). Cells have been labelled for 30 minutes and washed twice with the medium for analysis. Cell density was adjusted to 3×106cells per ml in the case of RPMI8866 cells and cat and to 2.2×106cells per ml in the case of cells alfabeta-293.

Humanized Fib504 variants disrupt binding alfaretta with MAdCAM

Adhesion of cells RPMI8866/MAdCAM-1-Ig

RPMI8866 cells Express alfaretta on the surface (Roswell Park Memorial Institute, Buffalo, NY). Humanized Fib504 variants (hu504 variants) is brought into contact with a mixture of cells RMPI8866 and MAdCAM, hereditary with IgG deposited on a solid carrier. To measure the concentration of humanized Fib504 variants that cause 50% inhibition (IC50) binding RPMI8866 cells to MAdCAM-1, 96-well tablets cover Nunc Maxisorp™, 2 μg/ml in PBS, and 100 µl/well MAdCAM-1-Ig (Genentech, Inc., Ig in hybrid with MAdCAM-1 means plot Fc) over night at 4°C. Then the tablets blocked by incubation with 200 μl/well of a solution of BSA, 5 mg/ml, for one hour at room temperature, after which each link is added 50 μl of solutions of humanized Fib504 variants for analysis (RPMI medium 1640, Hyclone®, Logan Utah, USA, containing 5 mg/ml BSA), 150000 BCECF-labeled cells (BCECF, Molecular Probes, Eugene, OR) in 50 μl of medium to analyze and incubated for 15 minutes at 37°C. the Wells are washed twice with 150 μl of medium for analysis, to remove unbound cells. Associated cells solubilizer by adding 100 μl of 0.1% SDS in 50 mm Tris/HCl, pH 7.5. The number of fluorescent labels, released lysed cells was measured using a SPECTRAmax GEMINI™ (Molecular Devices, Sunnyvale, CA) at a wavelength of excitation 485 nm and the wavelength of emission of 530 nm. The values of fluorescence analyzed as a function of the concentration of humanized Fib504 variants added to each assay, using chetyrehjadernogo nonlinear approximation by the method of least squares and get the values of IC50for each gumanitarnogo Fib504 variants used in this analysis. The values of the IC50and IC90determine using chetyrehjadernogo approximation. On Fig shows a typical graph obtained using the results of the analysis. The values of the IC90and IC50for each test illustrated below in table 4.

Table 4
The binding of an antibody to human MAdCAM-1
Analyzed the antibody:
Fib504 variants and hu504
IC50(nm)
exp/exp*
IC90(nm)
exp/exp*
Rat Fib5040,098/0,1970,483/0,703
Option hu504.50,067/0,2480,361/0,880
Option hu504.160,0768/0,2060,244/0,551
Option hu504.32being 0.036/0,1190,150/0,396
6B11(nonblocking control)>100>100
* exp/exp means the average value from duplicate analyses.

Humanitarianly Fib504 variant disrupts the binding alfaretta with VCAM

Adhesion of cells RPMI8866/7dVCAM-1

Analysis of the RPMI8866/7dVCAM-1 is performed according to the method used for analysis of the RPMI8866/MAdCAM-1-Ig, except that the coating of the tablets use 7dVCAM-1 (ADP5, R&D Systems, Minneapolis, MN) at a concentration of 2 µg/ml. the Results analyzed as described above for analysis of binding of MAdCAM. The values of the IC50for each of the tested variants are shown below in table 5.

Table 5
The binding of an antibody to human VCAM
Analyzed the antibody:
Fib504 variants and hu504
IC50(nm)
exp/exp*
IC90(nm)
exp/exp*
Rat Fib5040,107/0,1930,396/0,580
Option hu504.50,088/0,2700,396/0,726
Option hu504.160,098/0,2230,261/0,774
Option hu504.320,059/0,1100,183/of 0.337
6B11(nonblocking control)>100>100
* exp/exp means the average value from duplicate analyses.

Humanitarianly Fib504 variant disrupts the binding alphabet with human E-cadherine

Adhesion of cellsalphabet-293/huE-cadherin

The 293 cells (Graham et al. (1977) supra) transferout algae and beta (Genentech, Inc.). The analysis is performed according to the method used to analyze the RPMI8866/MAdCAM-1-Ig, except that to cover the planche is tov use huE-cadherin (648-EC R&D Systems, Minneapolis, MN) at a concentration of 2 µg/ml. Then tablets are blocked by incubation with 5 mg/ml BSA, as described above, after which each well add 50 ál of FIB504 variants in the environment for analysis (F-12:DMEM (50:50)containing 5 mg/ml BSA), 110000 BCECF-labeled cells in 50 µl of medium to analyze and incubated for 15 minutes at 37°C. the Wells are washed twice with 150 ál of medium to analyze, measure the amount fluorescent label, released lysed cells, and the results analyzed as described above. The analysis results obtained from three experiments are shown below in table 6.

