Methods of treatment that include the administration of bispecific antibodies

Patent metadata

Field	Value
Publication number	US-10450381-B2
Application number	US-201615132996-A
Country	US
Kind code	B2
Filing date	Apr 19, 2016
Priority date	Nov 17, 2010
Publication date	Oct 22, 2019
Grant date	Oct 22, 2019

Abstract

Various bispecific antibodies that specifically bind to both blood coagulation factor IX/activated blood coagulation factor IX and blood coagulation factor X and functionally substitute for the cofactor function of blood coagulation factor VIII, that is, the function to promote activation of blood coagulation factor X by activated blood coagulation factor IX, were produced. From these antibodies, multispecific antigen-binding molecules having a high activity of functionally substituting for blood coagulation factor VIII were successfully discovered.

First claim

The invention claimed is:

1. A method of treating bleeding or reducing the incidence of bleeding in a subject, the method comprising administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises a bispecific antibody that binds to blood coagulation factor IX and/or activated blood coagulation factor IX, and binds to blood coagulation factor X, wherein the bispecific antibody comprises:
a first antibody H chain comprising a variable region comprising complementarity determining regions (CDRs) 1, 2, and 3 that comprise SEQ ID NOs: 105-107, respectively;
a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 126-128, respectively; and
first and second antibody L chains, each L chain comprising a variable region comprising CDRs 1, 2, and 3 that comprise SEQ ID NOs: 156-158, respectively.

2. The method of claim 1 , wherein:
CDRs 1, 2, and 3 of the first antibody H chain consist of SEQ ID NOs: 105-107, respectively;
CDRs 1, 2, and 3 of the second antibody H chain consist of SEQ ID NOs: 126-128, respectively; and
CDRs 1, 2, and 3 of each L chain consist of SEQ ID NOs: 156-158, respectively.

3. The method of claim 1 , wherein the first antibody H chain variable region comprises SEQ ID NO: 45.

4. The method of claim 1 , wherein the second antibody H chain variable region comprises SEQ ID NO: 52.

5. The method of claim 1 , wherein the first and second antibody L chain variable regions each comprise SEQ ID NO: 62.

6. A method of treating bleeding or reducing the incidence of bleeding in a subject, the method comprising administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises a bispecific antibody comprising:
a first antibody H chain comprising a variable region comprising SEQ ID NO: 45;
a second antibody H chain comprising a variable region comprising SEQ ID NO: 52; and
first and second antibody L chains, each L chain comprising a variable region comprising SEQ ID NO: 62.

7. The method of claim 6 , wherein:
the first antibody H chain variable region consists of SEQ ID NO: 45;
the second antibody H chain variable region consists of SEQ ID NO: 52; and
the first and second antibody L chain variable regions each consist of SEQ ID NO: 62.

8. The method of claim 1 , wherein the first antibody H chain consists of SEQ ID NO: 20.

9. The method of claim 1 , wherein the second antibody H chain consists of SEQ ID NO: 25.

10. The method of claim 1 , wherein the first and second antibody L chains each consist of SEQ ID NO: 32.

11. The method of claim 6 , wherein:
the first antibody H chain comprises SEQ ID NO: 20;
the second antibody H chain comprises SEQ ID NO: 25; and
the first and second antibody L chains each comprise SEQ ID NO: 32.

12. The method of claim 11 , wherein:
the first antibody H chain consists of SEQ ID NO: 20;
the second antibody H chain consists of SEQ ID NO: 25; and
the identical first and second antibody L chains each consist of SEQ ID NO: 32.

13. The method of claim 1 , wherein the bispecific antibody:
(a) has increased activated blood coagulation factor X generation-promoting activity as compared to a reference antibody that consists of: (i) an antibody H chain consisting of SEQ ID NO: 165, (ii) an antibody H chain consisting of SEQ ID NO: 166, and (iii) two antibody L chains, each consisting of SEQ ID NO: 167; or
(b) exhibits less inhibition of F.Xase activity as compared to the reference antibody; or
(c) both (a) and (b).

