MXPA00009667A

MXPA00009667A - Humanized antibody against human tissue factor (tf) and process for constructing humanized antibody

Info

Publication number: MXPA00009667A
Application number: MXPA/A/2000/009667A
Authority: MX
Inventors: Koh Sato; Hideki Adachi; Naohiro Yabuta
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1998-04-03
Filing date: 2000-10-02
Publication date: 2001-07-09

Abstract

A humanized antibody against tissue factor (TF) which comprises:A. a humanized H chain containing (1) an H chain V region containing the H chain CDR of a mouse monoclonal antibody against TF and the H chain FR of a human antibody, and (2) the H chain C region of a human antibody;and B. a humanized L chain containing (1) an L chain V region containing the L chain CDR of a mouse monoclonal antibody against TF and the L chain FR of a human antibody, and (2) the L chain C region of a human antibody. The mouse monoclonal antibody CDR is grafted into the human antibody to construct the humanized V region. Next, the FR thereof is replaced by the corresponding FR of another human antibody with a high homology, thus detecting a highly active humanized antibody.

Description

HUMANIZED ANTIBODY AGAINST HUMAN TISSUE FACTOR (TF) AND HUMANIZED ANTIBODY PRODUCTION PROCESS TECHNICAL FIELD _ The present invention relates to a human / mouse chimeric antibody comprising a variable region (region V) of a mouse monoclonal antibody against a human tissue factor (TF) and a constant region (region C) of a human antibody; a humanized antibody in which regions of co-complementarity determination (CDRs) of V region of light chain (L chain) and region V of heavy chain (H chain) of a mouse monoclonal antibody against human TF have been grafted onto an antibody human; the L chain and the H chain of said antibody; and a fragment of region V which constitutes the L chain or the H chain of said antibody. The present invention also relates to a process for the production of humanized antibody against human TF. The present invention relates to the DNA encoding the aforementioned antibody, specifically to a V region fragment thereof, and to the DNA encoding an L chain or an H chain containing a V region. The present invention also relates to a recombinant vector comprising said DNA, and a host transformed with said vector. The present invention also relates to a process for the preparation of a chimeric antibody and a humanized antibody against human TF. The present invention also relates to a pharmaceutical composition and a therapeutic agent for disseminated intravascular coagulation syndrome (DIC) which comprises as an active ingredient a humanized antibody against human TF. BACKGROUND OF THE ART Tissue factor (TF), a coagulation factor VII receptor expressed on the surface of the cell, plays an indispensable role in the activation of coagulation factors IX and X through the formation of a complex with coagulation factor VII, and has been defined as a factor of practical initiation of blood coagulation reactions. It is known that TF is expressed in fibroblasts, smooth muscle cells, etc. That constitute the blood vessel, and performs a hemostatic function by activating the coagulation system at the time of injury to a blood vessel. DIC is a disease in which the activation of the coagulation system in a blood vessel leads to multiple systemic occurrences of blood clots, mainly in microvessels. It is not uncommon for the reduction of platelets and clotting factors due to consumption to cause bleeding which is the opposite phenomenon of blood coagulation. Multiple microthrombi can cause deficient microcirculation in the main organs which, once developed, causes irreversible functional deficiency and poor prognosis of DIC, and in that sense DIC is considered an important disease. The incidence of underlying diseases estimated from the research reports of 1990 and 1992 prepared by the Ministry of Health and Welfare Specified Diseases Blood Coagulation Disorders Survey and Study Group (Study Group and Evaluation of Blood Coagulation Disorders and Specific Diseases of the Ministry of Health and Welfare) is: hematological malignancies, approximately 30%; solid tumors, approximately 20%; infections, approximately 15%; obstetric diseases, approximately 10%; liver diseases, approximately 6%; shocks, approximately 5%; and cardiovascular diseases, approximately 3%. The incidence of DIC is up to approximately 15% in leukemia and approximately 6 to 7% in malignant lymphoma, and approximately 3% in solid tumors. DIC develops accompanied by several diseases mentioned above, but the causative agent is the same, ie TF. Thus, the mechanism of initiation of DIC is considered to be the following: abnormally high formation and / or abnormally high expression of TF in cancer cells in acute leukemia, malignant lymphoma, and solid tumors, the formation and / or enhanced expression of TF in monocytes and / or endothelial cells in infections (particularly, sepsis caused by gram-negative bacilli); Influence of TF binding from hepatic tissue necrotized in fulminating hepatitis; expression of TF in lumens of the blood vessel in aortic aneurysm, cardiac aneurysm, and giant emangioma; and also influence of TF in the blood in obstetric diseases (amniotic fluid embolism and abrupt placenta) as well as surgical interventions, injuries and burns. The treatment of the original (underlying) diseases is of special concern, however it is not easy in practical terms. As a current method for the treatment of DIC, anticoagulant therapies and substitution therapies are used. Heparin preparations (fractionated heparin, low molecular weight heparin) are mainly used for anticoagulant therapies. Synthetic protease inhibitors (gabexate mesylate, nafamostato mesylate) as well as concentrated plasma (activated protein C preparations, antithrombin III) are also used. . As substitution therapy there are platelet concentrates, fresh frozen plasmas (source of fibrinogen), washed red blood cells, and the like. However, current therapeutic agents are not satisfactory in terms of efficacy and side effects, and in most cases, a total withdrawal of DIC is impossible. Accordingly, there is a need to use drugs with high therapeutic effects and low side effects. On the other hand, as new attempts in the treatment of DIC, we can mention preparations of thrombomodulin, hirudin, and anti-FAP agents, inhibitor of the tissue factor pathway (TFPI). Selective inhibitors for Fxa are attracting a lot of attention as anti-coagulant and anti-thrombotics agents that can be administered orally. Also as an agent that neutralizes TF activity, WO / 88/07543 discloses a mouse antihuman TF monoclonal antibody, and O96 / 40921 discloses humanized antihuman TF antibody. It is thought that mouse anti-human TF monoclonal antibodies constitute a safe and effective therapeutic agent insofar as it does not present a symptom of bleeding associated with the main efficacy in DIC. However, mouse monoclonal antibodies are highly immunogenic (they are sometimes referred to as "antigenic"), and therefore the medical therapeutic value of mouse antibodin humans is limited. For example, the half-life of mouse antibodin humans is relatively high and, therefore, can not fully exhibit their anticipated effects, In addition, a human anti-mouse antibody (HAMA) that develops in response to mouse antibody administered causes immune reactions that are unfavorable and dangerous for patients. Accordingly, mouse monoclonal antibodcan not be administered repeatedly to humans. In order to solve these problems, methods have been developed that aim to reduce the immunogenicity of antibodderived from non-humans, such as mice (monoclonal antibodderived from mice). One of them is a method for making chimeric antibody wherein a variable region (region V) of the antibody is derived from mouse monoclonal antibody, and a constant region (region C) thereof is derived from a suitable human antibody. Since the obtained chimeric antibody contains a variable region of an original mouse antibody in the complete form, it is expected to bind an antigen with the affinity identical to the affinity of the original mouse antibody. In addition, in chimeric antibody, the ratio between the amino acid sequences derived from non-humans is substantially reduced, and therefore it is expected to have a reduced immunogenicity compared to the original mouse antibody. However, it remains possible for an immune response to emerge to the mouse variable region (LoBuglio, AF et al., Proc. Nati, Acad. Sci. USA, 86: 4220-4224, 1989). A second method for producing the Immunogenicity of mouse antibody, even though it is a more complicated method, can provide a drastic reduction in the potential immunogenicity of the mouse antibody. In this method, the region of complementarity determination (CDR) alone of a mouse antibody is grafted onto a human variable region to form a "reform" human variable region. As desired, however, some amino acid sequences of the framework regions (FRs) that support the CDRs can be grafted from the variable region of a mouse antibody into the human variable region in order to obtain the closest approximation possible of the original mouse antibody structure. Then, the humanized reformed human variable region is ligated over the human constant region. In the finally reformed humanized antibody, portions derived from non-human amino acid sequences are only the CDRs and a small portion of the FRs. The CDRs comprise hypervariable amino acid sequence and do not show specific sequences for spec In the case of humanized antibody, see also, Riechmann, L. et al., Nature 332: 323-327, 1988; Verhoeye, M. et al., Scienece 239: 1534-1536, 1988; Kettleborough, C A. et al., Protein Engng., 4: 773-783, 1991; Maeda, H., Human Antibodand hybridoma, 2: 124-134, 1991; Gorman, S. D. et al., Proc. Nati Acad. Sci. USA, 88: 4181-4185, 1991; Tempest, P R., Bio / Technology, 9: 226-271, 1991; Co, M. S. et al., Proc. Nati Acad. Sci. USA 88: 2869-2873, 1991; Cater, P. et al., Proc. Nati Acad. Sci. USA, 89: 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148: 1149-1154, 1992; and Sato, K. et al., Cancer Res., 53: 851-856, 1993. In conventional humanization technology part of the framework region (FR) includes an amino acid sequence grafted from the variable region of a Mouse antibody on the human variable region. Thus, when administered as a therapeutic agent in humans, there is a risk that antibodwill be formed against a site having an amino acid sequence not present in humans, even if it is simply a few amino acids. In order to avoid this risk, a third humanization technology was invented. A) Yes, the method includes, for four FRs (FR1-4) required to support the three-dimensional structure of three CDRs, the replacement of the FR of a human antibody having a high homology with the FR of the mouse antibody present in the database using a FR as a unit. In this case, several FRs are selected from human antibodies present in the database, and are sequentially replaced to prepare a humanized antibody having a high activity. In this way, it is possible to construct humanized antibodies wherein all of the FRs except the CDRs in the variable region have amino acid sequences derived from human antibody. Thus, the humanized antibody carrying the mouse CDR no longer has a greater immunogenicity than a human antibody containing the human CDR. Even though a humanized antibody is expected to be useful for the purpose of the treatment, as mentioned above, there is currently no fixed process universally applicable to any antibody in the humanized antibody production method, and therefore various artifices are required to construct a humanized antibody that exhibits sufficient binding activity and sufficient neutralization activity relative to a specific antigen (see, for example, Sato, K. et al., Cancer Res., 53: 851-856, 1993). DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a human / mouse chimeric antibody comprising the variable region (V region) of a mouse monoclonal antibody against a human tissue factor (TF) and the constant region (region). C) of a human antibody, a humanized antibody wherein the regions of complementarity determination (CDRs) of the V region of light chain (L chain) and the heavy chain region V (H chain) of a mouse monoclonal antibody against Human TF have been grafted onto a human antibody, the L chain and the H chain of said antibody, and a V region fragment constituting the L chain or the H chain of said antibody. It is a further object of the present invention to provide a process for making a humanized antibody against human TF. It is a further object of the present invention to provide a DNA encoding the above-mentioned antibody, specifically a fragment of the D region thereof and DNA encoding an L chain or an H chain containing a V region. It is a further object of the invention. present invention provides a recombinant DNA vector comprising said DNA, and a host transformed with said vector. It is a further object of the present invention to provide a pharmaceutical composition and a therapeutic agent for disseminated intravascular coagulation syndrome (DIC) which comprises as an active ingredient a humanized antibody against human TF. After an intensive study to solve the aforementioned problems, the inventors of the present invention successfully obtained an antibody in which the human immunogenicity of the mouse monoclonal antibody against human TFs is reduced and they also developed a process to make a novel humanized antibody , and in these ways achieved the present invention. Thus, the present invention relates to a chimeric HL chain containing the H chain C region of a human antibody and a fragment of the H chain B region of a mouse monoclonal antibody against human TF. H may be mentioned a region comprising an amino acid sequence presented in SEQ ID NO: 9, and like region C, a region derived from the C 4 4 region may be mentioned.In addition, the present invention relates to an L chain chimeric region containing the H chain C region of a human antibody and a fragment of the L chain region B of a mouse monoclonal antibody against human TF As the H chain region B, a region comprising a sequence of amino acids in accordance with the provisions of SEQ ID NO: 15, and like region C of chain L, a region derived from the Ckappa region can be mentioned. In addition, the present invention relates to a human / mouse chimeric monoclonal antibody against human TF, said antibody comprises the chimeric H chain and the above chimeric L chain. The present invention also relates to a H chain V region fragment of a humanized antibody, said fragment comprises the structure regions (FRs) 1-4 of the H chain V region of a human antibody and the regions of complementarity determination (CDRs) 1-3 of the H chain V region of a mouse monoclonal antibody against human TF. As the CDR 1-3, there can be mentioned a region that includes the amino acid sequence presented in SEQ ID NO: 133-135, respectively. As the FR1 of the H chain V region of the human antibody, the FR1 of human antibody having a homology of 40% or more can be mentioned with the FR1 of the H chain V region of a mouse antibody; as the FR2, the FR2 of human antibody having a homology of 40% or more can be mentioned with the FR2 of the V region of H chain of a mouse antibody; like the FR3, the FR3 of human antibody having a homology of 40% or more can be mentioned with the FR3 of the V region of H chain of a mouse antibody; such as FR4 can be mentioned the FR4 of human antibody having a homology of 40% or more with the FR4 of the V region of H chain of a mouse antibody. Preferably, as the FR1 of the H chain V region of a human antibody, the FR1 of human antibody having a homology of 50% or more can be mentioned with the FR1 of the H chain V region of a mouse antibody.; such as FR2, there can be mentioned the FR2 of human antibody having a homology of 70% or more with the FR2 of the V region of H chain of a mouse antibody; such as FR3, the FR3 of human antibody having a homology of 65% or more can be mentioned with the FR3 of the H chain V region of a mouse antibody; and like FR4, there can be mentioned the FR4 of human antibody having a homology of 80% or more with the FR4 of the V region of H chain of a mouse antibody. As specific examples, as the FR1 of the H chain V region of a human antibody, the human antibody L39130 can be mentioned; as the FR2 of the H chain V region of a human antibody, the human antibody L39130 can be mentioned; in human antibody P01742, and the human antibody Z80844; such as FR3 can be mentioned human antibody L39130, human antibody Z62723, human antibody P01825, human antibody M62723, human antibody Z80844, human antibody L04345, human antibody S78322, human antibody Z26827, human antibody U95239 , the human antibody L03147; and like FR4, the human antibody L39130 can be mentioned. As preferred examples, as the FR1 of the H chain V region of a human antibody, the human antibody L39130 can be mentioned. As the FR2, the human antibody L39130 can be mentioned, and as the human antibody Z80844; as the FR3 can be mentioned the human antibody Z34963, the human antibody M62723, and the human antibody U95239: like the FR4, the human antibody L39130 can be mentioned. As preferred examples, as the FR1 of the H chain V region of a human antibody, the human antibody L39130 can be mentioned; as the FR2, the human antibody L39130 can be mentioned; like FR3, mention may be made of the human antibody Z34963 and the human antibody U95239: like FR4, the human antibody L39130 may be mentioned. Furthermore, as used herein, the numbers in the structure regions are based on the definition of (Kabat, E.A. et al., US Dept. Health And Services, US Government Printing Offices, 1991). The present invention also relates to a fragment of the H chain V region of a humanized antibody, said fragment comprising any of the established amino acid sequences SEQ ID NO: 30, 40, 42, 50, 52, 58, 60, 64 , 70, 72, 76, 78, 82 and 84. The present invention also relates to a fragment of the L chain V region of a humanized antibody, said fragment comprises any of the FRs 1-4 of the region L chain V of a human antibody and the CDRs 1-3 of the L chain V region of a mouse monoclonal antibody against human TF. As CDRs 1-3, a region including the amino acid sequence presented in SEQ ID NO: 136-138, respectively, may be mentioned. As the FR1 of the L chain region V of the human antibody, a region having a homology of 40% or more can be mentioned with the region FR1.