8,8/24,5/11,73
Table 6
The binding of an antibody to human E-Katharina
Analyzed the antibody:
Fib504 variants and hu504
IC50(nm)IC90(nm)
Rat Fib5042,047/7,89/4,198,80/24,5/9,95
Option hu504.52,132/10,18/4,777,99/28,7/10,19
Option hu504.161,957/of 10.05/4,587,07/33,7/13,51
Option hu504.321,814/6,99/3,47
6B11(nonblocking control)>100/>100/>100>100/>100/>100

Humanitarianly Fib504 variant disrupts the binding beta with MAdCAM

Analysis of cell adhesion cat/muMAdCAM-1-Ig

Analysis BEA/muMAdCAM-1-Ig carried out according to the method used to analyze the RPMI8866/MAdCAM-1-Ig, except that the coating of the tablets use muMAdCAM-1-Ig (Genentech, Inc.) at a concentration of 2 µg/ml Cells mouse lymphoma 38C13 alpha+ (Crowe, D.T. et al., J. Biol. Chem. 269:14411-14418 (1994)) transferout DNA that encodes the integrin beta, so that alfaretta expressively on the cell surface. The ability of the variants of antibodies to disrupt the interaction alfaretta associated with the cellular membrane, with MAdCAM analyzed by the above method. The results are shown in table 7 (shown values IC50and IC90obtained from 2 experiments).

Table 7
The activity of variant antibodies hu504 against cells expressing 38C13-beta
The binding of murine MAdCAM
Analyzed the antibody:
Fib504 variants and hu504
IC50(nm)IC90(nm)
Rat Fib5040,682/0,3062,869/1,51
Option hu504.50,8587/0,4662,322/2,61
Option hu504.16is 0.998/0,6103,717/4,08
Option hu504.320,718/0,4584,08/1,51

Humanitarianly Fib504 variant disrupts the binding beta mouse VCAM

Analysis of cell adhesion cat/muVCAM-1-Ig

Analysis BEA/muVCAM-1-Ig is performed according to the method used previously for analysis of binding of murine MAdCAM-1-Ig with RPMI8866 cells, except that the coating of the tablets use a VCAM-1-Ig (Genentech, Inc.) at a concentration of 2 µg/ml. the results of the analyses are given in table 8 shows the values of the IC50and IC90obtained from 2 experiments).

Table 8
The activity of variant antibodies hu504 against cells expressing 38C13-beta
Binding to murine VCAM-1-Ig
Analyzed the antibody:
Fib504 variants and hu504
IC50(nm)IC90(nm)
Rat Fib504 0,845/0,4472,903/2,30
Option hu504.50,763/0,4073,074/2,30
Option hu504.160,835/0,5842,857/1,84
Option hu504.320,562/0,330at 2,004/1,84

The results of binding gumanitarnogo Fib504 variants show that humanitariannet antibody according to the invention is associated with its target - subunit of integrin beta, as well as integrins alfaretta and alfabeta, with an affinity that is approximately identical to the original affinity of rat antibodies, and in some embodiments greater than his. Thus, humanitariannet antibody against beta according to this invention can be used in methods of treatment directed against integrin beta, especially for the treatment of humans.

Comparative activity options hu504.32 according to the invention

The ability of the different amino acid variants of antibodies hu504.32 to inhibit the binding beta-containing receptor with its ligands tested by analysis of adhesion of human and mouse cells using the methods described in this document for analysis of cell adhesion. Analysis RPMI886/MAdCAM-1-Fc carried out by the above method. For analysis alfabeta-293/huE-cadherin using a modified method using human E-cadherin-Fc as a ligand (human E-cadherin-Fc, 648-EC, R&D Systems, Minneapolis, MN). Also analyze the relative ability options hu504.32 to inhibit the interaction of human fibronectin (huFN40) with the human receptor alfaretta on cells PRMI8866. Analysis of the RPMI8866/hu fibronectin (huFN40) this study was conducted according to the method used to analyze the RPMI8866/MAdCAM-1-Ig, opened in this description, except that the coating of the tablets use a fragment of human fibronectin alpha size 40 kDa, obtained by treatment with chymotrypsin (F1903, Chemicon International, Temecula, CA)at a concentration of 2 µg/ml.

Analyze the ability of the variants hu504.32 to inhibit the interaction of murine beta-containing receptors with murine MAdCAM-1 or murine VCAM-1. Options hu504.32 inhibit the interaction of hybrids murine MAdCAM-1-Fc and mouse VCAM-1-Fc cells mouse lymphoma Alfa+expressing murine subunit beta (cells BEA). Analyses of cell adhesion using murine MAdCAM-1-Fc and VCAM-1-Fc carried out according to the method described above for human MAdCAM and VCAM. If the ligands hybridized with Fc-plots, Fc receptors on cells blocking with 0.5 μg of antibodies against CD16/32 (antibody against the receptor is Fcγ III/II, catalog No. 553142, BD Biosciences, San Jose, CA) per million cells for 5 minutes at room temperature. To each well add 150000 labeled cells in 50 µl of medium to analyze and incubated for 13 minutes at 37°C. the Wells are washed and using the above method measures the amount of fluorescent label, released from lysed cells. When analyzing the adhesion of human cells as a control antibody using mouse monoclonal antibody against human serum albumin, 6B11 (catalog No. ab 10244, Novus Biologicals, Littleton, CO, USA). When analyzing the adhesion of mouse cells as a control antibody using rat antibody against mouse integrin beta, M293 (BD Biosciences, San Jose, CA), which does not compete with the ligand or Fib504 for binding to the integrin beta.

Results adhesion of human and mouse cells with triple repetitions are presented in tables 9 and 10, respectively.