14. The method of claim 1 , wherein, when activated blood coagulation factor X generation-promoting activity of a test sample comprising the bispecific antibody is determined in an assay in which the readout is absorbance at 405 nm, the test sample produces an absorbance readout that is more than 0.4 greater than the absorbance at 405 nm produced from an identically-assayed control sample, wherein the control sample is identical to the test sample except lacking any antigen-binding molecule, and wherein the assay comprises steps (a)-(e):
(a) incubating for 30 minutes at room temperature a first reaction mixture comprising: either (i) 5 μL of the test sample in which 300 μg/mL of the bispecific antibody is diluted in Tris-buffered saline containing 0.1% bovine serum albumin, or (ii) 5 μL of the control sample; 2.5 μL of 27 ng/mL human factor IXa beta in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 ; and 2.5 μL of 6 IU/mL human blood coagulation factor IX in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 ;
(b) immediately after the incubation of (a), adding to the first reaction mixture 5 μL of 24.7 μg/mL human factor X in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 , to generate a second reaction mixture, and incubating the second reaction mixture for 10 minutes at room temperature;
(c) immediately after the incubation of (b), adding to the second reaction mixture 5 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) in water, to generate a third reaction mixture;
(d) immediately after (c), adding to the third reaction mixture 5 μL of 1.47 mg/mL N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroaniline hydrochloride in purified water, to generate a fourth reaction mixture, and incubating the fourth reaction mixture for 50 minutes at room temperature; and
(e) immediately after the incubation of (d), determining the absorbance of the fourth reaction mixture at 405 nm.

15. The method of claim 1 , wherein, when inhibition of F.Xase activity of a test sample comprising the bispecific antibody encompasses inhibition as determined in an assay in which the readout is absorbance at 405 nm, the test sample produces an absorbance readout that is greater than the absorbance at 405 nm produced from an identically-assayed control sample, wherein the control sample is identical to the test sample except lacking any antigen-binding molecule, and wherein the assay comprises steps (a)-(f):
(a) incubating for 30 minutes at room temperature a first reaction mixture comprising: either (i) 5 μL of the test sample in which 300 μg/mL of the bispecific antibody is diluted in Tris-buffered saline containing 0.1% bovine serum albumin, or (ii) 5 μL of the control sample; and 2.5 μL of 80.9 ng/mL human factor IXa beta in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 ;
(b) immediately after the incubation of (a), adding to the first reaction mixture 2.5 μL of 1.8 IU/mL F.VIIIa in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 , to generate a second reaction mixture, and incubating the second reaction mixture for 30 seconds at room temperature;
(c) immediately after the incubation of (b), adding to the second reaction mixture 5 μL of 24.7 μg/mL human factor X in Tris-buffered saline containing 0.1% bovine serum albumin, 93.75 μM synthetic phospholipid solution, 7.5 mM CaCl 2 , and 1.5 mM MgCl 2 , to generate a third reaction mixture, and incubating the third reaction mixture for six minutes at room temperature;
(d) immediately after the incubation of (c), adding to the third reaction mixture 5 μL of 0.5 M EDTA, to generate a fourth reaction mixture;
(e) immediately after (d), adding to the fourth react

Assignees

• Chugai Pharmaceutical Co Ltd

Inventors

• Igawa Tomoyuki
• Sampei Zenjiro
• Kojima Tetsuo
• Soeda Tetsuhiro
• Muto Atsushi
• Kitazawa Takehisa
• Nishida Yukiko
• Imai Chifumi
• Suzuki Tsukasa
• Yoshihashi Kazutaka

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• C07K2317/51

  Complete heavy chain or Fd fragment, i.e. VH + CH1 · CPC title

• C07K2317/565

  Complementarity determining region [CDR] · CPC title