V of L chain of a human antibody; like FR2, the FR2 of human antibody having a homology of 40% or more can be mentioned with the FR2 of the V region of L chain of a mouse antibody; such as FR3, there can be mentioned the FR3 of human antibody having a homology of 40% or more with the FR3 of the V region of L chain of a mouse antibody; and as the FR4 one can mention the FR4 of human antibody having a homology of 40% or more with the FR4 of the V region of L chain of a mouse antibody. Preferably, as the FR1 of the L chain region V of a human antibody, the FR1 of human antibody having a homology of 75% or more with the FR1 of the L chain V region of a mouse antibody can be mentioned.; such as FR2, there can be mentioned the FR2 of human antibody having a homology of 80% or more with the FR2 of the V region of L chain of a mouse antibody; such as FR3, the FR3 of human antibody having a homology of 70% or more can be mentioned with the FR3 of the V region of L chain of a mouse antibody; and like FR4, FR4 of human antibody having a homology of 80% or more can be mentioned with FR4 of the V region of L chain of a mouse antibody. As specific examples, as the FR1 of the L chain V region of a human antibody, the human antibody Z37332 can be mentioned; as the FR2, the human antibody Z37332 and the human antibody X93625 can be mentioned; such as FR3 can be mentioned human antibody Z37332, human antibody S68699, and human antibody P01607; and like FR4, the human antibody Z37332 can be mentioned. As more preferred examples, as the FR1 of the L chain V region of a human antibody, the human antibody Z37332 can be mentioned; as the FR2, the human antibody X93625 can be mentioned; as the FR3 can be mentioned the human antibody S68699 and like the FR4, the human antibody Z37332 can be mentioned. Furthermore, as used herein, the numbers in the structure regions are based on the definition of (Kabat, E.A. et al., US Dept. Health And Services, US Government Printing Offices, 1991). The present invention also relates to a fragment of the L chain V region of a humanized antibody, said fragment comprising the amino acid sequences presented in SEQ ID NO: 93, 99, 101, 107, and 109. The present invention is also refers to the H chain of a humanized antibody, against human TF, said chain comprises a H chain V region fragment of the above humanized antibody and a H chain C region fragment of a human antibody. We can mention the region C? 4 as the region C; as the FRs 1-4 derived from a human antibody, there can be mentioned the derivatives of the human antibody L39130 (FRl), the human antibody L39130 (FR2), the human antibody Z34963 (FR3), or the human antibody U95239 (FR3) , the human antibody L39130 (FR4); and as the CDRs 1-3, we can mention the regions derived from the amino acid sequences according to that established in SEQ ID NO: 133-135, respectively. The present invention also relates to the L chain of a humanized antibody against human TF, said chain comprises a fragment of L chain region of the humanized antibody above and a previous fragment of region C of L chain of a human antibody. We can mention the Ckappa region as the C region; as FRs 1-4 derived from human antibody, we can mention the regions derived from the human antibody Z37332 (FRl), the human antibody X93625 (FR2), the human antibody S68699 (FR3), and the human antibody Z37332 (FR4); and as the CDRs 1-3, we can mention the regions derived from the amino acid sequences presented in SEQ ID NO: 136-138, respectively. The present invention also relates to a humanized antibody against human TF, said antibody comprises the L chain and the H chain of the above humanized antibody. The present invention also relates to a process for making a humanized antibody against human TF. The humanization process refers to the method of selecting the FRs 1-4 that support the structure of the CDRs 1-3 which are the antigen recognition site for the H chain or the L chain. Thus, the present invention relates to to the method of selecting some of the capital letters FRs of a human antibody having a high homology with the FR of a mouse antibody with each FR as a unit, and generating a humanized antibody having the desired activity by a sequential change of the FR . More specifically, an example of a process for preparing a natural humanized antibody having a region of complementarity determination (CDR) derived from non-humans and a structure region (FR) derived from a natural human antibody and having a reduced immunogenicity , said method comprises the steps of: (1) preparing a non-human monoclonal antibody that responds to the antigen of interest; (2) preparing some of the human antibodies that have a high homology with the amino acid sequence of the FR in the above monoclonal antibody (1); (3) replacing the 4 FRs of a human antibody in (2) above with the corresponding FRs of the non-human monoclonal antibody of (1) above to generate the first humanized antibody; (4) determining the ability of the humanized antibody generated in (3) above to bind with the antigen or to neutralize the biological activity of the antigen; (5) replacing one to three RFs of the humanized antibody generated in (3) above with the corresponding RFs of a human antibody that is different from the human antibody used in (3) among the human antibodies prepared in (2) to generate the second humanized antibody; (6) compare the capacity of the second humanized antibody generated in (5) above and the first humanized antibody generated in (3) above to determine its ability to bind to the antigen or to neutralize the biological activity ~ of the antigen thus selecting a humanized antibody having a favorable activity; (7) carrying out the above steps of (3) to (6) for the humanized antibody selected in (6) above; (8) repeating the above steps of (3) to (6) until obtaining a humanized antibody having an activity equivalent to the non-human monoclonal antibody in (1) above. Once the humanized antibody having a certain degree of neutralizing activity of human TF is obtained, a search for additional homology is carried out for the specific FR in the V region of H chain and L chain in such a way that it can be select a human antibody that has a high homology. By adding the human antibody obtained in this way to a group of some antibodies. humans in the previous step (2) and repeating further steps (3) to (6) a humanized antibody having the desired activity can be obtained. The present invention also relates to DNA encoding an H chain V region fragment or an L chain V region fragment of a mouse monoclonal antibody against human TF. As the amino acid sequence and the coding DNA of the H chain V region fragment or L chain V region fragment we can mention a sequence that includes the nucleotide sequence presented in SEQ ID NO: 9 or 15, respectively. The present invention also relates to DNA encoding the chimeric L chain or the chimeric H chain above. As the DNA encoding said H chain, we can mention a chain that includes the nucleotide sequence presented in SEQ ID NO: 9, and as DNA encoding said L chain, we can mention a DNA that includes the nucleotide sequence presented in SEQ ID NO: 15. The present invention also refers to DNA that encodes a fragment of V region of H chain or a fragment of L chain region V of the humanized antibody above. As the DNA encoding the H chain region V fragment, we can mention a DNA that includes any of the nucleotide sequences presented in SEQ ID NO: 29, 39, 41, 49, 51, 57, 59, 63, 69, 71, 75, 77, 81, or 83, and as the DNA encoding the L-region V region fragment we can mention a DNA that includes any of the nucleotide sequences presented in SEQ ID NO: 92, 98, 100, 106 or 108.

The present invention also relates to DNA encoding the H chain of a humanized antibody. It also refers to the H chain DNA of a humanized antibody, said DNA comprises DNA encoding any of the amino acid sequences according to that presented in SEQ ID NO: 30, 40, 42, 50, 52, 58, 60, 64 , 70, 72, 76, 78, 82, or 84. As said DNA, we can mention a DNA that includes any of the nucleotide sequences presented in SEQ ID NO: 29, 39, 41, 49, 51, 57, 59 , 63, 69, 71, 75, 77, 81, or 83. The present invention also relates to DNA encoding the L chain of the above humanized antibody. The present invention also relates to the L chain DNA of a humanized antibody, said DNA comprises DNA encoding the amino acid sequences presented in SEQ ID NO: 93, 99, 101, 107, or 109. As said DNA, we can mentioning a DNA that includes any of the nucleotide sequences presented in SEQ ID NO: 92, 98, 100, 106, or 108. The present invention also relates to a recombinant DNA vector containing any of the DNAs described above. The present invention also relates to a transformant transformed with a recombinant DNA vector described above.

The present invention also relates to a process for generating a chimeric antibody or a humanized antibody against human TF, said method comprises culturing the above transformant and obtaining a chimeric antibody or a humanized antibody against human TF from the harvested culture. . The present invention also relates to a pharmaceutical composition and a therapeutic agent for DIC, comprises the above humanized antibody as an active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized H chain version a / chimeric chain L antibody, and a chimeric chain antibody H / humanized chain L antibody version a. Figure 2 is a graph comparing the neutralizing activity against human TF of an H-chain chimeric antibody / L-chain chimeric antibody, a humanized antibody of H chain version / L chain chimeric antibody, and a chain chimeric antibody H / humanized chain L antibody version a. Figure 3 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody and an H chain version humanized version / L chain chimeric version. Figure 4 is a graph comparing the neutralizing activity against human TF of an anti-TF mouse monoclonal antibody ATR-5, an H chain chimeric antibody / L chain chimeric antibody, and a humanized antibody of H chain version a / Humanized chain L antibody version a. Figure 5 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized version B chain b antibody / chimeric L chain antibody and an H chain antibody version b humanized / chain antibody L humanized version. Figure 6 is a graph comparing the antigen-binding activity of an H-chain chimeric antibody / L-chain chimeric antibody, a humanized version H chain c antibody / chimeric L-chain antibody and an H chain antibody version d humanized / chimeric L-chain antibody. Figure 7 is a graph comparing the neutralizing human TF activity of an H chain chimeric antibody / L chain chimeric antibody, an H chain b humanized / chimeric chain L antibody. , an H chain humanized version c / chimeric chain L antibody, and an H chain humanized version d / chimeric chain L antibody. Figure 8 is a graph comparing the neutralizing human TF activity of a chimeric antibody H chain / L chain chimeric antibody and an H chain humanized version b / L chain antibody humanized version. Figure 9 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a chimeric chain H antibody / humanized version B L chain antibody, and a chimeric H chain antibody / L chain version humanized version c. Figure 10 is a graph comparing the neutralizing human TF activity of an H chain chimeric antibody / L chain chimeric antibody, an H chain chimeric antibody / humanized version B L chain antibody, and an H chain chimeric antibody / chain antibody L version c humanized. Figure 11 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, an H chain b humanized version / L chain b humanized antibody, and a chain antibody H humanized version b / chain antibody L version c humanized.