Table 9
The activity of variant antibodies hu504.32 in the analysis of the adhesion of human cells
Variant antibodiesIC50
AVG. +SD
RPMI8866/
huMAdCAM-1-Fc
RPMI8866/
hu7dVCAM-1
αEβ7-293/
huE-cadherin-Fc
RPMI8866/
huFN40
hu504.320,088±0,0350,101±0,0213,970±kzt1.6640,100±0,046
hu504.32M94Q0,090±0,0450,111±0,0354,130±1,2120,124±0,056
hu504.32M94R0,075±0,0340,089±0,0093,963±1,7760,119±0,056
Control (V)>100>100>100>100

Table 10
The activity of variant antibodies hu504.32 in the analysis of adhesion of murine cells
Variant antibodiesIC50
AVG. +SD
Cat/muMAdCAM-1-FcCat/mu7dVCAM-1-Fc
hu504.320,270±0,0410,228±0,065
hu504.32M94Q 0,370±is 0.1020,264±0,083
hu504.32M94R0,391±0,1120,228±of 0.081
Control (M293)>100>100

Antibody hu504.32 contains methionine at position 94 of the heavy chain CDR3. Options M94Q (or hu504.32Q) and M94R (or hu504.32R) contain glutamine or arginine, respectively, in position 94 variant antibodies hu504.32. Antibodies hu504.32M, Q and R significantly reduce the interaction of the receptor integrin beta with ligand in all analyses and, therefore, are powerful inhibitors beta-mediated cell adhesion.

The activity of antibodies hu504.32R in vivo

Analyze the ability of the variant antibodies hu504.32R to reduce the interaction of the receptor integrin beta with ligand in vivo and reduce the recruitment of lymphocytes in the inflamed colon in vivo in murine models of inflammatory bowel disease. BALB/c mice and mice CB 17 SCID get from Charles River Laboratories International, Inc. (Wilmington, MA, USA). Mice with SCID colitis containing T cells, enriched CD4+CD45Rb, obtained by selection of T cells, enriched CD4+CD45Rb, from donor BALB/c mice, followed by the intravenous injection of 3×105cells in 100 µl PBS. Control SCID mouse do not receive T cells, enriched CD4+CD45Rb. Consider, Thu is the CD4+-converted mice, meet the criteria set in the experimental group, that is, having a weight loss of 10% compared to baseline levels, or 15%, compared with the maximum level at 4 week, is induced by inflammatory bowel disease, such mice are selected for processing.

On the day of processing the test antibodies are harvested cells of mesenteric lymph node (MLN) BALB/c mice and mark radioactive isotope Cr51. Processing includes pre intravenous antibodies against GP120 antibodies against beta hu504.32, antibodies against beta hu504.32R containing no antibody (control), 200 μg/100 μl PBS. Thirty minutes after administration of the antibody is administered Cr51-labeled cells in MLN, 4×106cells/100 μl. One hour after injection of labeled cells in mice subjected to Athanasii, collect spleen, large intestine and Peyer's plaques, weigh them and determine the total amount of Cr51in each organ. On Fig shows a histogram of the results, showing the relative ability of antibodies to block homing radiolabelled T-cells in the colon of mice suffering from inflammatory bowel disease. Antibodies against beta hu504.32 and hu504.32R inhibit homing T-cells in the inflamed colon compared with the negative control antibody is Rotel GP120. All antibodies have the same distribution in the spleen. Thus, antibodies against beta hu504.32 and hu504.32R effectively inhibit homing T-cells in the inflamed colon in vivo.

The glycation of antibody does not affect the ability of the variant hu504.32R to block the binding of MAdCAM-1 receptor alfaretta.

The glycation, nonenzymatic glycosylation of proteins may influence the interaction of the antibody with the ligand (see, for example, Kennedy, D.M. et al., Clin Exp Immunol. 98(2):245-51 (1994)).

