RU2004113373A

RU2004113373A - ANTIBODIES AGAINST HUMAN TISSUE FACTOR

Info

Publication number: RU2004113373A
Application number: RU2004113373/13A
Authority: RU
Inventors: Пер-Ола ФРЕСКГОРД (SE); Пер-Ола ФРЕСКГОРД; Ес Торн КЛАУСЕН (DK); Ес Торн КЛАУСЕН; Брит Биноу СЕРЕНСЕН (DK); Брит Биноу СЕРЕНСЕН; Марианне КЬЯЛЬКЕ (DK); Марианне КЬЯЛЬКЕ
Original assignee: Ново Нордиск А/С (DK); Ново Нордиск А/С
Priority date: 2001-10-02
Filing date: 2002-09-30
Publication date: 2005-03-27
Also published as: CN1575302A; HUP0401658A2; WO2003029295A1; CZ2004454A3; EP1434802A1; BR0213046A; IL160998A0; MXPA04003051A; ZA200402303B; CA2460917A1; PL368989A1; JP2005512970A; KR20040045478A; NO20041802L

Claims

1. An isolated human antibody that immunologically interacts with an epitope present on human TF.

2. The isolated human antibody according to claim 1, characterized in that it inhibits the binding of human coagulation factor VIIa to human TF.

3. The selected human antibody according to any one of paragraphs. 1 and 2, characterized in that it is a monoclonal antibody.

4. The selected human antibody according to claim 1, characterized in that it is a recombinant antibody.

5. The selected human antibody according to claim 1, characterized in that said antibody is selected from the group consisting of a Fab fragment, an F (ab) ₂ fragment, an F (ab ') ₂ fragment and a single chain Fv fragment.

6. The selected human antibody according to claim 1, characterized in that said antibody is characterized by K _d binding to human TF in the range of 10 ^-15 -10 ^-8 M, in the range of 10 ^-15 -10 ^-10 M.

7. A pharmaceutical composition comprising a therapeutically effective amount of a human antibody that immunologically interacts with an epitope present on human TF.

8. The pharmaceutical composition according to claim 7, characterized in that said antibody is an antibody according to any one of claims 1 to 6.

9. A method of treating a disorder associated with FVIIa / TF in humans, comprising administering to said person a therapeutically effective amount of a human antibody that immunologically interacts with an epitope present on human TF.

10. The method according to claim 9, characterized in that said antibody is an antibody according to any one of paragraphs. 1-6.

11. A method for producing a human antibody, comprising a) obtaining human antibodies against human TF, b) testing antibodies in an TF-induced coagulation assay and selecting a human antibody that inhibits clot formation in this assay with an IC ₅₀ value lower than the IC ₅₀ value of FFR- rFVIIa + 1 nM, such as less than an IC ₅₀ value of FFR-rFVIIa + 500 pM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 200 pM, preferably less than an IC value of ₅₀ FFR-rFVIIa + 100 pM, preferably less than IC ₅₀ FFR-rFVIIa + 50 pM, preferred but less than an IC ₅₀ value of FFR-rFVIIa + 10 pM, more preferably less than an IC ₅₀ value of FFR-rFVIIa + 5 pM, more preferably less than an IC ₅₀ value of FFR-rFVIIa, or antibody testing in an FXa generation assay and selection a human antibody that inhibits the generation of FXa with an IC ₅₀ value less than an IC ₅₀ value of FFR-rFVIIa + 100 nM (using 0.1 nM FVIIa), such as less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 5 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 1 nM, more preferably less than an IC ₅₀ value of FFR-rFVIIa + 0.1 nM, more preferably less than an IC ₅₀ value of FFR-rFVIIa, or antibody testing in an FVIIa / TF amidolytic assay and selection of a human antibody that inhibits TF-induced amidolytic activity of FVIIa with an IC ₅₀ value less than an IC ₅₀ value of FFR-rFVIIa + 100 nM (using 10 nM FVIIa in this assay), such as less than an IC ₅₀ value of FFR-rFVIIa + 40 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 20 nM, preferably lower than the IC ₅₀ value of FFR-rFVIIa + 10 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa, if testing antibodies in a competitive assay c FVIIa and selecting human antibodies which competes for binding to FVIIa, or testing antibodies in ELISA TF assay comprising TF, and selecting human antibody which immunologically reacts with human TF.