Figure 12 is a graph comparing the neutralizing human TF activity of an H-chain chimeric antibody / L-chain chimeric antibody, a humanized version B-chain antibody / humanized version B L-chain antibody, and a chain antibody H humanized version b / chain antibody L version c humanized. Figure 13 is a graph comparing the antigen binding activity of an H chain chimeric antibody, L chain chimeric antibody, humanized chain antibody. H version b / humanized antibody of chain L version b, and a humanized antibody of chain H version d / humanized antibody of chain L version b. Figure 14 is a graph comparing the neutralizing human TE activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H version b / humanized chain L version b antibody, and a humanized antibody of chain H version d / humanized antibody of chain L, version b. Figure 15 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H version e / chimeric L chain antibody, and a humanized antibody of H chain version e / humanized antibody chain L version b. Figure 16 is a graph comparing the neutralizing human TF activity of an H chain chimeric antibody / L chain chimeric antibody, and a humanized chain H version e / chimeric chain antibody. Figure 17 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody and a humanized chain H version g / humanized antibody of L chain b version. Figure 18 is a graph comparing the neutralizing human TF activity of an H chain chimeric antibody / L chain chimeric antibody and a humanized chain H version g / humanized antibody of L chain b version. Figure 19 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H antibody b3 version / humanized chain L antibody b version, and an antibody humanized chain H version d3 / humanised antibody chain L version b. Figure 20 is a graph comparing the neutralization activity of human TF of an H-chain chimeric antibody / L-chain chimeric antibody, a humanized H-chain version b3 / humanized L-chain version b antibody, and a humanized antibody of chain H version d3 / humanized antibody of chain L version b. Figure 21 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized H chain version i / chimeric L chain antibody, and a humanized H chain antibody version j / chimeric chain L antibody. Figure 22 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized H chain version i / humanised antibody chain L version b, and a humanized antibody of chain H version j / humanized antibody of chain L version b. Figure 23 is a graph comparing the neutralization activity of human TF of an H chain chimeric antibody / L chain chimeric antibody, a humanized H chain version i / chimeric L chain antibody, and a humanized chain antibody H version j / L chain chimeric antibody. Figure 24 is a graph comparing the neutralization activity of human TF of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H antibody b version / humanized antibody of chain L version b, a humanized antibody of chain H version i / humanised antibody of chain L version b, and a humanized antibody of chain H version j / humanized antibody of chain L version b. Figure 25 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, an H chain chimeric antibody / humanized L chain version bl, and an H chain chimeric antibody / Humanized chain L antibody b2 version. Figure 26 is a graph comparing the neutralization activity of human TF of an H-chain chimeric antibody, L-chain chimeric antibody, an H-chain chimeric antibody / L-chain humanized antibody version Bl, and a chimeric chain antibody H / humanized chain L antibody b2 version. Figure 27 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody and a humanized H chain antibody version b / humanized chain H antibody b version / humanized chain L antibody b2 version. Figure 28 is a graph comparing the neutralization activity of human TF of an H chain chimeric antibody / L chain chimeric antibody, a humanized H chain version I / humanized antibody of L chain b version, a humanized antibody of chain H version b / humanized antibody of chain L version b, and a humanized antibody of chain H version b / humanized antibody of chain L version b2. Figure 29 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized H chain version i / humanized chain L version bl antibody, and a humanized antibody of H chain version i / humanized chain L antibody b2 version. Figure 30 is a graph comparing the neutralization activity of human TF of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H version i / humanized chain L version b antibody, a humanized antibody of H chain version i / humanized antibody chain L version bl, and a humanized antibody H chain version i / humanized antibody chain L version b2. Figure 31 is a graph comparing the antigen binding activity of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H antibody b version / humanized chain L version b antibody, a humanized chain antibody H version i / humanized antibody of chain L version b, and a humanized antibody of chain H version i / humanised antibody of chain L version b2. Figure 32 is a graph comparing the neutralization activity of human TF (the activity to inhibit the production of factor Xa by TF) of an H chain chimeric antibody / chimeric L chain antibody, a humanized chain H antibody b version / humanized chain L antibody version b, a humanized antibody of chain H version i / humanized antibody of chain L version b, and a humanized antibody of chain H version i / humanized antibody of chain L version b2. Figure 33 is a graph comparing the neutralization activity of human TF (the activity to inhibit factor X binding) of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H antibody b version / antibody humanized chain L version b, a humanized antibody of chain H version i / humanized antibody of chain L version b, and a humanized antibody of chain H version i / humanized antibody of chain L version b2. Figure 34 is a graph comparing the neutralization activity of human TF (the activity to inhibit plasma coagulation by TF) of an H chain chimeric antibody / L chain chimeric antibody, a humanized chain H antibody b version / humanized chain L antibody version b, a humanized antibody of H chain version i / humanised antibody of L chain version, b, and a humanized antibody of chain H version i / humanized antibody of chain L version b2. Figure 35 is a figure comparing reactivity with human TF treated under various conditions of the chimeric chain H antibody / L chain chimeric antibody, a humanized chain H antibody b version / humanized chain L b version antibody, a humanized antibody of chain H version i / humanized antibody of chain L version b, and a humanized antibody of chain H version i / humanized antibody of chain L version b2. PREFERRED MODE OF THE INVENTION The present invention will be explained below with additional details. 1. Preparation of a mouse monoclonal antibody against human TF A mouse monoclonal antibody against TF can be generated by the generation of a hybridoma by a fusion of cells that produce antibodies obtained from animals immunized with the antigen with myeloma cells , then selecting from the obtained hybridoma a clone that produces antibody that specifically inhibits TF activity. Thus, spleen cells from a mouse immunized with TF that was purified from human placenta as an antigen were fused with myeloma cells to prepare a hybridoma. For the purpose of screening the hybridoma, the binding capacity of the antibody on TF was determined by means of an ELISA assay for cells using the line of cells with high expression of TF J82, and its TF neutralization activity was determined in a trial that he used as. Index the activity of inhibition of the activation of the coagulation factor X (Factor X: FX). As a result, hybridomas that produce 6 antibodies that strongly inhibit FX activation of the TF / VIIa complex were successfully established. (1) Antigen preparation As TF for animal immunization, there may be mentioned a peptide that is part of the TF amino acid sequence generated by recombinant DNA technology or chemical synthesis, or TF derived from human placenta. For example, TF purified from human placenta according to the method Ito et al. (Ito T. et al., J. Biochem. 114: 691-696, 1993) can be used as an antigen. The TF obtained is mixed with an adjuvant and then the mixture is used as an antigen. As an adjuvant, Freund's complete adjuvant or incomplete Freund's adjuvant can be mentioned, any of them can be mixed. (2) Immunization and harvesting of cells that produce antibodies The antigen obtained according to the above mentioned can be administered to non-human mammals, for example, mammals such as mice, rats, horses, monkeys, rabbits, goats, sheep and the like. The immunization can be carried out using any of the existing methods, and is carried out mainly by intravenous, subcutaneous and intraperitoneal injection and the like. The period of immunization is still from a few days to a few weeks, preferably at an interval of 4 to 21 days, but it is not limited to this period. The cells that produce antibodies can be collected 2-3 days after the last day of immunization. As cells that produce antibodies, we can mention spleen cells, lymphatic cells and peripheral blood cells, and spleen cells are generally used. The amount of the antigen to be used for each immunization is 0.1 to 100 microg per mouse. (3) Determination of antibody titer In order to confirm the level of immune response of the immunized animal and to select the desired hybridoma of the cells after the cell fusion treatment, the antibody titer is determined in the blood of the immunized animal and the titre of antibodies in the supernatant of cultures of the cells that produce antibodies. As methods to detect antibodies, we can mention known methods such as enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and the like. (4) Fusion of cells Like the myeloma cells fused to the cells that produce antibodies, coated cells derived from mice, rats, humans, etc. are used. and generally available to a person skilled in the art. As lines of cells to be used we can mention some lines of cells that have the properties of being resistant to drugs, unable to survive in the selection medium (for example the HAT medium) in the non-fused state, and capable of surviving only in the merged state. Generally strains resistant to 8-azaguanine are employed, and these cell lines do not have hypoxanthine-guanine phosphoribosyltransferase and consequently can not survive in medium containing hypoxanthine, aminopterin and thymidine (HAT). As myeloma cells, several known cell lines such as P3 (P3X63Ag8.653) (J. Immunol., 123: 1548-1550, 1979), P3X63Ag8.653 (Current Topics in Microbiology and Immunology 81: 1-) are preferably used. 7, 1978), NS-1 (Kohler, G. And Milstein, C, Eur. J. Immunol., 6: 511-519, 1976), MPC-11 (Margulies, DH, Cell 8: 405-415, 1976) , SP2 / 0 (Schulman, M. et al., Nature 276: 269-270, 1978), FO (from St. Groth, SF et al., J. Immunol. Methods (1980) 35: 1-21, 1980 ), S194 (Trowbridge, IS, J. Exp. Med. 148: 313-323, 1978), R210 (Galfre, G. et al., Nature 277: 131-133, 1979) and the like. Cells that produce antibodies can be obtained from spleen cells, lymphatic cells, and the like, thus, from the above animals, spleens, lymph nodes, etc. are removed or collected, and said tissues are dispersed. The dispersed cells are suspended in a medium or buffer PBS, DMEM, and RPMI1640, filtered with stainless mesh, etc., and centrifuged to prepare the cells that produce the antibodies. The aforementioned myeloma cells and the cells that produce the antibodies are subjected to cell fusion. Cell fusion can be carried out by contacting the myeloma cells and the cells that produce antibodies at temperatures within a range of 30 to 37 degrees C for a period of 1 to 15 minutes in the presence of an accelerator. fusion in a mixed ratio of 1: 1 to 1:10 in a culture medium for culture of animal cells such as for example MEM, DMEM, RPMI1640, and the like. In order to accelerate the fusion of cells, a fusion accelerator of cells or fusion viruses such as polyethylene glycol (PEG) of a molecular weight of 1,000 to 6,000, polyvinyl alcohol, or Sendai virus (HVJ) can be used. In addition, commercially available cell fusion instruments (e.g., electroporation) employing electrical stimulation can be used to fuse the cells that produce antibodies on the myeloma cells. (5) Selection and cloning of a hybridoma The desired hybridoma can be selected from the fused cells. By way of example we can mention methods that employ the selective growth of cells in a selection medium. In this way, after dilution of a cell suspension with a suitable medium is placed in a microtitre plate and added to a selection medium (such as for example HAT medium) which is cultivated with adequate replacement of the selection medium. As a result, growing cells can be obtained as the hybridoma. Screening of a hybridoma is carried out by the limiting dilution method, the method of classifying fluorescence activated cells and the like, and finally a hybridoma producing a monoclonal antibody can be obtained. The selection of the hybridoma that produces a monoclonal antibody can be carried out by combining several test systems. For example, an antigen recognition system such as, for example, Cell-ELISA, a TF neutralization activity assay system that employs the activity of Factor Xa as an index, and a neutralizing assay assay system such as for example the assay system that measures the inhibition activity of plasma coagulation are combined to obtain a hybridoma that produces monoclonal antibody having the desired activity. In this way a hybridoma producing monoclonal antibody can be obtained such as for example ATR-2, ATR-3, ATR-4, ATR-5, ATR-6, ATR-7, ATR-8. (6) Collection of monoclonal antibody As a method for collecting monoclonal antibodies from the obtained hybridoma, we can mention a conventional cell culture method, an ascites formation method, and the like. In the cell culture method, a hybridoma is cultured in a culture medium for animal cell culture such as, for example, RPMI1640 supplemented with 10-20% fetal calf serum, DMEM medium, a serum-free medium or the like , under culture conditions (e.g., 37 degrees C, 5% C02 concentration) for 2-14 days, and then the antibody is obtained from the culture supernatant. In the ascites formation method, a hybridoma is administered intraperitoneally to an animal species similar to the mammal from which the myeloma cells are derived, and the hybridoma is proliferated in large quantities. Afterwards, the ascites or serum is collected 1-4 weeks later. When purification of antibody is required in the above method of obtaining antibody, the purification is carried out by selecting, as appropriate, a meted known as, for example, fractionation of ammonium sulfate, ion exchange chromatography, and affinity chromatography , or by combining these methods. 2. Cloning of DNA encoding the V region of a monoclonal antibody to human TF (i) Preparation of mRNA In order to clone the DNA encoding the V region of H chain and L chain of a mouse monoclonal antibody against human TF , the total RNA is isolated from the harvested hybridoma by the method known as for example guanidine ultracentrifugation method (Chirgwin, JM et al., Biochemistry, 18: 5294-5299, 1979), and the AGPC method (Chomczynski, P. et al., 162: 156-159, 1987), and then mRNA is purified by a rotating oligo (dT) -cellulose column fixed on the mRNA purification kit (Pharmacia Biotech), and the like. The mRNA can also be purified without total RNA extraction using the QuickPrep mRNA Purification Kit (Pharmacia Biotech). (ii) Preparation and amplification of 'cDNA. From the mRNA obtained above (i), the cDNA is synthesized in the V region of L chain and H chain, respectively using reverse transcriptase. The cDNA synthesis can be carried out using a suitable oligo-dT primer or primer (for example, the synthetic cDNA primer attached to the Kit) that hybridizes to the C chain region C or the C chain H region. The amplification of cDNA can be carried out by polymerase chain reaction based on the 5 '-Race method (Forman, MA et al., Proc. Nati, Acad. Sci. USA 85: 8998-9002, 1988; Belyavsky , A. et al., Nucleic Acids Res. 17: 2919-2932, 1989) using the Kit 5 '-Ampie FINDER RICE (CLONETECH) together with the L chain and the H chain. Thus, a cDNA adapter is linked to the ends of the double-stranded cDNA synthesized above, and then a polymerase chain reaction (PCR) is carried out for "cDNAs encoding the V region of the H chain and the V region of the L chain (DNA encoding a fragment of the V region of the L chain is then referred to as * 'V-region DNA of the L-chain "or" DNA coding for the V-region of the CH In L ", the same is true of region V of the H chain, etc.). As an initiator for the amplification of DNA from an H chain V region, the adapter primer 1 can be used for the 5 'end primer, and the H chain constant primer MHC-G1 of mouse antibody (SEQ ID NO: 1) (ATR-2, ATR-3, ATR-4, and ATR-5) (region C range 1) or initiator MHC-G2a (SEQ ID NO: 2) (ATR-7, and ATR-8) ( region C range 2a) (ST Jones et al., Biotechnology, 9: 88, 1991). For example, in the case of the 5 'end primer, the adapter primer 1 attached to the kit, and for the 3' end primer, the L-chain chain constant region initiator (Ckappa region) of an antibody of mouse (as, for example, the MKc primer having the nucleotide sequence presented in SEQ ID NO: 3) can be used. (iii) DNA purification and determination of the nucleotide sequence Polymerase chain reaction products are subjected to agarose electrophoresis according to the known method. After removing the desired DNA fragment, the DNA is recovered and purified, and then ligated onto a vector DNA. DNA can be purified either by extraction with phenol and chloroform (J. Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989) or by using a commercially available kit (eg, GENECLEAN II: BI0101) . A vector DNA used to retain DNA fragments can be any known vector DNA (pUC19 and Bluescript, etc.). The aforementioned DNA and a vector DNA are ligated using a known ligation kit (manufactured by Takara Shuzo) to obtain a recombinant vector. After, . The recombinant DNA vector is produced before it is introduced into a competent cell of Escherichia coli JM109 (Nippongene), etc., ampicillin-resistant colonies are selected, and then a vector DNA is prepared based on a known method (J. Sambrook et al., "Molecular Cloning," Cold Spring Harbor Laboratory Press, 1989). After digestion of the aforementioned vector with a restriction enzyme, the DNA nucleotide sequence of interest (for example the dideoxy method) can be determined by a known method (J. Sambrook et al., "Molecular Cloning" , Cold Spring Harbor Laboratory Press, 1989). In accordance with the present invention, an automatic instrument for determining the nucleotide sequence can be used (DNA Sequencer 373A Perkin-Elmer). (iv) Region of determination of complementarity (CDR) Region V of chain H and region V of chain L form an antigen binding site, and the global structure is similar between them. Thus, every four structure regions (FRs) are linked by three hypervariable regions, that is, regions of complementarity determination (CDRs). The amino acid sequences of FRs are well conserved whereas the amino acid sequences of the CDRs are highly variable (Kabat, E.A. et al., "Sequence of Proteins of Immunological Interest", North American Department of Health and Human Services, 1983). Many regions of the four previous FRs take the form of a beta sheet structure with the result that the CDR forms a loop. The CDR can sometimes be part of a beta sheet structure. Thus, the three CDRs are conserved in close proximity between them three-dimensionally, and the FRs constitute an antigen binding site together with the three CDRs. Based on these facts, by adjusting the amino acid sequence of a mouse monoclonal antibody against human TF in the database in the amino acid sequences of antibodies generated by Kabat ("Sequence of Proteins of Immunological Interest", North American Department Health and Human Services, 1983), its homology can be examined and therefore CDRs can be found. Sequences of a CDRs altered by insertion, substitution or removal may be included in the present invention insofar as it retains the binding or neutralizing activity of human TF when a humanized antibody is generated using it. For example, we can mention those that have a 90-100% homology with each CDRs for SEQ ID NO: 133-138 or with each CDR in region V of SEQ ID NO: 139-141, 143-144, 145- 147, and 149-150. Preferably, we can mention the sequences that have a homology of 95-100%. With greater preference we can mention the sequences that have a homology of 98-100%. 3. Preparation of a chimeric antibody expression vector Once a DNA fragment encoding a mouse L chain (L chain or H chain of antibody can be referred to below as "mouse L chain" in the case of antibody to mouse and "human H chain" in the case of the H chain of human antibody), and the V region of the H chain of mice have been cloned the DNA encoding the mouse V region is ligated to the DNAs encoding the constant region of a human antibody and is expressed to obtain a chimeric antihuman TF antibody. Basic methods for generating a chimeric antibody comprise the binding of a mouse leader sequence and a V region sequence in the cDNA cloned to a sequence encoding the human antibody constant region already in an expression vector for mammalian cells. Alternatively, it comprises the binding of a mouse leader sequence and a V region sequence in the cDNA cloned to a sequence encoding the human antibody C region, and then its binding to an expression vector for mammalian cells. Fragments of the human C antibody regions can be fragments of the H chain C region and fragments of the L chain C region of any human antibody. For example, can we mention 'C? L, C? 2, C? 3, or C? 4 for those of the human H chain and C? or CK for the L string respectively. For the production of chimeric antibody, an expression vector containing DNA encoding a mouse H chain V region and a human H chain C region under the control of an expression regulatory region such as an enhancer system is prepared. promoter and a single expression vector (see, for example, WO 94/11523) containing DNA encoding a V region of L chain of mouse and a C region of human L chain under the control of a regulatory region of expression as enhancer / promoter system. Then, the expression vector is used to cotransform a cell or host such as a mammalian cell and the transformed cells are cultured in vitro or in vivo to produce a chimeric antibody (see, for example, WO 91/16928). As a single vector, an IgGl?, N5KG1 (V) -type antibody expression vector and an IgG4 ?, N5KG4P antibody expression vector can be used. (i) Construction of an H chain of chimeric antibody An expression vector for a chimeric antibody H chain can be obtained by introducing cDNA encoding a mouse H chain V region into a suitable expression vector containing DNA encoding the H chain C region of a human antibody. As the region C of chain H, we can mention, for example, the region C? L, C? 2, C? 3, or C? 4. As used herein, for the purpose of introducing a cDNA encoding a mouse H chain v region into an expression vector, a suitable nucleotide sequence can be introduced into said cDNA by the polymerase chain reaction method. For example, said suitable nucleotide sequence can be introduced into an expression vector by performing a polymerase chain reaction using polymerase chain reaction primers designed to have a recognized sequence of a suitable restriction enzyme at the 5 'end of the polymerase chain reaction. said cDNA and for improved transcription efficiency, the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196: 947-950, 1987) immediately from the initiation codon of said cDNA, and primers of polymerase chain reaction designed in such a way that they have a recognized sequence of suitable restriction enzyme at the 3 'end of said cDNA. After treating a cDNA encoding the V region of H chain of mouse constructed in this way with a suitable restriction enzyme, it is inserted into the expression vector mentioned above and then a chimeric H chain expression vector containing DNA is constructed. coding for region C of chain H (C? or C? 4). (II) Construction of an expression vector containing cDNA encoding the L-chain K chain of a chimeric antibody. An expression vector for an L chain of chimeric antibody can be obtained by introducing cDNA encoding a mouse L chain V region into a suitable expression vector containing DNA encoding the L chain C region of a human antibody. As the region C of chain L we can mention, for example, the region CK and C?). As used herein, in order to construct an expression vector containing cDNA encoding a mouse L chain V region, a suitable nucleotide sequence can be introduced into said cDNA by the polymerase chain reaction method. For example, said suitable nucleotide sequence can be introduced into said cDNA by performing a polymerase chain reaction using polymerase chain reaction primers designed to have a recognized sequence of a suitable restriction enzyme at the 5 'end of said cDNA. , and for improved transcription efficiency, the Kozak consensus sequence and the polymerase chain reaction primers designed to have a recognized sequence of a suitable restriction enzyme at the 3 'end of said cDNA. After treating a cDNA encoding the V region of L chain of mouse constructed in this way with a suitable restriction enzyme, it is inserted into the aforementioned expression vector and then an L-chain expression vector containing DNA that is constructed is constructed. encodes the C region of L chain (CK region). 4. Preparation of humanized antibody (1) search for human antibody homology In order to generate a humanized antibody wherein the CDRs of a mouse monoclonal antibody are grafted onto a human antibody, it is preferable to handle a high homology between the FRs of an antibody monoclonal mouse and the FRs of a human antibody. Thus, the V chain H and L chain regions of a mouse antihuman TF monoclonal antibody are compared to the V region of all known antibodies whose structure has been published using a Data Bank. At the same time they are purchased with human antibody subgroups (HSG: human subgroup) (Kabat, EA et al., US Department of Health and Human Services, US Government Printing offices, 1991) classified by Kabat et al based on length of FR and amino acid homology. Based on the HSG classification by Kabat et al, the human H chain V regions can be grouped into HSG and III; for example, the H chain V region of the mouse antihuman TF monoclonal antibody ATR-5 have a homology of 67.8% with the HSGI consensus sequence. On the other hand, the V chain K regions of human chain L can be grouped in HSGI to IV; for example, the V chain K chain L region of the mouse antihuman TF monoclonal antibody ATR-5 have a homology of 72.3% with the HSGI consensus sequence. When a mouse antibody is humanized by a conventional technology, the amino acid sequences of some of the V region FRs of a CDR-bearing mouse antibody can be grafted to the FR of a human V region, as desired, of such that the CDR structure of a humanized V region may more closely resemble the structure of the original mouse antibody. However, there are no fixed rules as to which amino acids of the V region FR of a mouse antibody graft to the V region FR of a human antibody. Therefore, many efforts are required to specify amino acids that are essential to preserve the CDR structure. There is also a risk in the sense that a human antibody against the amino acid sequence grafted to the human V region from the V region of a mouse antibody can be formed in part from the FR. According to the present invention, in order to change all the amino acid sequences except the CDR in the humanized antibody in amino acid sequences derived from human antibody, the FRs of a human antibody having a high homology with the FR of mouse antibodies present In the database, we searched, with an FR as a unit, for the four FRs (FR1-4) that are required to conserve the three-dimensional structure of the CDR. Next we present a result of the search of homology with the database for each FR of the V region of H chain and of the V region of L chain of a monoclonal antibody ATR-5. Table 1 FR No. Homology No. with each SEQ ID NO: access FR region of H chain of mouse antibody (%) FRl of H chain L39130 53. .0 110 FR2 of H chain L39130 92. .9 111 P01742 71. .4 112 Z80844 78. .6 113 FR3 chain H L39130 62. .5 114 Z34963 71, .9 115 P01825 53, .1 116 M62723 68,, 8 117 Z80844 68,, 8 118 L04345 65. , 6 119 S78322 75., 0 120 Z26827 56. .3 121 U95239 65.. 6 122 L03147 65., 6 123 FR4 of chain H L39130 90. 9 124 Table 2 No of FR No. of Homology with each SEQ ID NO access FR of V region of L chain of ra-tonal antibody (%) FRL of chain L Z37332 78.3 125 FR2 of chain L Z37332 80.0 126 S65921 80.0 127 X93625 80.0 128 Chain FR3 L Z37332 71.9 129 S68699 75.0 130 P01607 71.9 131 FR4 of chain L Z37332 90.0 132 (2) DNA design encoding a humanized antibody V region The first step in the design of DNA encoding a humanized antibody V region is to select each FR of a human antibody V region that forms the basis of the design. In replacement of FR, it is required to select for each FR a highly variable FR of the region V of human antibody. For monoclonal antibody ATR-5, according to the present invention, three FRs were selected from human antibody region V for FR2 and 10 for FR3 based on the results of homology search between the V region of H chain of all mouse antibodies and each FR for the E chain. ' For the L chain, three FRs of human antibody region V for FR2 and three FRs for FR3 can be selected based on the result of the homology search between the L chain V region of mouse antibodies and each FR. For region V of H chain and region V of humanized L chain, it is possible to select V regions of L chain L39130 and Z37332, which have a high homology with V region of H chain and V region of L chain of mouse antibody ATR-5, respectively. In order to allow easy replacement of FR in the generation of these humanized antibodies, it is possible to design suitable restriction enzyme recognition sites at suitable sites in each CDR and FR. In this way, only one of the FRs can easily be replaced. Examples of such sites include an EcoT22I restriction enzyme recognition site in the humanized H chain CDR1, a restriction enzyme recognition site Ball in CDR2, a recognition site for the restriction enzyme Ncol in CDR3 and a Xhol restriction enzyme recognition site in FR3, for example, an AflII restriction enzyme recognition site in humanized L chain CDR1, a Spel restriction enzyme recognition site in CDR2, a recognition site of the restriction enzyme PstI in CDR3, and a recognition site of the restriction enzyme AccIII in FR3. Based on the a version designed in this way, FR replacement can be carried out for each FR to obtain a humanized antibody having the desired activity. (3) Preparation of a fragment of region V of humanized antibody The humanized antibody of the present invention is such that the FRs of the C region and of the V region of said antibody are derived from a human antibody and the CDR from the V region. it is derived from a mouse antibody. Fragments of region V of the humanized antibody of the present invention can be generated by a method known as CDR grafting through the polymerase chain reaction method, if DNA fragments of the human antibody are available as Co-tempering or used herein, "CDR grafting" is a method in which a DNA fragment encoding the CDR of a mouse antibody is generated, which is exchanged by the CDR of a human antibody as an antibody. When DNA fragments of a human antibody are not available as a template, the nucleotide sequence registered in the database can be synthesized using a DNA synthesizer, and the V region of humanized antibody can be generated using the chain reaction method of polymerase In addition, when the amino acid sequence is recorded only in the database, the entire nucleotide sequence can be deduced based on the codon usage frequency of antibodies reported by Katat, E.A. et al. (US Department of Health and Human Services, US Government Printing Offices, 1991). The nucleotide sequence can be synthesized using a DNA synthesizer, and fragments of humanized antibody V region can be generated using the polymerase chain reaction method. (i) Construction of DNA and an expression vector encoding the humanized H chain V region In accordance with the present invention, the DNA encoding the humanized H chain V region can be constructed by obtaining the gene encoding the H chain V region of a human antibody to be used as a template for humanized antibody and then by synthesis of the entire nucleotide sequence of DNA encoding the humanized H chain V region using a DNA synthesizer, followed by the polymerase chain reaction method. For example, L39130 which has a high homology to the H chain V region of mouse antihuman TF monoclonal antibody ATR-5 can be generated as the humanized H chain V region "a" version. In order to generate the humanized chain H region V version "a", for example, 5 primers are used separately in accordance with the provisions of SEQ ID NOS: 22-26 and two exogenous primers in accordance with that established in SEQ. ID NOS: 27-28. The CDR graft primers hR5HvlS (SEQ ID NO: 22), Hr5Hv2S (SEQ ID NO: 23), and hR5Hv4S (SEQ ID NO: 24) have a sense DNA sequence and the CDR hR5Hv3A graft primers (SEQ. ID NO: 25) and hR5Hv5A (SEQ ID NO: 26) have an anti-sense DNA sequence, each having a complementary sequence of 18-35 base pairs at both ends of the primers. HR5HvlS is designed in such a way that it has the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol. 196: 947-950, 1987) and a Sali recognition site, and hR5Hv5A is designed in such a way that has a Nhel recognition site. The exogenous Initiators hR5HvPrS (SEQ ID NO: 27) and hR5HvPrA (SEQ ID NO: 28) also have homology with the CDR graft primers hR5HvlS and hR5Hv5A. Using the polymerase chain reaction method, five primers are assembled to synthesize a full-length cDNA, and after adding an exogenous primer, DNA is amplified. The assembly by the polymerase chain reaction method as used herein means that hR5HvlS, hR5Hv2S, hR5Hv4S, hR5Hv3A, and hR5Hv5A are fused by their complementary sequences and the DNA from the full length humanized H chain V region is synthesized. The H chain C region of human antibody can be any C region of human H chain, and there may be mentioned for example C? I, C? 2, C? 3, or C? 4 of human H chain. The -DNA of the H chain V region of humanized antibody constructed in accordance with the aforementioned can be linked to the DNA of any H chain C region of human antibody, e.g., C? Lo C? 4 of H chain C region. human In accordance with that described in the construction of an H chain of chimeric antibody, after treatment with a suitable restriction enzyme, it binds to the DNA encoding the C region of human H chain under the control of a regulatory region of expression as for example, an enhancer / promoter system for generating an expression vector containing the humanized H chain V region DNA and human H chain C region. (ii) Construction of DNA and an expression vector encoding the humanized L chain V region. As in the case of DNA encoding the H chain V region, according to the present invention, a DNA encoding the H chain can be constructed. humanized L-chain region V by obtaining a gene of the L chain V region of a human antibody to be used as an annealing and then synthesizing the entire nucleotide sequence of the DNA encoding the humanized L chain V region using a DNA synthesizer. DNA, followed by the polymerase chain reaction method. For example, Z37332 which has high homology to the L chain V region of the mouse anti-human TF monoclonal antibody ATR-5 can be generated as the "a" version of humanized L chain V region. In order to generate the "a" version of humanized chain 1 V region, the CDR graft primers h5LvlS (SEQ ID NO: 85) and h5Lv4S (SEQ ID NO: 86) have a sense DNA sequence and the CDR graft initiators h5Lv2A (SEQ ID NO: 87), h5Lv3A (SEQ ID NO: 88), and h5Lv5A (SEQ ID NO: 89) have an anti-sense DNA sequence, each having a complementary sequence of 20 base pairs at both ends of the primer. The h5LvlS primer is designed in such a way that it has the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol. 196: 947-950, 1987) and a recognition site of the restriction enzyme BglII, and h5Lv5A is also designed in such a way that it has a recognition site for the restriction enzyme SplI. The exogenous primers h5LvS (SEQ ID NO: 90) and h5LvA (SEQ ID NO: 91) also have a homology with the CDR graft primers h5LvlS and h5Lv5A. As in the case of the humanized H chain V region, using the polymerase chain reaction method, 5 primers are assembled to synthesize full-length cDNA, and after adding the exogenous initiator, the DNA can be amplified.