The glycation of lysine at position 49 504.32R

The glycation of lysine at position 49 of the light chain variant hu504.32R (relative position B1 HVR-L2) is observed, but no significant effect on the ability of the variant antibodies to block the binding of MAdCAM-1 with RPMI8866 cells expressing the receptor alfaretta. The glycation and its level is determined by standard mass spectrometry with ionization by elektrorazpredelenie (ESI-MS) and boronate affinity chromatography. Description of the method boronate affinity HPLC, ispolzuemogo for analysis of glycation can be found, for example, in Quan CP.et al., Analytical Chemistry 71(20): 4445-4454 (1999) and Li Y.C. et al., J. Chromatography A, 909: 137-145 (2001). Analysis of cell adhesion is performed according to the method, opened in this description for the analysis of cell adhesion RPMI8866/MAdCAM-1-Fc. In alternative embodiments according to the invention, glycation Polo the attachment 49 is reduced or does not occur, if in position 49 is an amino acid other than lysine. This invention encompasses polypeptides or antibodies containing in position 49 (relative position B1 HVR-L2) any of the amino acids A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y, where each letter indicates the amino acid in accordance with the standard single letter designation system for amino acids. Alternatively, the amino acid at position 49 of the light chain variant 504.32R (or other option 504) may be selected from the group consisting of R, N, V, A, F, Q, H, P, I or L. the Amino acid at position 49 is chosen, for example, by display (get ragovoy library) Fab hu504.32R on the phage (variant) and separately by replacing codons encoding all 20 natural amino acids, plot, encoding the position 49. Expressed by phage variants hu504.32R modified at position 49, tested for binding to integrin beta and/or receptors containing integrin beta, such as receptors alfaretta or alphabet. Variants that are associated with integrin beta or receptors alfaretta or alphabet, is subjected to further screening for the ability to inhibit the binding of receptor integrin beta with ligand and efficacy in vivo, as described herein. Using standard methods of mutagenesis at position 49 can be replaced by an alternative natural or prirodnoy amino acid and then to test the obtained antibodies by analysis of cell adhesion and in vivo analyses, described in this document. Alternatively, the amino acid at position 49 of the light chain is an amino acid other than lysine (K), and amino acids in any other position (or in any other provisions) HVR or frame area light chain and/or heavy chain are replacements and then spend the breeding option beta-binding polypeptide or antibody that has affinity binding, biological activity in vitro and in vivo, pharmacokinetic characteristics excretion of drugs and immunogenicity, allowing to reduce inflammation by reducing the biological activity of integrin beta. Mutagenesis and selection of such a variant polypeptide or antibody is carried out using the methods disclosed herein, and in accordance with other standard methods. This option beta-binding polypeptide, or antibody has an affinity to the integrin beta that 10,000 times, 1000 times, alternative 100 times, alternative 10 times, alternative 5 times, alternative 2 times greater than the affinity of the binding disclosed in this description of the humanized Fib504 variants.

Using the above description, the person skilled in the art can realize the present invention. Scope of the present invention is not limited by this description, because the described embodiment the Oia are intended only to illustrate certain aspects according to the invention and any functionally equivalent embodiments are included in the scope according to the invention. Herein, the material eliminates the assumption that the above description is not enough to implement any aspect according to the invention, including the best way to implement it, in addition, this description should not be construed as limiting the scope of the claims, the specific examples. In fact, on the basis of the above description specialists in this field can perform, in addition to the shown and described herein, various other modifications according to the invention, which are also included in the scope the appended claims.

1. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDDLLH (SEQ ID NO: 9), KYASQSIS (SEQ ID NO: 2), QQGNSLPNT (SEQ ID NO: 3), GFFITNNYWG (SEQ ID NO: 4), GYISYSGSTSYNPSLKS (SEQ ID NO: 5) and ARTGSSGYFDF (SEQ ID NO: 64).

2. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDDLLH (SEQ ID NO: 9), KYASQSIS (SEQ ID NO: 2), QQGNSLPNT (SEQ ID NO: 3), GFFITNNYWG (SEQ ID NO: 4), GYISYSGSTSYNPSLKS (SEQ ID NO: 5) and MTGSSGYFDF (SEQ ID NO: 6).

3. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDDLLH (SEQ ID NO: 9), KYASQSIS (SEQ ID NO: 2), QQGNSLPNT (SEQ ID NO: 3), GFFITNNYWG (SEQ ID NO: 4), GYISYSGSTSYNPSLKS (SEQ ID NO: 5) and AMTGSSGYFDF (SEQ ID NO: 63).

4. International narcotics the new antibody against beta person, contains
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDDLLH (SEQ ID NO: 9), KYASQSIS (SEQ ID NO: 2), QQGNSLPNT (SEQ ID NO: 3), GFFITNNYWG (SEQ ID NO: 4), GYISYSGSTSYNPSLKS (SEQ ID NO: 5) and RTGSSGYFDF (SEQ ID NO: 66).

5. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDSLLH (SEQ ID NO: 7), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5) and (SEQ ID NO: 64).

6. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDSLLH (SEQ ID NO: 7), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5) and (SEQ ID NO: 6).

7. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDTLLH (SEQ ID NO: 8), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5) and (SEQ ID NO: 64).

8. Humanitariannet antibody against beta person, containing
HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, each of which is in the order has RASESVDTLLH (SEQ ID NO: 8), (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5) and (SEQ ID NO: 6).

9. Humanitariannet antibody against beta person containing HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 antibodies against beta according to any one of claims 1 to 8 and a variant HVR-L1, which is a variant of the sequence A1-A11, where A1-A11 is a RASESVDTYLH (SEQ ID NO: 1) and contains one replacement in any of the following positions: A2 (A, G, S, T or V), A3 (S, G, I,K, N, P, Q, R or T); A4 (E, A, D, G, H, I, K, L, N, Q, R, or V), A5 (S, A, D, G, H, I, K, N, P, R, T, V or Y), A6 (V, A, G, I, K, L, M, Q or R), A7 (D, A, E, G, H, I, K, L, N, P, S, T or V), A8 (S, D, E, G, P, T or N), A9 (L, Y, I or M), A10 (L, A, I, M or V) or A11 (H, F, S, or Y).

10. Humanitariannet antibody against beta person containing HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 antibodies against beta according to any one of claims 1 to 8 and a variant HVR-L2, which is a variant of the sequence B1-B8, where B1-B8 is a KYASQSIS (SEQ ID NO: 2) and contains one replacement in any of the following positions: B1 (K, N or Y), B6 (S, R or L) or V7 (I, T, E, Or V).