12. The method according to claim 11, characterized in that human antibodies against human TF are obtained by a method comprising a) immunizing a mammal with human TF, b) isolating antibodies produced by an immunized mammal, such as a mouse.

13. The method according to any one of paragraphs. 11 and 12, characterized in that it includes antibody testing in the TF-induced coagulation assay and selection of a human antibody that inhibits clot formation in this assay with an IC ₅₀ value lower than an IC ₅₀ value of FFR-rFVIIa + 1 nM, such as a lower than an IC ₅₀ value of FFR-rFVIIa + 500 pM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 200 pM, preferably less than an IC value of ₅₀ FFR-rFVIIa + 100 pM, preferably less than an value of IC ₅₀ FFR-rFVIIa + 50 pM, preferably less than IC ₅₀ FFR-rFVIIa + 10 pM, more preferably less than the value of the IC ₅₀ FFR-rFVIIa + 5 PM, more preferably less than the value of the IC ₅₀ FFR-rFVIIa.

14. The method according to p. 11, characterized in that it includes antibody testing in the analysis of the generation of FXa and selection of a human antibody that inhibits the generation of FXa with an IC ₅₀ value lower than the IC ₅₀ value of FFR-rFVIIa + 100 nM (using 0 in the analysis , 1 nM FVIIa), such as less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 5 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 1 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa + 0.1 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa.

15. The method according to p. 11, characterized in that it includes testing the antibodies in the FVIIa / TF amidolytic assay and selecting a human antibody that inhibits TF-induced amidolytic activity of FVIIa with an IC ₅₀ value lower than the IC ₅₀ value of FFR-rFVIIa + 100 nM ( c in the assay using 10 nM FVIIa), such as less than an IC ₅₀ value of FFR-rFVIIa + 40 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 20 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa.

16. The method according to p. 11, characterized in that it includes testing the antibodies in a competitive analysis with FVIIa and the selection of a human antibody that competes for binding to FVIIa.

17. The method according to p. 11, characterized in that it includes testing the antibodies in the ELISA TF analysis, including TF, and the selection of a human antibody that immunologically interacts with human TF.

18. A human antibody that immunologically interacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtained by a method comprising a) obtaining human anti-human TF antibodies, b) testing antibodies in an analysis of TF induced clotting and selection of a human antibody that inhibits clot formation in this assay with an IC ₅₀ value less than an IC ₅₀ value of FFR-rFVIIa + 1 nM, such as less than an IC ₅₀ value of FFR-rFVIIa + 500 pM, presumably significantly less than IC ₅₀ FFR-rFVIIa + 200 pM, preferably less than IC ₅₀ FFR-rFVIIa + 100 pM, preferably less than IC ₅₀ FFR-rFVIIa + 50 pM, preferably less than IC ₅₀ FFR -rFVIIa + 10 pM, more preferably less than the IC ₅₀ value of FFR-rFVIIa + 5 pM, more preferably less than the IC ₅₀ value of FFR-rFVIIa, or antibody testing in the FXa generation assay and selection of a human antibody that inhibits the generation of FXa co IC ₅₀ value less than the IC ₅₀ value of FFR-rFVIIa + 100 nM (using 0.1 nM in the assay FVIIa), such ka smaller than the value of IC ₅₀ FFR-rFVIIa + 10 nM, preferably lower than the IC ₅₀ FFR-rFVIIa + 5 nM, preferably lower than the IC ₅₀ FFR-rFVIIa + 1 nM, more preferably lower than the IC ₅₀ FFR -rFVIIa + 0.1 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa, or antibody testing in the FVIIa / TF amidolytic assay and selection of a human antibody that inhibits TF-induced amidolytic activity of FVIIa with an IC ₅₀ value less than the IC value ₅₀ FFR-rFVIIa + 100 nM (using 10 nM FVIIa), such as a lower value than IC ₅₀ FFR-rFVIIa + 40 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 20 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, more preferably less than an IC ₅₀ value of FFR-rFVIIa or antibody testing in competitive analysis with FVIIa and selection a human antibody that competes for binding to FVIIa, or antibody testing in an ELISA TF including TF, and selection of a human antibody that immunologically interacts with human TF.