The L chain region C of human antibody can be any human L chain C region and, for example, we can mention C? and human L chain CK. The L chain V region of humanized antibody constructed in accordance with the above can be linked to the DNA of any human L chain C region, for example, a region derived from the CK O C region? of human L chain. After treatment with a suitable restriction enzyme, it is linked to DNA encoding a human L chain C region under the control of an expression regulatory region such as an enhancer / promoter system in order to generate an expression vector that contains DNA encoding the humanized L chain V region and the human L chain K chain C region. Even when a V region fragment is generated as a humanized antibody, as mentioned above, it is not always clear whether said V region fragment has an activity as an antibody (ie, antigen binding activity, antigen neutralization activity, etc.) . Thus, it is necessary to investigate the presence of the activity by a combination with a humanized H chain and expressed in an animal cell, for example COS-7. (iii) FR replacement of L-chain region V and humanized antibody H chain The present inventors have performed the transient expression of a humanized antibody containing a V region of L chain and humanized H chain in an animal cell, e.g. COS-7 to investigate antigen binding activity and neutralizing activity, and have found that the antibody has antigen binding and neutralization activity but that the activity is not adequate as compared to a chimeric antibody. The present inventors can solve this problem by sequentially replacing each FR of the V region of L chain and humanized H chain. The antibody used in FR replacement can be selected from the existing databases. The FR of the selected human antibody can be synthesized based on the nucleotide sequence shown in the database using a DNA synthesizer. At the same time, as mentioned above, by the addition of the restriction enzyme recognition sequences designed for CDR or FR, it can easily be replaced with the FR of the L chain region V and H chain of the humanized antibody generated above. By investigating the activity of the humanized antibody generated in this way, a humanized antibody having antigen binding and neutralizing activity can be obtained. For example, H chain FR3 of humanized antibody region V can be replaced by FR3 derived from human antibody Z34963 (GenbBank, Borrentzen M. et al., Proc. Nati, Acad. Sci. USA, 91: 12917-12921, 1994 ). The FR Replacement Initiator F3RFFS (SEQ ID NO: 35) and F3RFBS (SEQ ID NO: 36) have a sense DNA sequence, and F3RFFA (SEQ ID NO: 37) and F3RFBA (SEQ ID NO: 38) have an anti-sense DNA sequence. The FR replacement primers F3RFFS, F3RFBS, F3RFFA, and F3RFBA can be synthesized using a DNA synthesizer. F3RFFS and F3RFFA, and F3RFBS and F3RFBA were fused, were digested with Ball i Xhol, and Ncol and Xhol, respectively.

