| US 7,494,647 B2 | ||
| Humanized antibody against human tissue factor (TF) and process of production of the humanized antibody | ||
| Koh Sato, Gotenba (Japan); Hideki Adachi, Gotenba (Japan); and Naohiro Yabuta, Gotenba (Japan) | ||
| Assigned to Chugai Seiyaku Kabushiki Kaisha, Tokyo (Japan) | ||
| Filed on Jun. 16, 2003, as Appl. No. 10/462,062. | ||
| Application 10/462062 is a division of application No. 09/647468, granted, now 6,677,436, previously published as PCT/JP99/01768, filed on Apr. 02, 1999. | ||
| Claims priority of application No. 10-91850 (JP), filed on Apr. 03, 1998. | ||
| Prior Publication US 2004/0044187 A1, Mar. 04, 2004 | ||
| Int. Cl. A61K 39/395 (2006.01) | ||
| U.S. Cl. 424—133.1 [424/139.1; 424/141.1; 424/145.1; 435/7.1; 435/7.92] | 5 Claims |
| 1. A process of preparing a natural humanized antibody that has complementarity determining regions (CDRs) derived from non-humans
and a framework region (FR) derived from a natural human antibody and that has a reduced immunogenicity, said method comprising
the steps of:
(1) preparing a non-human monoclonal antibody responsive to an antigen of interest;
(2) preparing a plurality of human antibodies having a high homology with the amino acid sequences of FRs of heavy chain (H
chain) or light chain (L chain) in the monoclonal antibodies of the step (1);
(3) preparing a first humanized antibody by steps comprising: a) replacing all FRs of H chain of the non-human antibody prepared in step (1) with corresponding FRs H chain of one human
antibody prepared in step (2);b) replacing all FRs of L chain of the non-human antibody prepared in step (1) with corresponding FRs of L chain of one human
antibody prepared in step (2); andc) changing constant region of the non-human antibody prepared in step (1) to constant region of a human antibody;
(4) generating second antibodies by: a) in an H chain V region of the first humanized antibody prepared on said (3)a), replacing 1 to 3 FRs having at least one
of FR2 and FR3, with corresponding FRs of a human antibody different from the antibody used in (3)a) selected from the human
antibodies prepared in the step (2);b) in an L chain V region of the first humanized antibody prepared on said (3)b), replacing 1 to 3 FRs having at least one
of FR2 and FR3, with corresponding FRs of a human antibody different from the antibody used in (3)b) selected from the human
antibodies prepared in the step (2); andc) obtaining a second humanized antibody comprising an H chain obtained in a), an L chain obtained in b, and a constant region
of a human antibody in (3)c);
(5) determining ability of the first and the second humanized antibodies to bind to the antigen or ability of the first and
the second humanized antibodies to neutralize a biological activity of the antigen;
(6) comparing the ability of the first and the second antibodies determined in step (5), and selecting antibodies which have
a higher antigen binding ability or higher ability to neutralize a biological activity of an antigen; and
(7) repeating the steps of (4) to (6) while using the antibody selected in step (6) as a first humanized antibody in step
(4) until a humanized antibody is selected having the same level or more of ability as the non-human monoclonal antibody in
step (1), wherein said ability is to bind to the antigen or to neutralize a biological activity of the antigen.
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