19. A method for producing a human antibody, comprising a) immunizing a mouse with human TF, b) isolating an antibody-producing cell from an immunized mouse and obtaining immortalized cells, such as a hybridoma cell, to secrete human antibodies, c) separating from the immortalized cells a culture medium containing produced antibodies, d) antibody testing in indirect ELISA TF analysis, including TF in solution, and selection of human antibodies that immunologically interact in solution with human TF, e) test vanie antibodies in a competitive assay with a FVIIa and selecting human antibody which compete for binding to FVIIa, f) testing antibodies in the amidolytic assay FVIIa / TF and selecting human antibodies which inhibits induced TF amidolytic activity of FVIIa with IC value ₅₀ lower than the IC ₅₀ FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), such as less than an IC ₅₀ value of FFR-rFVIIa + 40 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 20 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, more preferably less, than the IC ₅₀ value of FFR-rFVIIa, g) antibody testing in the analysis of FXa generation and selection of a human antibody that inhibits the generation of FXa with an IC ₅₀ value lower than the IC ₅₀ value of FFR-rFVIIa + 100 nM (using 0.1 nM in the analysis FVIIa), such as less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 5 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 1 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa + 0.1 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa, h) antibody testing in the analysis of inducers clotting TF and selection of a human antibody that inhibits clot formation in this assay with an IC ₅₀ value lower than an IC ₅₀ value of FFR-rFVIIa + 1 nM, such as a lower value than an IC ₅₀ value of FFR-rFVIIa + 500 pM, preferably lower than the IC ₅₀ value of FFR-rFVIIa + 200 pM, preferably less than the IC ₅₀ value of FFR-rFVIIa + 100 pM, preferably less than the value of IC ₅₀ FFR-rFVIIa + 50 pM, preferably less than the value of IC ₅₀ FFR-rFVIIa + 10 pM, more preferably less than the IC ₅₀ value of FFR-rFVIIa + 5 pM, more preferably less than the IC ₅₀ value of FFR-rFVIIa, i) o collecting and culturing in a suitable culture medium an immortalized cell producing the selected antibody, in accordance with steps dh, j) isolating the selected antibody from the culture medium of the selected immortalized cell.

20. The method according to claim 19, characterized in that it further includes testing the antibodies in a direct ELISA TF analysis, including immobilized TF, and the selection of a human antibody that immunologically interacts with immobilized human TF.

21. The method according to any one of paragraphs. 19 and 20, characterized in that it further includes testing the antibodies in the analysis of the generation of FXa on the TF expressing cell and the selection of a human antibody that inhibits the generation of Fxa on the TF expressing cell with an IC ₅₀ value lower than the IC ₅₀ value of FFR-rFVIIa + 500 nM ( using 1 nM FVIIa in the assay), such as less than an IC ₅₀ value of FFR-rFVIIa + 100 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 50 nM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 10 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa + 5 nM, more Preference Tel'nykh less than the IC ₅₀ value of FFR-rFVIIa.

22. The method according to p. 19, characterized in that it further includes testing the antibodies in the analysis of binding of TF to whole cells and the selection of human antibodies that compete for binding of FVIIa to human TF expressed on the cell surface of whole cells.

23. The method according to p. 19, characterized in that it further includes testing the antibodies in a biosensor analysis and selecting human antibodies with K _d binding of human TF less than 100 nM, such as less than 10 nM, preferably less than 5 nM preferably less than 1 nM, more preferably less than 0.5 nM.

24. The method according to p. 19, characterized in that it further includes testing the antibodies in the analysis of MAPK signal transmission and the selection of human antibodies that inhibit FVIIa-induced activation of MAPK signal transmission.

25. The method according to p. 19, characterized in that it further includes testing the antibodies in the analysis of epitope mapping and selection of human antibodies that immunologically interact with preferred epitopes on TF.