Through its introduction in the hATRSHva / CVIDEC plasmid (Ball / Ncol) prepared by digestion with Ball and Ncol, and confirming the nucleotide sequence thereof, a plasmid having the correct sequence can be obtained. The plasmid obtained in this way containing the H chain of humanized antibody was designated hATR5Hvb / CVIDEC, and the humanized H chain contained in the plasmid hATR5Hvb / CVIDEC was designated as version "b". The nucleotide sequence and corresponding amino acid sequences appear in SEQ ID NO: 39, and the amino acid sequence of version "b" appears in SEQ ID NO: 40. Similarly, FR is derived from chain region B H and L chain of another human antibody selected from the database can also be replaced by the FR of the V region of H chain and L chain of a humanized antibody.

In order to select a more preferred human antibody to replace the FR of the H chain V region and the L chain V region of a humanized antibody, the following can be carried out. Thus, a combination of the "B" version of H chain of humanized antibody and an L chain of chimeric antibody has a neutralizing activity equal to the neutralizing activity of a chimeric antibody or a mouse antibody. However, the combination of the "b" version of H chain of humanized antibody and the "a" version of L chain of humanized antibody has a lower neutralizing activity than the neutralizing activity of a chimeric antibody or a mouse antibody. In such cases, in order to select a human antibody to be a candidate for replacement of FR, a homology search may be carried out, for example, for FR3 (access No. Z34963: SEQ ID NO: 115) of the "b" version of H chain of humanized antibody and a human antibody having a high homology with this sequence can be obtained. For example, it can be mentioned as FR3 of region V of chain H of the human antibody selected in this way, U95239 (SEQ ID NO: 122) and L03147 (SEQ ID NO: 123) can also be mentioned. The amino acid sequences of the H chain of region V of humanized antibody generated in this way appear in Tables 3 and 4, and the amino acid sequence of the L chain of region V of the humanized antibody appears in Table 5. Table 3 Sequences of amino acids of the V chain region H FRl CDR1 1 2 3 123456789012345678901234567890 12345 L39130 (a) QVQLLESGAVLARPGTSVKISCKASGFNIK DYYMH Z34963 (b) M30885 (c) M62723 (d) Z80844 (e) 'L04345 (f) S78322 (g) Z26827 (h) U95239 (i) L03147 (j) P01742 (bl) P01742 (di) Z80844 (b3) Z80844 (d.3) FR2 CDR2 5 6 67890123456789 012A3456789012345 L39130 (a) WVKQRPGQGLE IG GNDPANGHSMYDPKFQG Z34963 (b) M30885 (c) M62723 (d) Z80844 (e) L04345 (f) S78322 (g) Z26827 (h) U95239 (i) L03147 (j) P01742 (bl) -RA M- P01742 (dl) -RA M- Z80844 (b3) --RA Z80844 (d3) -RA Table 4 Amino acid sequences of the V region of H chain (continued) FR3 CDR3 7 8 9 10 67890123456789012ABC345678901234 56789012 L39130 (a) RAKLTAATSASIAYLEFSSLTNEDSAVYYCAR DSGYAMDY Z34963 (b) -VTI-D-TNT-ML RS-TI M30885 (c) -VTMLVD - KNQFS-RL - V-AA-T M62723 (d) -VTI-DE-TT - ML RS F Z80844 ( e). -VSl-DE-TK M-LN-RS-TF L04345 (f) -VTI-DT-TT-M-LR-RSD-T S78322 (g) KT DE-ST-MQL RS S Z26827 (h) -VTMS -DK-SA QWT-KAS-TIF- U95239 (i) -VTI-D-T-TVFM-L RS-T L03147 (j) -VTF-D NT - M-LR - RSA-T P01742 (bl) -VTI-D-TNT-M-L RS-T-I P01742 (dl) -VTI-DE-T-T-M-L RS F- Z80844 (b3) -VTI-D - TNT - M-L RS-T-I- Z80844 (d.3) -VTI-DE-TT-ML RS F- FR4 11 34567890123 L39130 (a) WGQGTLVTVSS Z34963 (b) M30885 (c) M62723 (d) Z80844 (e) L04345 (f) S78322 (g) Z26827 (h) U95239 (i) L03147 (j) P01742 (bl) P01742 (dl) Z80844 (b3) Z80844 (d3) Table 5 Amino acid sequences of the L chain V region FRI CDR1 1 2 12345678901234567890123 45678901234 Z37332 (a) DIQMTQSPSSLSASVGDRVTITC KASQDIKSFLS S68699 (b) P01607 (c) S65921 (bl) X93625 (b2) FR2 CDR2 567890123456789 0123456 Z37332 (a) WYQQKPGKAPKLLIY YATSLAD S68699 (b) P01607 (c) S65921 (bl) X93625 (b2) FR3 CDR3 6 7 8 78901234567890123456789012345678 901234567 Z37332 (a) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQHGESPYT S68699 (b) and P01607 (c) S65921 (bi: Y X93625 (b2) Y FR4 10 8901234567 Z37332 (a) FGGGTKVEIK S68699 (b) P01607 (c) S65921 (bl) X93625 (b2) Each Version of the H chain V region and L chain of the humanized antibody constructed in this way may be linked to the DNA of any human H chain C region or human L chain C region, for example, the H chain C 4 regions. human and human L chain CK, respectively.After treatment with a suitable restriction enzyme, it binds to a DNA encoding the C? 4 region of human H chain and human L chain CK region under the control of a regulatory region of the expression as for example an enhancer / promoter system, and an expression vector containing a DNA encoding each version of the V region of H chain and humanized L chain and a DNA coding for the C? 4 region of human H chain and the human L chain CK region is generated. A DNA encoding the H chain V region of humanized antibody and the human H chain C region constructed as above and a DNA encoding the humanized L chain V region and the human L chain C region are entered into a single vector (see, for example, WO 94/11523) and then said vector is used to transform a host cell. Then, the transformed host can be cultured in vivo or in vitro to produce the desired humanized antibody. 5. Production of chimeric antibody and humanized antibody In order to produce a chimeric antibody or a humanized antibody, a DNA encoding a V region of H chain and a C region of H chain and a DNA encoding a V region can be ligated of L chain and a C chain region L to a unique vector, which is transformed into a suitable host cell to produce antibody. Thus, for the expression of chimeric antibody, a DNA encoding a mouse leader sequence in the cloned cDNA and a mouse H chain V region and a human H chain C region and a DNA encoding a mouse leader sequence and a mouse L-chain V region and a human H-chain C region are introduced into a single expression vector (see, for example, WO 94/11523) under the control of a regulatory region of expression as for example an enhancer / promoter system. For the expression of a humanized antibody, a DNA encoding a humanized H chain V region and a humanized H chain C region and a DNA encoding a humanized L chain V region and a human H chain C region are introduced into a unique expression vector (see, for example, WO 94/11523) under the control of a regulatory region of expression such as for example an enhancer / promoter system. These vectors are used to transform a host cell. Then, the transformed host cell can be cultured in vivo or in vitro, and therefore the chimeric antibody or the humanized antibody can be produced. Two expression vectors can also be generated, each containing a V region of H chain and a V region of L chain. Thus, for chimeric antibody, an expression vector containing a DNA encoding a V region of H chain of mouse and a human H chain C region under the control of an enhancer / promoter system and an expression vector containing DNA encoding a mouse L chain V region and a human L chain C region under the control of a system enhancer / promoter are generated, and for a humanized antibody an expression vector is generated which contains a DNA encoding a humanized H chain V region and a human H chain C region under the control of an enhancer / promoter system and a expression vector which contains a DNA encoding a humanized L chain V region and a human L chain C region under the control of an enhancer / promoter system.

Alternatively, for the chimeric antibody, an expression vector is generated which contains a DNA encoding a mouse H chain V region and a human H chain C region and a DNA encoding a mouse L chain V region and a human L chain C region under the control of an expression regulatory region such as an enhancer / promoter system, and for the humanized antibody an expression vector containing a DNA encoding a humanized H chain V region is generated and a human H chain C region and a DNA encoding a humanized L chain V region and a human L chain C region under the control of an expression regulatory region such as an enhancer / promoter system. Then, these expression vectors are used to cotransform host cells such as for example mammalian cells, and the transformed cells are cultured in vitro or in vivo to produce a chimeric antibody or a humanized antibody (see, for example, WO 94 / 16928). As mentioned above, a transformant transformed with a gene encoding the desired chimeric antibody or humanized antibody is cultured, and the chimeric antibody produced or the humanized antibody produced can be separated from the inner or outer part of the cell and purified until obtaining the homogeneity.

The isolation and / or purification of the chimeric antibody or humanized antibody, or the desired protein of the present invention, can be effected by the use of a Protein A Sepharose column. Other methods include, but are not limited to, separation and / or purification methods employed for common proteins. By way of example, chimeric antibodies or humanized antibodies can be isolated and / or purified by the combination, as appropriate, of various chromatographic methods, ultracentrifugation, precipitation by addition of salts, dialysis, and the like. In order to produce the chimeric antibody or the humanized antibody of the present invention against human TF, any expression system can be employed. For example, when eukaryotic cells are used, cells from animals (e.g., established mammalian cell lines), fungal cells or yeast cells can be used and, when prokaryotic cells are used, bacterial cells are used (e.g. Escherichia coli cells). Preferably, the chimeric antibody or the humanized antibody of the present invention is expressed in mammalian cells such as COS cells or CHO cells. In these cases, common common promoters can be used for expression in mammalian cells. For example, the human cytomegalovirus immediate early promoter (HCMV) is preferably used. Examples of expression vectors containing the HCMV promoter include HCMV-VH-HC1, HCMV-VL-HCK, and the like, and those derived from pSV2neo (WO 92-19759). Other promoters for gene expression in mammalian cells that can be employed in the present invention include viral promoters such as promoters of retroviruses, polyoma viruses, adenoviruses, and simian viruses (SV40), and promoters derived from mammalian cells such as a lalfa factor of human polypeptide chain elongation (HEFlalfa). For example, expression can be easily obtained by the method of Mulligan et al.

(Nature (1979) 277: 108) when the SV40 promoter is used, or by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18: 5322) when the HEFlalpha promoter is used. As the origin of replication, we can use the derivatives of SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like. In addition, amplification of the number of gene copies in the host cell system, expression vectors can include as selectable markers the phosphotransferase gene APH (3 ') II or I (neo), the thymidine kinase gene (TK), the E. coli gene xantinguaninphosforibosyltransferase (Ecogpt), the dihydrofolate reductase (dhfr) gene and the like. 6. Evaluation of antigen binding activity and neutralization activity of chimeric antibody and humanized antibody (1) Measurement of antibody concentration by ELISA The concentration of purified antibody obtained can be measured by ELISA. ELISA plates for measurement of antibody concentration can be prepared as follows. Each well of a 96-well ELISA plate (eg, Maxisorp, NUNC) is immobilized with 100 μl of IgG? anti-human goat (BíoSource) prepared at a concentration of 1 μg / ml.