11. Humanitariannet antibody against beta person containing HVR-L1, HVR-L2, HVR-H1, HVR-H2 and HVR-H3 antibodies against beta according to any one of claims 1 to 8 and a variant HVR-L3, which is a variant of the sequence C1-C9, where C1-C9 is a QQGNSLPNT (SEQ ID NO: 3), and contains a substitution at position C8 (W, Y, R, S, A, F, H, I, L, M, N, T or V).

12. Humanitariannet antibody against beta person containing HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H3 antibodies against beta according to any one of claims 1 to 8, and HVR-H2, which is a variant of the sequence E1-E17, where E1-E17 is a GYISYSGSTSYNPSLKS (SEQ ID NO: 5) and contains a single substitution in any of the following positions: E2 (Y, V, D, or F), E6 (S or G), E10 (S or Y), E12 (N, a, D, or T), E13 (P, D, or H), E15 (L or V) or E17 (S or G).

13. Humanitariannet antibody against beta person containing HVR-L1, HVR-L2, HVR-L3, HVR-H1 and HVR-H2 of the antibodies is against beta according to any one of claims 1 to 8, and HVR-H3, which is a variant of the sequence F2-F11, where F2-F11 is a MTGSSGYFDF (SEQ ID NO: 6) or RTGSSGYFDF (SEQ ID NO: 66) or sequence F1-F11, where F1-F11 is a AMTGSSGYFDF (SEQ ID NO: 63) or ARTGSSGYFDF (SEQ ID NO: 64) and contains one replacement in any of the following provisions: F2 (M, a, E, G, Q, R or S) or F11 (F or Y).

14. Antibody against beta man according to any one of claims 1 to 8, wherein the antibody against beta contains one or more sequences of human and/or human consensus sequences of frame section in the variable domain of their heavy and/or light chain.

15. Antibody against beta person under 14, where the antibody against beta contains at least part or all of the consensus sequence of frame section to subgroup I of man.

16. Antibody against beta person under 14, where the variable domain heavy chain antibodies against beta contains a consensus sequence of frame plot of subgroup III of the person.

17. Antibody against beta person on any of PP-16, where the amino acid at position 71 of the frame section of the heavy chain selected from the group consisting of R, a and T, and/or the amino acid at position 73 of the frame section of the heavy chain selected from the group consisting of N and T, and/or the amino acid at position 78 of the heavy chain selected from the group consisting of F, and L.

18. Antibody against the be the A7 person under 14 where the antibody against beta option contains a consensus sequence frame plot heavy chain subgroup III, containing one or more substitutions R71A, N73T, L78A and/or R94M.

19. Humanitariannet antibody against beta man according to any one of claims 1 to 8, wherein the monovalent affinity of the antibody or its binding fragment to human beta essentially identical to the monovalent affinity to or greater than monovalent affinity of the antibody containing the variable sequence of the light chain and heavy chain, as shown in figa (SEQ ID NO: 10) and figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12) and figv (SEQ ID NO: 13).

20. The antibody or its binding fragment according to claim 19, where the affinity to a maximum of 3 times higher than the affinity of the antibody containing the sequence of the light chain and heavy chain depicted in figa (SEQ ID NO: 10) and figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12) and figv (SEQ ID NO: 13).

21. The antibody or binding fragment according to claim 19, where the monovalent affinity of the antibody to human beta 3 times higher than the monovalent affinity of the antibody containing the sequence of the light chain and heavy chain depicted in figa (SEQ ID NO: 10) and figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12) and figv (SEQ ID NO: 13).

22. The antibody or binding fragment according to claim 19 or 20, where the antibody containing the sequence of the light chain and heavy chain depicted in figa (SEQ IDNO: 10) and figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12) and figv (SEQ ID NO: 13), is obtained using hybridoma cell line deposited in the American type culture collection under the number of ATSS HB-293.

23. The antibody or binding fragment according to claim 19, where the affinity of binding is expressed as Kd values.

24. The antibody or binding fragment according to claim 19, where the affinity of binding is measured by the method of Biacore (TM) or radioimmunoassay method of analysis.

25. Antibody against beta on any of PP-18 monovalent affinity of the antibody to human beta essentially identical to the monovalent affinity to or greater than monovalent affinity to human beta antibodies containing a variable sequence of light chain and heavy chain, as shown in figa (SEQ ID NO: 10) and figv (SEQ ID NO: 11), or figa (SEQ ID NO: 12) and figv (SEQ ID NO: 13).

26. Method of inhibiting the interaction of subunit beta human integrin with a second integrin subunit and/or ligand in vitro by bringing into contact with antibodies against beta according to any one of claims 1 to 25 with bet the integrin.

27. The method according to p, where the second integrin subunit is a subunit alpha integrin, and where the ligand is a MAdCAM, VCAM or fibronectin.

28. The method according to item 27, where alpha-integrin subunit is a human.

29. The method according to p, where the ligand is a human.

30. The method according to clause 29, where the second integrin subunit is algae-subunit of integrin and where the ligand is an E-cadherin.

31. The method according to item 30, where algae-integrin subunit is a human.

32. The method according to p, where the ligand is a human.

33. Antibody against beta according to any one of claims 1 to 25 for therapeutic and/or prophylactic treatment of inflammatory diseases associated with increased expression or activity of integrin beta or increase the interaction between integrin beta on one cell and receptor integrin beta to another cell.