26. The method according A.25, characterized in that the preferred epitope includes the remains of Trp45, Lys46 and Tyr94.

27. A human antibody that immunologically interacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtained by a method comprising a) immunizing a mouse with human TF, b) isolating an antibody-producing cell from an immunized mouse and obtaining immortalized cells for the secretion of human antibodies, c) separation from immortalized cells of the culture medium containing the produced antibodies, d) antibody testing in indirect analysis ELISA TF, including TF in solution, and selection of human antibodies that immunologically interact in solution with human TF, e) testing of antibodies in competition with FVIIa and selection of human antibodies that compete for binding to FVIIa, f) testing of antibodies in amidolytic analysis FVIIa / TF and selection of a human antibody that inhibits TF-induced amidolytic activity of FVIIa with an IC ₅₀ value less than the IC ₅₀ FFR-rFVIIa + 100 nM (using 10 nM FVIIa in the assay), such as less than the IC ₅₀ FFR -rFVIIa + 40 nM, preferably less than IC ₅₀ FFR-rFVIIa + 20 nM, preferably less than IC ₅₀ FFR-rFVIIa + 10 nM, more preferably less than IC ₅₀ FFR-rFVIIa, g) antibody testing in FXa generation assay and selection a human antibody that inhibits the generation of FXa with an IC ₅₀ value less than the IC ₅₀ value of FFR-rFVIIa + 100 nM (using 0.1 nM FVIIa in the assay), such as less than the IC ₅₀ value of FFR-rFVIIa + 10 nM preferably smaller than the value of IC ₅₀ FFR-rFVIIa + 5 nM, preferably lower than the IC ₅₀ value of FFR-rFVIIa + 1 nM, more prefer no less than the IC ₅₀ value of FFR-rFVIIa + 0,1 nM, more preferably less than the IC ₅₀ value of FFR-rFVIIa, h) testing antibodies in a TF-induced clotting assay and selecting human antibody which inhibit clot formation in this assay with an IC ₅₀ value less than an IC ₅₀ value of FFR-rFVIIa + 1 nM, such as less than an IC ₅₀ value of FFR-rFVIIa + 500 pM, preferably less than an IC ₅₀ value of FFR-rFVIIa + 200 pM, preferably less than IC ₅₀ value of FFR-rFVIIa + 100 pM, preferably lower than the IC ₅₀ value of FFR-rFVIIa + 50 pM, preferably less than h Achen IC ₅₀ FFR-rFVIIa + 10 pM, more preferably lower than the IC ₅₀ FFR-rFVIIa + 5 pM, more preferably lower than the IC ₅₀ FFR-rFVIIa, i) selection and cultivation in a suitable culture medium immortalized cell producing the selected antibody, in accordance with stages dh, j) the selection of the selected antibodies from the culture medium of the selected immortalized cells.

28. A human antibody that immunologically interacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF, obtained by the method according to any one of claims. 19-26.

29. A cell producing a human antibody that immunologically interacts with an epitope present on human TF and inhibits the binding of human coagulation factor VIIa to human TF.

30. The cell according to clause 29, wherein the specified cell is an isolated lymphoid cell.

31. A cell according to any one of paragraphs. 29 and 30, characterized in that said cell is isolated from a mouse.

32. The cell according to clause 29, wherein the specified cell is a hybridoma cell.

33. The cell according to clause 29, wherein said hybridoma cell is obtained by fusion of an antibody-producing lymphoid cell with an immortalized cell to obtain an antibody-producing hybridoma cell.

34. The cell of claim 29, wherein said antibody inhibits the binding of human coagulation factor VIIa to human TF.

35. The cell of claim 29, wherein said antibody immunologically interacts with a 3-dimensional surface, in the formation of which all Trp45, Lys46 and Tyr94 residues are involved.

36. The cell of claim 29, wherein said antibody is selected from the group consisting of a Fab fragment, an F (ab) ₂ fragment, an F (ab ') ₂ fragment, and a single chain Fv fragment.

37. The cell of claim 29, wherein said antibody is characterized by K _d binding to human TF in the range of 10 ^-15 -10 ^-8 M, in the range of 10 ^-15 -10 ^-10 M.