After blocking with 200 μl of the division regulator (hereinafter referred to as DB; 50 mM Tris-HCl, 1 mM MgCl2, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN3, 1% bovine serum albumin (BSA), pH 7.2), the culture supernatants of the COS-7 cells or CHO cells where the antibody was expressed chimeric or the humanized antibody, either the purified chimeric antibody or the purified humanized antibodies are serially diluted, and added to each well. Then, 100 μl of goat antihuman IgG antibody conjugated with alkaline phosphatase is added, 100 μl of a 1 mg / ml substrate solution (Sigmal04, p-nitrophenylphosphate, SIGMA) is added, and then the absorbance is measured at 405 / 655 nm using the Microplate Reader (Microplate reader) (Bio Rad). As a standard for the measurement of the concentration, can IgG4 be used? Human (The Link Site). (2) Measurement of antigen binding activity ELISA plates with cells for measurement of antigen binding activity are prepared as follows: human bladder carcinoma cells J82 (ATCC HTB-1) are inoculated at 60 wells of a 96-well cell culture plate with a cell count of 1 x 10 5 cells. This is cultured (RPMI1640 medium containing 10% fetal bovine serum (GIBCO)) for one day in a C02 incubator to allow fixation of the cells there. After discarding the culture liquid, each well is washed twice with 300 μl of PBS. 100 μl of PBS containing 4% formaldehyde (hereinafter referred to as PFA / PBS) is added to each well, and placed on ice for 10 minutes to immobilize the cells. PFA / PBS is discarded, and each well is washed twice with 300 μl of PBS, and then blocked with 250 μl of DB. 100 μl of culture supernatants containing a chimeric antibody or a humanized antibody, or a "purified chimeric antibody or humanized antibodies are serially diluted, and then added to each well, and incubated at room temperature for 2 hours. of wash with rinse buffer (hereinafter known as RB PBS containing 0.05% Tween 20), 100 μl of goat anti-human IgG? antibody conjugated with alkaline phosphatase (BioSource) diluted 1000 times with DB are added. incubation at room temperature for 1 hour, and after washing with RB, the substrate solution is added, and then the absorbance is measured at 405/655 nm using a Microplate Reader (Bio Rad). (3) ) Measurement of neutralizing activity The neutralizing activity of mouse antibody, chimeric antibody and humanized antibodies can be measured with the activity of inhibition against the production of or Xa by thromboplastin derived from human placenta, Thromborel S (Boehringer AG), as index. Thus, 60 μl of the buffer (TBS containing 5 mM CaCl 2 and 0.1% BSA) is added to 10 μl of 1.25 mg / ml Thromborel S and 10 μl of an appropriately diluted antibody, which is then incubated in a 96-well plate. wells at room temperature for 1 hour. Add 10 μl each of 3,245 μg / ml human factor X (Celsus Laboratories) and 82.5 ng / ml human factor Vlla (Enzyme Research), and then incubate at room temperature for an additional hour. After adding 10 μl of 0.5 M EDTA to suspend the reaction, 50 μl of the chromogenic substrate solution is added and the absorbance at 405/655 nm is determined. After reacting the room temperature for 1 hour, the absorbance is again determined at 405/655 nm. The neutralization activity can be determined by calculating the residual activity (%) from each change in absorbency with the change in absorbance without addition of antibody as 100% activity. The chromogenic substrate solution is prepared by dissolving the chromogenic substrate Testzyme S-2222 (Chromogenix) in accordance with the attached instructions, diluting 2 times with purified water and then mixing with a solution of pollbrene (0.6 mg / ml hexadimethylene bromide, SIGMA) at 1: 1. 7. Interaction kinetic analysis of humanized antibody and soluble TF Kinetic parameters, ie dissociation constants (KD), dissociation rate constants (kdiss), and binding rate constants (kass), of anti-TF antibody of the present invention can be determined by BIACORE. A recombinant G protein is immobilized on a sensor chip, to which the antibody is connected, and purified recombinant TF _ is used (a soluble TF 1-219 where the FLAG peptide was tagged) (hereinafter referred to as soluble TF) as the antigen while soluble TF prepared in various concentrations are used as analytes. From the obtained sensorgram, the kinetic parameters are calculated (kdiss dissociation speed constant, and kass junction speed constant), from which the dissociation constant can be determined. For kinetic analysis, see, for example, "Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system" (kinetic analysis of monoclonal antibody-antigen interactions with a new analytical system based on biosensors) (Karlsson, R. et al. al., (1991) J. Immunol Methods 145: 229-240). The anti-TF antibody of the present invention preferably has a smaller value of dissociation constants (KD) since it will have a greater neutralizing activity. In an anti-TF antibody of the present invention, KD values not greater than 2.30 x 10 ~ 8 [l / M], more preferably not greater than 2.30 x 10"9 [l / M], and especially not greater than 1.17 x 10 ~ 9 [l / M] In addition, KD s * values determine from two parameters, the dissociation velocity constant (kdiss) and the joint velocity constant (kass) (KD = kdiss / kass) Therefore, it is evident that when a kdiss value is small and a kass value is large, then a KD value becomes small, specifically, in the case of the anti-TF antibody of the present invention, kdiss values can not be greater than 0.52 x 10"3 [1 / sec]. Preferably, the kdiss values are not greater than 9.52 x 10"4 [1 / sec], more preferably not greater than 6.35 x 10 ~ 4 [1 / sec.] On the other hand, kass values can not be less than 4.15 x 104 [1 / M »sec]. Preferably, kass values are not less than 4.15 x 105 [1 / M »sec], and more preferably they are not greater than 4.65 x 105 [1 / M« sec]. In addition, anti-TF antibodies preferably have a kdiss value of no greater than 9.52 x 10 ~ 3 [1 / sec] and a kass value of not less than 4.15 x 104 [1 / M * sec]. More specifically, for the anti-TF antibody of the present invention, KD values are within a range of 1.09 x 10 -10 2.30 x iCT [l / M], preferably 1. 09 x 10 -9 2.30 x 10 ~ 9 [l / M], and especially 1.09 x 10 ~ 9 - 1.39 x 10"9 [l / M] In addition, kdiss values are within the range of 5.06 x 10" 4 - 9.52 x 10"3 [ 1 / sec], preferably 5.06 x 10 ~ 4 - 9.52 [1 / sec], and especially 5.06 x 10".49 x 10" [1 / sec] And, kass values are within the range of 4.15 x 104 - 5.44 x 105 [1 / M "sec], preferably 4.15 x 105 - 5.44 x 10b [1 / M" sec], and especially 4.65 x 10 ~ 5.44 10- [1 / M »sec]. Even though these KD values, kdiss values and kass values can be obtained, in addition to BIACORE, by Scatchard analysis, and the like, the use of BIACORE is preferred. 8. Measurement of Humanized Antibody Reactivity with Human TF The method of dot blot hybridization can be used to investigate the reactivity of undenatured TF, denatured TF under non-reduced condition, and denatured TF under reduced condition. TF can be a TF that was purified from human tissue, or that was expressed in mammalian cells such as CHO cells and purified, and can be used for research. As the denaturing agent, guanidine hydrochloride or SDS, etc. may be used in place of urea. As the reducing agent, an SH reducing agent such as 2-mercaptoethanol can be used instead of DTT. To detect a humanized antibody, a human IgG antibody labeled with several substances can be used. As used herein, the labeling agents can be radioisotopes, biotin, fluorogenic substances such as FITC, enzymes such as peroxidase and alkaline phosphatase, and the like. The anti-TF antibody of the present invention reacts with any of the non-denatured TF, denatured TF under non-reduced condition, and denatured TF under reduced condition. 9. Pharmaceutical compositions and therapeutic agents for DIC comprising a humanized antibody as active ingredient In order to confirm the therapeutic effect of a humanized antibody on human TF, a humanized anti-human TF antibody is administered to an animal having a symptom of elevated DIC, and then the DIC indices are measured to confirm the therapeutic effects. The antibody as used herein is a humanized antibody for human TF. The antibody neutralizes the activity of human TF by binding to human TF, and a humanized ATR5 antibody can preferably be mentioned. The method for generating a humanized ATR5 antibody is described in the examples. An antibody as used herein can be purified to a high degree of purity by the combination of common purification methods such as precipitation by addition of salts, gel filtration method such as HPLC, affinity chromatography using a Protein A column, and the like. Antibody purified in this way can be confirmed to recognize human TF with high precision using common immunological means such as for example radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), or immunofluorescent antibody method (immunofluorescence analysis), and the like . Pharmaceutical compositions or therapeutic agents for DIC of the present invention comprising as an active ingredient the humanized antibody against TF can be administered non-orally, either systemically or locally. For example, the method of administration may be selected from intravenous injection such as, for example, drop infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection, and may be selected, as appropriate, based on the age and conditions of the patient. The effective dosage is selected within the range of 0.01 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage of 10 mg / body, preferably 1 to 1000 mg / body per patient can be selected. Pharmaceutical compositions and therapeutic agents for DIC of the present invention containing a humanized antibody against human TF as an active ingredient may contain pharmaceutically acceptable carriers or additives according to the route of administration. Examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble dextran, sodium carboxymethylstarch, pectin, methylcellulose, ethylcellulose , xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like . The additives employed are selected, without being limited thereto, from the foregoing or combinations thereof, as appropriate, depending on the dosage form of the present invention. EFFECTS OF THE INVENTION According to the present invention, there is provided a chimeric antibody and a humanized antibody against human TF, and a process for generating a humanized antibody. These antibodies are useful as therapeutic agents due to their low antigenicity. EXAMPLES The present invention will be explained below with further details with reference to the following examples. EXAMPLE 1. CLONING OF DNA CODING REGION D OF A MONOCLONAL MOUSE ANTIBODY AGAINST HUMAN TF (1) Preparation of mRNA MRNA was prepared from hybridomas ATR-2, ATR-3, ATR-4, ATR-5 (IgGlK ), ATR-7, and ATR-8 (IgG2a?) Using the QuickPrep mRNA purification kit (Pharmacia Biotech). Each hybridoma cell was fully homogenized in the extraction buffer according to instructions attached to the kit and then mRNA was purified through the spin column of oligo (dT) -cellulose, followed by ethanol precipitation. The precipitate of mRNA was dissolved in the elution buffer. (2) Preparation and amplification of cDNA of the gene encoding a V region of mouse antibody, (i) Cloning of V-region cDNA of H chain. Cloning of the gene encoding the V region of H chain of a monoclonal antibody of Mouse against human TF was carried out using the 5 '-RACE method (Frohman, MA et al., Proc. Nati. Acad. Sci. USA 85: 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17: 2919-2932, 1989). For the 5 '-RACE method, the Marathon cDNA amplification kit (CLONTECH) was used and the procedure was carried out with the instructions attached to the kit. Employing about 1 μg of mRNA prepared in accordance with the above as quenched, the cDNA synthesis primer attached to the kit was added, which reacted with a reverse transcriptase at a temperature of 42 degrees C for 60 minutes to effect reverse transcription to cDNA . Reacted with DNA polymerase I, DNA ligase and RNaseH, at a temperature of 16 degrees C for 1.5 hours, and with T4 DNA polymerase at a temperature of 16 degrees C for 45 minutes thus synthesizing a double-stranded cDNA. The double-stranded cDNA was extracted with phenol and chloroform, and recovered by ethanol precipitation. By overnight reaction with T4 DNA ligase at a temperature of 16 degrees C a cDNA adapter was ligated to both ends of the double stranded cDNA. The reaction mixture was diluted 50 times with lOmM tricine-KOH (pH 8.5) containing 0.1 mM EDTA. Using this as an annealing, the gene coding for the H chain V region was amplified by polymerase chain reaction. The adapter primer 1 attached to the kit was used for the 5 'end primer and for the 3' primer, used the initiator MHC-G1 (SEQ ID NO: 1) (ATR-2, ATR-3, ATR-4 and ATR-5) or the initiator MHC-G2a (SEQ ID NO: 2) (ATR-7 and ATR -8) (ST Jones, et al., Biotechnology, 9: 88-89, 1991). Polymerase chain reaction solutions for the H chain V region of ATR-2,3,4 and 5 antibody contained in lOOμl, 120mM tris-HCL (pH-8.0), 10mM KCL, 6mM (NH4) 2S04 , 0.1% triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 mM MgCl2, 2.5 units KOD DNA polymerase (Toyo Boseki), 30-50 Pmol starter adapter 1, as well as an MHC-G1 primer, and l-5μl of a cDNA reaction mixture to which the cDNA adapter was ligated. All polymerase chain reactions were performed using the DNA thermal cycler 480 (Perkin-Elmer), and the polymerase chain reaction was carried out for 30 cycles at a temperature cycle of 94 degrees C for 30 seconds, 55 degrees C for 30 seconds, and 74 degrees c for 1 minute. (ii) cloning of L chain V region cDNA The cloning of the g coding for the L chain V region of a mouse monoclonal antibody against human TF was carried out using the 5 '-RACE method (Frohman, MA et al. , Proc. Nati, Acad. Sci. USA 85: 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res. 17: 2919-2932, 1989). For the 5 '-RACE method, the Marathón cDNA amplification kit (CLONTECH) was used and the procedure was carried out with the instructions attached to the kit. Using about 1 μg of mRNA prepared in accordance with the above as annealed, the cDNA synthesis primer attached to the kit was added, which reacted with a reverse transcriptase at a temperature of 42 degrees C for 60 minutes to effect reverse transcription to cDNA . Reacted with DNA polymerase I, DNA ligase and RNaseH, at a temperature of 16 degrees C for 1.5 hours, and with T4 DNA polymerase at a temperature of 16 degrees C for 45 minutes thus synthesizing a double-stranded cDNA. The double-stranded cDNA was extracted with phenol and chloroform, and recovered by ethanol precipitation. By overnight reaction with T4 DNA ligase at a temperature of 16 degrees C a cDNA adapter was ligated to both ends of the double stranded cDNA. The reaction mixture was diluted 50 times with lOmM tricine-KOH (pH 8.5) containing 0.1 mM EDTA. Using this as an annealing, the gene coding for the V region of the L chain was amplified by polymerase chain reaction. The adapter primer 1 attached to the kit was used for the 5 'end primer and for the 3' end primer, the MKC primer (SEQ ID NO: 3) was employed (ST Jones, et al., Biotechnology, 9: 88-89, 1991). The polymerase chain solutions contained, in lOOμl, 120 mM tris-HCL (pH-8.0), 10mM KCL, 6mM (NH4) 2S04, 0.1% triton X-100, 0.001% BSA, 0.2 mM dNTPs ( dATP, dGTP, dCTP, dTTP), 1 mM MgCl2, 2.5 units of KOD DNA polymerase (Toyo Boseki), 30-50 Pmol of adapter 1 primer, as well as an MKC primer, and 1 μl of a cDNA reaction mixture to which the cDNA adapter was ligated. All polymerase chain reactions were performed using the DNA thermal cycler 480 (Perkin-Elmer), and the polymerase chain reaction was carried out for 30 cycles at a temperature cycle of 94 degrees C for 30 seconds, 55 degrees C for 30 seconds, and 74 degrees c for 1 minute. (3) Purification and fragmentation of polymerase chain reaction products The above polymerase chain reaction mixture was extracted with phenol and chloroform, and the amplified DNA fragments were recovered by ethanol precipitation. DNA fragments were digested with the restriction enzyme Xmal (New England Biolabs) at a temperature of 37 degrees C for 1 hour. The Xmal-digestion mixture was separated by agarose gel electrophoresis using 2% -3% NuSieve GTG agarose (FMC BioProducts) and the agarose strips containing approximately 500 base pairs of long DNA fragments as the V region of hya chain approximately 500 base pairs of long DNA fragments as the V region of L chain were removed. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol, which were then dissolved in lOμl of lOmM Tris-HCL (pH 8.0) containing 1 mM EDTA (hereinafter known as TE). The DNA fragments digested with X to the preparations according to the above contained genes coding for a V region of H chain of mouse and a V region of L chain of mouse and plasmid vector Pucl9 prepared by Xmal digestion were ligated using the kit of DNA ligation ver. 2 (Takara 'Shuzo) by reaction at a temperature of 16 degrees C for 1 hour in accordance with the instructions attached to the kit. The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 degrees C., 300 μl of the Hi-Competence Broth (high competition broth) (Nippongene) was added, being incubated at a temperature of 37 degrees C for 1 hour. Escherichia coli was then plated on an LB agar medium (Molecular cloning: A Laboratory Manual, (J. Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989). contained 100 μg / ml of ampicillin (hereinafter referred to as LBA agar medium), and incubated overnight at 37 ° C to obtain an E. coli transformant.The transformant was grown overnight in 3 ml or 4 ml of LB medium containing 50 μg / ml of ampicillin (hereinafter referred to as the LBA medium) at a temperature of 37 degrees C, and from the cell fractions, plasmid DNA was prepared using the QIAPrep Spin Plasmid Kit (Rotating Plasmid Kit) (QIAGEN), and then the nucleotide sequence was determined. (4) Determination of the nucleotide sequence of the gene encoding a V region of mouse antibody The nucleotide sequence of the region encoding cDNA in the previous plasmid was determined by using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (FS Reaction Kit sequencing cycle of dye terminator) (Perkin-Elmer) through the use of the DNA sequencer 373A (Perkin-Elmer) . The initiator M13 M4 (Takara) 1 was used as sequencing primer.