34. Antibody against beta according to any one of claims 1 to 25 for therapeutic and/or prophylactic treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory liver disease, CNS inflammation, chronic pancreatitis, systemic lupus erythematosus, Sjogren syndrome, psoriasis and skin inflammation, asthma, chronic obstructive pulmonary diseases, interstitial pulmonary, Allergy, autoimmune diseases, transplant rejection, kidney transplant rejection, graft versus host disease, diabetes or cancer.

35. The use of antibodies against beta according to any one of claims 1 to 25 for the preparation of a medicinal product for therapeutic and/or prophylactic L. the treatment of inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory liver disease, CNS inflammation, chronic pancreatitis, systemic lupus erythematosus, Sjogren syndrome, psoriasis and skin inflammation, asthma, chronic obstructive pulmonary diseases, interstitial pulmonary, Allergy, autoimmune diseases, transplant rejection, kidney transplant rejection, graft versus host disease, diabetes or cancer.

36. Antibody against beta according to any one of claims 1 to 25 for use in the method of inhibiting the interaction of subunit beta human integrin with a second integrin subunit and/or ligand by contacting with beta the integrin, where inhibition reduces or alleviates symptoms of disorders selected from the group consisting of inflammation, asthma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, diabetes, inflammation accompanying organ transplantation, graft versus host and inflammation associated with violations of allotransplantation.

37. The use of antibodies against beta according to any one of claims 1 to 25 for the preparation of a medicinal product for use in the method of inhibiting the interaction of subunit beta human integrin with a second integrin subunit and/or ligand by contacting with beta what negrino, where inhibition reduces or alleviates symptoms of disorders selected from the group consisting of inflammation, asthma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, diabetes, inflammation accompanying organ transplantation, graft versus host and inflammation associated with violations of allotransplantation.

38. A composition comprising an effective amount of antibodies against beta according to any one of claims 1 to 25 and a pharmaceutical carrier for use in the method of inhibiting integrin beta-mediated adhesion and/or recruitment of cells in a mammal suffering from a disorder associated with abnormal or unwanted signal transmission mediated by integrin beta.

39. The composition according to § 38, where the violation is selected from the group consisting of inflammation, asthma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, diabetes, inflammation accompanying organ transplantation, graft versus host and inflammation associated with violations of allotransplantation.

40. The composition according to § 38 or 39, where the mammal is man.

41. Composition according to any one of p-39, where the composition further comprises a second biopharmaceutical agent or chemotherapeutic agent.

42. Composition according to any one of p-41, where the way of Saint who is associated with the inhibition of the interaction beta integrin with alpha integrin, alphae integrin, MAdCam, VCAM, E-cadherine and/or fibronectin.

43. The use of a composition containing an antibody against beta according to any one of claims 1 to 25 and a pharmaceutical carrier for the preparation of a medicinal product for use in the methods of modulating integrin beta-mediated adhesion and/or recruitment of cells in a mammal suffering from a disorder associated with abnormal or unwanted signal transmission mediated by integrin beta.

44. Use item 43, where the violation is selected from the group consisting of inflammation, asthma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, diabetes, inflammation accompanying organ transplantation, graft versus host and inflammation associated with violations of allotransplantation.

45. Use item 43 or 44, where the mammal is man.

46. The use according to any one of p-45, where the composition further comprises a second biopharmaceutical agent or chemotherapeutic agent.

47. The use according to any one of p-46, where the modulation is associated with inhibition of the interaction beta integrin with alpha integrin, alphae integrin, MAdCam, VCAM, E-cadherine and/or fibronectin.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: this invention relates to biotechnology and immunology. One proposes: JAM-A protein antibody or functional fragment thereof, hybridoma secreting such antibody, nucleic acid, expression vector and host cell as well as a method for the antibody and composition production. One considers application of the JAM-A protein antibody or functional fragment thereof.

EFFECT: invention usage ensures creation of new JAM-A protein antibodies which may be further applied in treatment or prevention of diseases related to proliferation of tumour cells extracting JAM-A protein.

34 cl, 31 dwg, 5 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: there are presented a polynucleotide coding an antibody, an expression vector, a host cell, compositions containing the antibody, and also a method for preparing the antibody and methods for using the antibody.

EFFECT: invention may be used for preparing therapeutic and diagnostic agents to be used for the purpose of detecting the pathological conditions associated with expression or activity of ephrine B2 ligand pathways.

27 cl, 10 dwg, 3 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a polynucleotide encoding the antibody, an expression vector, a host cell, compositions containing the antibody, methods of producing the antibody, a method of detecting EphB4, a method of diagnosing disorders associated with EphB4 expression, as well as use of the antibody to produce a drug.

EFFECT: invention can be effectively used to diagnose and treat diseases associated with EphB4 expression.

36 cl, 8 dwg, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is an antibody or its antigen-binding fragment which specifically coupling hlL-4R with KD less than 200-pM measured with using surface plasmon resonance. What is described is a recovered nucleic acid molecule coding the antibody, and a based vector for producing the antibody. There are disclosed a host-vector system for producing the antibody or its antigen-binding fragment, and a method for producing the substances stated above with using such system. What is disclosed is using the antibody or antigen-binding fragment for preparing a drug for relieving (inhibiting) hlL-4R mediated diseases. What is disclosed is a composition on the basis of the antibody or antigen-binding fragment to be used in a method for treating a hlL-4R mediated disease or disorder in humans.