Shuzo) (SEQ ID NO: 4) and the M13 RV primer (Takara Shuzo) (SEQ ID NO: 5) and the sequence was determined by confirmation of the nucleotide sequence in both directions. The plasmids obtained in this way containing the gene coding for the V region of H chain of mouse derived from the hybridomas ATR-2, ATR-3, ATR-4, ATR-5, ATR-7, and ATR-8) were designated as ATR-xLv / pUC19 (x = 2, 3, 4, 5, 7, or 8), and plasmids obtained in this manner containing the gene encoding a V region of L-chain of mouse derived from hybridomas ATR-2, ATR-3, ATR-4, ATR-5, ATR-7, and ATR-8) were designated as ATR-xLv / pUC19 (x = 2, 3, 4, 5, 7, or 8) . The nucleotide sequences of the genes encoding the H chain V region of each mouse antibody contained in the plasmid ATR-xHv / pUC19 (x = 2, 3, 4, 5, 7, or 8) (including the sequences of corresponding amino acids) is shown in SEQ ID NO: 6 to 11, respectively, and the nucleotide sequences of the genes encoding the L chain V region of each mouse antibody contained in the plasmid ATR-xLv / pUC19 (x = 2, 3, 4, 5, 7, or 8) (including the corresponding amino acid sequences) are shown in SEQ ID NO: 12 17, respectively. EXAMPLE 2 CONSTRUCTION OF CHEMOTERIC ANTIBODY A chimeric ATR-5 antibody was generated in which the V region of mouse ATR-5 antibody was ligated onto the C region of human antibody. A chimeric antibody expression vector was constructed by ligating the gene encoding the V region of the ATR-5 antibody to an expression vector encoding the human antibody C region. (1) Construction of an H Chain V Region of Chimeric Antibody The H chain V region of ATR-5 antibody was modified by polymerase chain reaction method in order to ligate it on an expression vector encoding the region H chain of human antibody. The 5 'end primer ch5HS (SEQ ID NO: 18) was designed in such a way that it hybridizes to the 5' end of the DNA encoding the V region and in such a way that it has the Kozak consensus sequence (Kozak, M et al., J. Mol. Biol. 196: 947-950, 1987) and a recognition sequence of the restriction enzyme Salí. The 3 'end primer ch5HS (SEQ ID NO: 19) was designed in such a way that it hybridizes to the 3' end of the DNA encoding the V region and such that it has an Nhel restriction enzyme recognition sequence. . The polymerase chain reaction solutions contained, in lOOμl, 120 mM tris-HCL (pH-8.0), 10mM KCL, 6mM (NH4) 2S04, 0.1% triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 mM MgCl2, 2.5 units of KOD DNA polymerase (Toyo Boseki), 50 pmol primer ch5HS and initiator ch5HA and a 1 .mu.l of plasmid ATR5Hv / pUC19 as DNA tempered. For the polymerase chain reaction, a DNA thermal cycler 480 (Perkin-Elmer) was used, and the polymerase chain reaction was carried out for 30 cycles at a temperature cycle of 94 degrees C for 30 seconds, 55 degrees C for 30 seconds, and 74 degrees C for 1 minute. The mixture of the polymerase chain reaction was extracted with phenol and chloroform, and the amplified DNA fragments were recovered by precipitation with ethanol. DNA fragments were digested with the Nhel restriction enzyme (Takara Shuzo) at a temperature of 37 degrees C for 1 hour, and then with the restriction enzyme Sali (Takara Shuzo) at a temperature of 37 degrees C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using 3% NuSieve GTG agarose (FMC BioProducts) and the agarose strips containing DNA fragments of approximately 450 base pairs long were removed. The agarose strips were extracted with phenol and chloroform, and the DNA fragments were precipitated with ethanol, which were then dissolved in 20 μl of TE. As a cloning vector, an altered promoter vector (known below as CVIDEC) was used where the recognition sequences of the restriction enzymes Nhel, Sali, and Spll, BglII were introduced. The gene fragment prepared as above coding for the H chain V region of the mouse and the CVIDEC vector prepared by digestion with Nhel and Sali were ligated using the DNA ligation kit ver. 2 (Takara Shuzo) by reaction at a temperature of 16 degrees C for 1 hour in accordance with the instructions attached to the kit. The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 degrees C. Afterwards, 300 μl of Hi-Competence was added. Broth (high competition broth) (Nippongene), was incubated at a temperature of 37 degrees C for 1 hour, and then plated on an LBA agar medium and incubated overnight at a temperature of 37 degrees C for get an E.coli transformant. The transformant was cultured overnight at a temperature of 37 degrees C in 3 ml of LBA medium, and from the cell fractions, plasmid DNA was prepared using the QIAPrep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the region encoding cDNA in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer) DNA sequencer 373A (Perkin-Elmer). The M13 M4 primer (Takara Shuzo) (SEQ ID NO: 4) and the M13 RV primer (Takara Shuzo) (SEQ ID NO: 5) were used as sequencing primer, and the sequence was determined by confirming the sequence of nucleotides in both directions. The plasmid containing the gene encoding the H chain V region of ATR-5 antibody, and having a Sali recognition sequence and the Kozak consensus sequence at the 5 'end and an Nhel recognition sequence in the 3 'end was designated as ChATR5Hv / CVIDEC. (2) Construction of a V region of L chain of chimeric antibody. The V chain region V of ATR-5 antibody was modified by polymerase chain reaction method in order to ligate it with an expression vector encoding the C region of L chain of human antibody. The 5 'end primer ch5LS (SEQ ID NO: 20) was designed to hybridize to the 5' end of the DNA encoding the V region and in such a way that it has the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol. 196: 947-950, 1987) and a recognition sequence of the restriction enzyme SplI.

The 3 'end primer ch5LA (SEQ ID NO: 21) was designed in such a way that it hybridizes to the 3' end of the DNA encoding the J region and in such a way that it has a recognition sequence of the restriction enzyme SplI . The polymerase chain reaction solutions contained, in 100 μl, 120 mM tris-HCL (pH-8.0), 10 mM KCL, 6 mM (NH4) 2 SO4, 0.1% triton X-100, 0.001% BSA, 0.2 mM of dNTPs (dATP, dGTP, dCTP, dTTP), 1 mM of MgCl2, 2.5 units of KOD DNA polymerase (Toyo Boseki), 50 Pmol of the ch5LS primer and of the ch5LA primer, as well as 1 μL of the plasmid ATR5Lv / pUC19 as Tempering DNA For the polymerase chain reaction, a DNA thermal cycler 480 (Perkin-Elmer) was used, and the polymerase chain reaction was carried out for 30 cycles at a temperature cycle of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 74 ° C for 1 minute. The mixture of the polymerase chain reaction was extracted with phenol and chloroform, and the amplified DNA fragments were recovered by precipitation with ethanol. DNA fragments were digested with the restriction enzyme Spll (Takara Shuzo) at a temperature of 37 ° C for 1 hour, and then with the restriction enzyme BglII (Takara Shuzo) at a temperature of 37 ° C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using 3% NuSieve GTG agarose (FMC BioProducts) and the agarose strips containing DNA fragments approximately 400 base pairs long were removed. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol, which were then dissolved in 20 μl of TE. The gene fragment prepared according to the above coding for the V region of L chain of mouse and the vector CVIDEC prepared by digestion with Spll and BglII were ligated using the DNA ligation kit ver. 2 (Takara Shuzo) by reaction at a temperature of 16 ° C for 1 hour in accordance with the instructions enclosed with the kit. The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. Then, 300 μl of the Hi-Competence Broth (Nippongene) was added, incubated at a temperature of 37 ° C for 1 hour, and then coli was placed on a 100 μg / ml LBA agar medium and the incubated overnight at a temperature of 37 ° C to obtain an E.coli transformant. The transformant was cultured overnight at a temperature of 37 ° C in 3 ml of the LBA medium, and from the cell fractions, plasmid DNA was prepared using the QTAPrep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the region encoding cDNA in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer) through the use of the DNA sequencer 373A (Perkin-Elmer). The M13 M4 primer (Takara Shuzo) and the M13 RV primer (Takara Shuzo) were used as sequencing primer, and the sequence was determined by confirmation of the nucleotide sequence in both directions. The plasmid containing the gene encoding the L chain V region of ATR-5 antibody, and having a BglII recognition sequence and the Kozak consensus sequence at the 5 'end and a Spll recognition sequence at the 3 'end was designated as ChATR5Lv / CVIDEC. (3) Construction of a chimeric antibody expression vector A chimeric antibody expression vector was constructed using an antibody expression vector introduced from IDEC Pharmaceuticals. As a vector, the vector was used. of antibody expression of IgGl type H5KG1 (v) and the antibody expression vector of IgG4 type N5KG4P. The chimeric ATR-5 antibody expression vector was generated by ligating a gene encoding the H chain V region of ATR-56 over the Sall-Nhel site located immediately before the H chain C region of human antibody of the HTR vector. expression N5KG1 (v) or N5KG4P and ligand a gene encoding the V chain region V DE ATR-5 to the BglII-Spll site located immediately before the human antibody L chain C region of the N5KG1 expression vector (v) or N5KG4P. (i) Introduction of the V chain region H The plasmid chATR5Hv / CVIDEC was digested with the restriction enzyme Nhel (Takara Shuzo) at a temperature of 37 ° C for 3 hours and with the restriction enzyme Salí (Takara Shuzo) a a temperature of 37 ° C for three hours. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMC BioProducts), and agarose strips containing DNA fragments approximately 450 base pairs long were removed. The agarose strips were extracted with phenol and chloroform, and the DNA fragments were precipitated with ethanol, which were then dissolved in 20 μl of TE. The N5KG1 expression vector (V) and the N5KG4P expression vector were digested with the restriction enzyme Nhel (Takara Shuzo) at a temperature of 37 ° C for three hours, and with the restriction enzyme Sali (Takara Shuzo) at a temperatures of 37 ° C for three hours. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose strips containing DNA fragments of approximately 9,000 base pairs long were removed. The agarose strips were extracted with chloroform and phenol, and the DNA fragments were precipitated with ethanol, and were then dissolved in 20 μl of TE.