EFFECT: inventions can find application in therapy of the hlL-4R mediated diseases.

15 cl, 3 dwg, 5 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a drug for multiple sclerosis which is presented in the form of a pharmaceutical composition and contains an activated potentiating form of human gamma-interferon (IFN-γ) antibodies and an activated potentiating form of brain-specific protein S-100 antibodies as an additive intensifying component. Using the drug in a method for treating multiple sclerosis involves in introduction of the activated potentiating form of human gamma-interferon (IFN-γ) in combination with the intensifying component in the form of the activated potentiating form of very-low-dose affinity purified brain-specific protein S-100 antibodies.

EFFECT: use of the inventions allows higher clinical effectiveness in multiple sclerosis.

9 cl, 1 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: described is antibody or one of its functional fragments, which have high affinity to receptor of human insulin-like growth factor (IGF-IR) and inhibiting binding of IGFI with IGF-IR and binding of IGF2 with IGF-IR. Claimed is hybridoma, producing described antibody. Invention also includes application of antibody as therapeutic means for preventive and/or therapeutic treatment of cancer, during which takes place overexpression of IGF-IR, stimulated either by IGF-1, and/or by IGF-2, or any other pathology, connected with overexpression of said receptor, as well as in methods or in sets for diagnostics of diseases, associated with overexpression of receptor IGF-IR and/or hybrid receptor IGF-1/Insulin.

EFFECT: invention makes it possible to obtain antibody, which specifically with high affinity recognises IGF-IR and which also inhibits not only binding of IGF1 with IGF-IR, but also binding of IGF2 with IGF-IR.

51 cl, 11 dwg, 4 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology and biotechnology. Claimed are: versions of antibody and antigen-binding fragments of antibody to receptor Il-6 of humans. Considered are: isolated molecule of nucleic acid and vector which contains it. Described are: system "host-vector" and method of obtaining antibody or its antigen-binding fragment, as well as application of antibody or its antigen-binding fragment for obtaining medication.

EFFECT: invention application provides novel antibodies to receptor IL-6 of humans, which can be applied in therapy of IL-6- mediated diseases.

11 cl, 5 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, and represents a polyclonal Nogo antibody. Also, there are presented a pharmaceutical composition and application of the antibody for preparing a drug.

EFFECT: invention can effectively used for treating central nervous system disorders.

6 cl, 79 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: what is described is an antibody molecule which selectively binds with a cellular surface ICAM-1 and if bound with ICAM-1, induces apoptosis in a target cell, and a nucleic acid molecule coding said antibody. Also, methods and pharmaceutical compositions for apoptosis induction and application thereof are presented.

EFFECT: invention allows producing molecules for cell apoptosis induction.

33 cl, 11 dwg, 3 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to an amine compound of formula (I), pharmaceutically acceptable addition salts, hydrates or solvates thereof, having immunodepressive effect , where R - H or P(=O)(OH)2; X - O or S; Y denotes -CH2CH2- or -CH=CH-; Z denotes C1-5-alkylene, C2-5-alkenylene or C2-5-alkynylene; R1 denotes CF3, R2 denotes C1-4-alkyl, substituted with OH or halogen; R3 and R4 independently denotes H < or C1-4-alkyl; A denotes optionally substituted C6-10-aryl, heteroaryl containing 5-10 ring atoms, where 1 or 2 atoms are selected from N, O and S, C3-7-cycloalkyl optionally condensed with optionally substituted benzene, or heterocycloalkyl containing 5-7 ring atoms, where 1 or 2 atoms are selected from N and O, where said substitutes are selected from C1-4-alkylthio, C1-4-alkylsulphanyl, C1-4-alkylsulphonyl, C2-5-alkylcarbonyl, halogen, cyano, nitro, C3-7-cycloalkyl, C6-10-aryl, C7-14-aralkyloxy, C6-10-aryloxy, optionally substituted with oxo or halogen, C2-3-alkyleneoxy, C3-4-alkylene or C1-2-alkylenedioxy, optionally substituted with halogen C1-4-alkyl or C1-4-alkoxy.

EFFECT: novel compound which is effective in reducing the level of lymphocytes in peripheral blood, suppresses tissue breakdown and exhibiting less side effects, such as bradycardia, is disclosed.

20 cl, 237 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely reflex therapy. A method involves biologically active zone exposure to a flame generated by burning of a wet cotton tincture. It is preceded by steaming the cotton in a herbal infusion, rolling it and used to massage. Then the cotton is smoothed down and placed on an exposure area. It is covered with bands impregnated with an alcoholate and set fire. A burning time is 7 to 30 seconds. The burning time and a number of setting fire is specified on the basis of individual tolerance.

EFFECT: method provides reduced length of body exposure required in various pathological conditions and ensured by improved skin penetration of biologically active substances contained in the herbal infusions.

2 tbl, 3 cl

Polycyclic compound // 2451685

FIELD: medicine, pharmaceutics.

SUBSTANCE: described is a new polycyclic compound with general formula (I-1) and (1-3) or a pharmaceutically acceptable salt thereof where X1- -CR1 =CR2 - where R1 and R2 independently stand for hydrogen or C1-6 alkyl while Het stands for a radical of the following formulae: that may be substituted 1-3 times additionally described is a pharmaceutical composition containing such compound and intended for prevention or treatment of diseases caused by β-amyloid.

EFFECT: production of a pharmaceutical composition prevention or treatment of diseases caused by β-amyloid.

7 cl, 392 ex, 12 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new compounds of formula

, where X1, X2, Y, R1a, R1b, R2a, R2b, A1, A2, A3 and A4 have the values specified in the description, which are vanilloid receptor subtype 1 (VR1) antagonists.

EFFECT: preparing a pharmaceutical composition on the basis of the compounds of formula 1 and developing methods of managing pain, neurotic pain, allodynia, inflammation or inflammatory disease associated pain, inflammatory hyperalgesia, bladder hyperactivity and urine incontinence.

22 cl, 21 ex

Gsk-3 inhibitors // 2449998

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns applying urea derivatives or their pharmaceutically acceptable salts characterised by formula , wherein RB is specified in: while R3, R4, R'2, R'3, R'4, R'5, and R'6 represent hydrogen as GSK-3 inhibitors, pharmaceutical compositions containing them, and using them for treating and/or preventing disorders the development of which involves GSK-3.

EFFECT: preparing the pharmaceutical compositions containing them, and using them for treating and/or preventing disorders the development of which involves GSK-3.

14 cl, 2 ex, 1 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are offered a combination for preventing or treating a proliferative disease, containing a microtubular active agent epothilone B and a pharmaceutically active agent, 1H-pyrazolo[3,4-d]pyrimidine-4-amine 3-(4-chloriophenyl)-1-(1,1-dimethylethyl)-(9CI); the related pharmaceutical composition containing said combination, methods of preventing and treating (versions), a commercial packing including said combination (versions). Particularly, the proliferative disease can represent ovarian cancer, carcinoma of lung and melanoma.

EFFECT: what is shown is synergism of the combination in growth inhibition of non-small cell carcinoma of lung in relation to independent action of its ingredients.

17 cl, 3 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and may be applied as an agent for prosthetic stomatitis accompanying a denture adaptation period. It involves mouth rinsing with 0.2% aqueous acetylsalicylic acid. The regimen is at least three times a day before meals. The length is at least five days.

EFFECT: method enables decreasing saliva viscosity and surface force, enhancing its wetting properties, providing a shorter denture adaptation period.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to preparing drugs in the form of a herbal ointment for fibrocystic disease (mastopathy). As active substances, the ointment contains an alcoholic extract of milk-wax green walnut and pericarp, walnut and buckeye leaves in equal proportions of the active substances, an ointment base containing vegetable oil, emulsion wax, interior fat, purified water and parabens.

EFFECT: ointment provides hormone balance control in females.

3 cl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically cardiosurgery, and may be used for treating thromboembolia of pulmonary artery. For this purpose a surgical intervention accompanied by artificial circulation involves mechanical thrombus removal from greater branches of the pulmonary artery. It is followed by exposing a left atrium and conducting a retrograde pulmonary perfusion by sequential perfusion of each of the four pulmonary veins for 4 minutes by a combination solution. The combination solution is prepared by mixing blood from a pump oxygenator and a protective solution in proportions 3:1. The protective solution consists of 0.9% sodium chloride 800 ml, dexamethasone 16 ml, actovegin 400 ml and 15% mannitol 100 ml. The perfusion procedure is conducted at volumetric speed 200-250 ml/min and perfusion pressure 20 mm Hg. Thromboemboles are removed from peripheral parts of the pulmonary artery by aspiration of blood stream from the incised pulmonary artery. The procedure is terminated with hermetically closed wound of the pulmonary artery. A left atrium cavity is closed with recovering cardiac activity, and the incisional wound is closed in layers.

EFFECT: method provides considerably reduced death rate and risk of postoperative complications in the case patients, as well as provided effective removal of small thromboembols from the peripheral parts of the pulmonary artery and prevented developing air embolism of the pulmonary artery.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely ophthalmology. A method involves introducing a photosensitiser (PS) in a patient's body that is followed by retinal exposure to laser light. Chlorine photosensitiser 0.4-0.5 mg/kg is intravenously injected for 10 minutes. Immediately after the PS introduction, ischemic areas and retinal neovascularisation areas are exposed to transpupillary laser light. That is ensured by laser light at wave length matched to maximum PS adsorption of optical emission. Power density makes 50-75 J/cm2.

EFFECT: method provides the absence of damaging action on the surrounding functional retinal areas, the absence of hemorrhagic complications and developing exudative retinal detachment, stabilises proliferative diabetic retinopathy and enables conducting the whole therapeutic volume for one session ensured by graduated retinal exposure to laser light that covers ischemic areas and retinal neovascularisation areas.

1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to peptide-based compounds containing three-member rings containing a heteroatom, which efficiently and selectively inhibit specific activity of N-terminal nucleophilic (Ntn) hydrolase, bonded with a proteasome. The peptide-based compounds contain epoxide and are functionalised at the N-end.

EFFECT: peptide-based compounds exhibit anti-inflammatory properties and cell proliferation inhibition, oral administration of said peptide-based proteasome inhibitors is possible owing to bioavailability thereof.

23 cl, 14 ex

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