The Sall-Nhel DNA fragment prepared as above contained the gene coding for the V region of H chain and N5KG1 (V) or N5KG4P digested with Sali and Nhel were ligated using the DNA ligation kit ver. 2 (Takara Shuzo) by reaction at a temperature of 16 ° C for 1 hour in accordance with the attached instructions. The ligation mixture was added to 100 μl of competent E.coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for one minute at a temperature of 42 ° C. then 300 μl of the Hi-Competence Broth v (High Competence Broth) (Nippongene) was added, incubated at a temperature of 37 ° C for one hour, and then E, coli plated on a 100 μg LBA agar medium was placed. / ml and incubated overnight at a temperature of 37 ° C to obtain an E. coli transformant. The transformant was grown overnight at a temperature of 37 ° C in 3 ml of medium LBA, and from the cell fractions, a Plasmid DNA using the QIAprep Spin Plasmid Kit (QIAGEN). These plasmids containing the genes encoding the H chain of chimeric ATR-5 antibody were designated as chATR5Hv / N5KGl (V) and chATR5Hv / N5KG4P, respectively. (ii) Introduction of the V chain region L The plasmid chATR5Lv / CVIDEC was digested with the restriction enzymes BglII (Takara Shuzo) and Spll (Takara Shuzo) at a temperature of 37 ° C for 1.5 hours. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG Agarose (FMC BioProducts), and agarose strips containing DNA fragments approximately 400 base pairs long were removed. The agarose strips were extracted with phenol and chloroform, and the DNA fragments were precipitated with ethanol, which were then dissolved in 20 μl of TE. Plasmids chATR5Hv / N5KGl (V) and chATR5Hv / N5KG4P were digested with the restriction enzymes BglII (Takara Shuzo) and Spll (Takara Shuzo) at a temperature of 37 ° C for 1.5 hours. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose strips containing DNA fragments of approximately 9400 base pairs were removed. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol, which were then dissolved in 20 μl of TE. The SplI-BglII DNA fragment prepared according to the above indicated contained the gene coding for the V region of L chain and chATR5Hv / N5KGl (V) or chATR5Hv / N5KG4P digested with Spll and BglII were ligated using the Ligation Kit DNA see 2 (Takara Shuzo) by reaction at a temperature of 16 ° C for one hour in accordance with the enclosed instructions. The ligation mixture was added to 100 μl of competent E.coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. After, 300 μl of the Hi-Competence Broth (Nippongene) was added, incubated at a temperature of 37 ° C for one hour, and then E. coli was placed on 100 μg / LBA agar medium plates. ml and incubated overnight at a temperature of 37 ° C to provide E. coli transformant. The transformant was cultured overnight at a temperature of 37 ° C in 1 1 of 2xT medium containing 50 μg / ml of ampicillin, and from the cell fractions, plasmid DNA was prepared using the Plasmid Maxi Kit (QIAGEN) . These plasmids containing the gene encoding the chimeric ATR-5 antibody were designated chATR5 / N5KGl (V) and chATR5 / N5KG4P, respectively. (4) Transfection in COS-7 cells In order to evaluate the antigen binding activity and the chimeric antibody neutralizing activity, the aforementioned expression plasmid was transfected to COS-7 cells and the antibody was expressed transiently . The plasmid chATR5 / N5KGl (V) or chATR5 / N5KG4P was transduced in COS-7 cells by electroporation using the Gene Pulser instrument (Bio Rad). 50 μg of the plasmid was added to 0.78 ml of the COS-7 cells suspended in PBS from Dulbecco (-) (hereinafter referred to as PBS) at a cell concentration of 1 × 10 7 cells / ml, which was subjected to pulses of 1,500 V and 25 μF capacity. After 10 minutes of the recovery period at room temperature, the cells subjected to electroporation were suspended in a DMEM medium containing 5% ultra-low IgG fetal bovine serum (GIBCO), and cultured using a 10 cm culture dish. in an incubator with 5% C02. After culturing for 24 hours, the culture supernatant was removed by aspiration, and then a serum free HBCHO medium (Irvine Scientific) was added. After cultivating for 72 hours, the culture supernatant was collected and centrifuged to recover the cell debris. (5) Purification of antibody From the culture supernatant of COS-7 cells, a chimeric antibody was purified using rProtein A Sepharose Fast Flow (Pharmacia Biotech) in the following manner. One ml of rProtein A Sepharose Fast Flow was filled into a column and the column was equilibrated by 10 volumes TBS. The culture supernatant of the COS-7 cells was applied to the equilibrated column, which was then washed with 10 volumes of TBS.

The adsorbed antibody fraction was then eluded by 13.5 ml of 2.5 mM HCL (pH 3.0), and the eluate was immediately neutralized by the addition of 1.5 ml of 1M Tris-HCL (pH 8.0). By performing an ultrafiltration twice for the purified antibody fraction using Centriprep 100 (Amicon), the solvent was replaced by 50 mM Tris-HCL (pH 7.6) with 150 mM NaCl (then TBS), and was finally concentrated to approximately 1.5 ml. (6) Establishment of a CHO cell line of stable production. In order to establish a cell line stably producing a chimeric antibody, the aforementioned expression plasmid was introduced into CHO cells (DG44) acclimated to the serum free medium CHO-S-SFMII (GIBCO). The plasmid chATR5 / N5KG1 (V) or chATR5 / N5KG4P was dissociated with the restriction enzyme SspI (Takara Shuzo) to linearize DNA, and after extraction with phenol and chloroform, the DNA was recovered by precipitation with ethanol. The linearized plasmid was transduced in DG44 cells by electroporation using the Gene Pulser instrument (Bio Rad). 10 μg of the plasmid was added to 0.78 ml of DG44 cells suspended in PBS at a cell concentration of 1 × 10 7 cells / ml, and pulsed at 1,500 V and 25 μF capacity.

After 10 minutes of the recovery period at room temperature, the electroporated cells were suspended in a CHO-S-SFMII (GIBCO) medium containing hypoxanthine / thymidine (GIBCO), and cultured using two 96-well plates (Falcon) in a 5% C02 incubator. The day after the start of the culture, the medium was changed to a selection medium containing the medium CHO-S-SFM-Il (GIBCO) containing hypoxanthine / thymidine (GIBCO) and 500 μg / ml of GE ETICIN (G418Sulfate, GIBCO) to select cells in which the antibody gene was introduced. After changing the selection medium, the cells were examined under a microscope after approximately 2 weeks. After observing favorable cell growth, the amount of antibody produced was measured by the ELISA method described below to determine the concentration of antibodies, and cells having a high antibody yield were selected. EXAMPLE 3. CONSTRUCTION OF THE HUMANIZED ANTIBODY (1) Construction of the humanized antibody H chain (i) Construction of the humanized "H" chain version "H" The humanized ATR-5 antibody chain H was generated using CDR grafting through of the polymerase chain reaction method. In order to generate the "a" version of H chain of humanized antibody having the FRs derived from human antibody L39130 (DDBJ, Gao L. et al., Unpublished, 1995), 7 chain reaction primers were employed. polymerase The CDR graft initiators hR5Hvls (SEQ ID NO: 22), hR5Hv2s (SEQ ID NO: 23), and hR5Hv4s (SEQ ID NO: 24) have a sense DNA sequence, and the CDR graft initiators hR5Hv3A (SEQ ID NO: 25) and hR5Hv5A '(SEQ ID NO: 26) have an antisense DNA sequence, each primer having a complementary sequence of 18-35 base pairs at both ends. HR5Hvls was designed to have the Kozak consensus sequence (Kozak, M. Et al., J. Mol. Biol. 196: 947-950, 1987) and a Sali recognition site, and hR5Hv5A was designed in such a way that have a Nhel recognition site. The exogenous initiator hR5HvPrS (SEQ ID NO: 27) has a homology with the CDR graft initiator hR5HvlS, and hR5HvPrA (SEQ ID NO: 28) has a homology with the CDR graft initiator hR5Hv5A. The CDR graft initiators hR5HvlS, hR5Hv2S, hR5Hv3A, hR5Hv4S, and hR5Hv5A, and the exogenous primers hR5HvPrS and hR5HvPrA were synthesized and purified by Pharmacia BioTech. A polymerase chain reaction was carried out using the KOD DNA polymerase (Toyo Boseki) and using the attached buffer under the condition of containing 120 mM Tris-HCl (pH 8.0), 10 mM KCl, 6 mM (NH4) 2S04, 0.1% Triton X100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 mM MgCl2, 2.5 units KOD DNA polymerase ( Toyo Boseki), and 5 pmol each of the CDR graft primers hR5HvlS, hR5Hv2S, hR5Hv3A, hR5Hv4S, and hR5Hv5A in 98 μl, for 5 cycles at a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute. After addition of 100 pmol of exogenous primers hR5HvPrS and hR5HvPrA, a polymerase chain reaction was carried out for 25 cycles in a 100 μl system with the same temperature cycle. DNA fragments amplified by the polymerase chain reaction method were separated by agarose gel electrophoresis using 2% NuSieve GTG agarose (FMC BioProducts). Agarose strips containing DNA fragments approximately 430 base pairs long were removed, 3 volumes (ml / g) of TE were added to them and then extracted with phenol, phenol / chloroform, and chloroform to purify the fragments. of DNA. After precipitating the purified DNA with ethanol, one third of the volume was dissolved in 17 μl of water. The polymerase chain reaction mixture obtained was digested with Nhel and Sali, and was ligated to the vector of the CVIDEC plasmid prepared by digestion with Nhel and Sali, using the DNA ligation kit ver. 2 (Takara Shuzo) in accordance with the instructions enclosed with the kit. The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. Then, 300 μl of the Hi-Competence Broth (Nippongene) was added, incubated at a temperature of 37 ° C for 1 hour, and then E. coli was plated on the LBA agar medium and it was incubated overnight at a temperature of 37 ° C to obtain an E. coli transformant. The transformant was cultured overnight at a temperature of 37 ° in 3 ml of LBA medium, and from the cell fractions, plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the cDNA coding region in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer) by the DNA sequencer 373A (Perkin-Elmer). As the sequencing primer, the M13 M4 primer (Takar.a Shuzo) and the M13 RV primer (Takara Shuzo) were used, and the sequence was determined by confirmation of the nucleotide sequence in both directions. Since mutation and / or removal was observed before or after the EcoT221 recognition site, each of the fragments having the correct sequence was ligated and then subcloned again into CVIDEC to determine the nucleotide sequence. The plasmid that had the correct sequence was designated hATR5Hva / CVIDEC. The nucleotide sequence and the corresponding amino acid sequence of the humanized "H" chain version "a" contained in the plasmid hATR5Hva appear in SEQ ID NO: 29. The amino acid sequence of the "a" version also appears in SEQ ID NO: 30. (ii) Construction of humanized H chain versions The "b" and "c" versions were generated by replacing FR3 version "a" with FR3 derived from another human antibody using the FR replacement method. To replace FR3 in version "b" with a derivative of human antibody Z34963 (DDBJ, Borretzen M. et al., Proc. Nati, Acad. Sci. USA, 91. 12917-12921, 1994), the four initiators of DNA encoding the FR3 were generated. The FR replacement primers F3RFFS (SEQ ID NO.31) and F3RFBS (SEQU ID NO: 32) have a sense DNA sequence and F3RFFA (SEQ ID NO: 33) and F3RFBA (SEQ ID NO: 34) have an anti-sense DNA sequence. F3RFFS and F3RFFA have a complementary sequence between them, and have Ball and Xhol recognition sequences at both ends. F3RFBS and F3RFBA have a complementary sequence between them, have Xhol and Ncol recognition sequences at both ends. In order to replace FR3 in version "c" with a derivative of human antibody P01825 (SWISS-PROT, Poljak RJ. Et al., Biochemistry, 16: 3412-3420, four DNA primers were generated that encode FR33 The replacement primers of FR F3NMFS (SEQ ID NO: 35) and F3NMBS (SEQ ID NO: 36) have a sense DNA sequence and F3NMFA (SEQ ID NO: 37) and F3NMBA (SEQ ID NO: 38) have An anti-sense DNA sequence: F3NMFS and F3NMFA have a sequence complementary to each other, and have Ball and Xhol recognition sequences at both ends.F3NMBS and F3NMBA have Xhol and Ncol recognition sequences at both ends.F3RFFS were synthesized , F3RFBS, F3RFFA, F3RFBA, F3NMFS, F3NMBS, F3NMFA, and F3NMBA by Pharmacia Biotech, F3RFFS and F3RFFA, and F3RFBS and F3RFBA were fused, and were digested with Ball and Xhol, and Ncol and Xhol, respectively, were introduced into the plasmid. hATR5Hva / CVIDEC (Ball / Ncol) prepared by digestion with Ball and Nco 1, and the sequence of a nucleotide was determined. The plasmid having the correct sequence was designated hATR5Hvb / CVIDEC. The nucleotide sequence and the corresponding amino acid sequence of the "b" version of the humanized H chain contained in the plasmid hATR5Hvb / CVIDEC appear in SEQ ID NO. 39. The amino acid sequence of version "b" also appears in SEQ ID NO. 40 F3NMFS and F3NMFA, and F3NMBS and F3NMBA were fused, and then digested with Ball and Xhol, and Ncol and Xhol, respectively. They were introduced to the plasmid hATR5Hva / CVIDEC (Ball / Ncol) prepared by digestion with Ball and Ncol, and the nucleotide sequence was determined. The plasmid having the correct sequence was designated hATR5Hvc / CVIDEC. The nucleotide sequence and the corresponding amino acid sequence of the "c" version of humanized H chain contained in plasmid hATR5Hvc / CVIDEC appear in SEQ ID NO: 41. The amino acid sequences of version "c" also appear in SEQ ID NO: 42. (iii) Construction of "d" and "e" versions of humanized H chain The "d" and "e" versions were generated by replacing FR3 from version "a" with FR3 derived from another antibody human using the FR replacement method. In order to replace the FR3 in the "d" version with a derivative of the human antibody M62723 (DDBJ, Pascual v. Et al., J. Clin. Invest., 86. 1320-1328, 1990), four primers were generated. of DNA encoding the 'FR3. The FR F3EPS replacement primer (SEQ ID NO: 43) has a sense DNA sequence and F3EPA (SEQ ID NO: 44) has an anti-sense DNA sequence, and the 3 'end of the primers has a complementary sequence of 18 base pairs. Exogenous initiators F3PrS (SEQ ID NO: 45) and F3PrA (SEQ ID NO: 46) have a homology with the FR3EPS and F3EPA replacement primers, and can also be used for FR replacement of other FRs. In order to replace FR3 in version "e" with a derivative of human antibody Z80844 (DDBJ, Thomsett AR et al., Unpublished), two DNA primers were generated that encode FR3. The replacement primers of FR F3VHS (SEQ ID NO: 47) have a sense DNA sequence and F3VHA (SEQ ID NO: 48) has an anti-sense DNA sequence, and the 3 'end of the primers has a complementary sequence of 18 base pairs. F3EPS, F3EPA, F3PrS, F3PrA, F3VHS and F3VHA were synthesized by Pharmacia Biotech. A polymerase chain reaction was performed using KOD DNA polymerase (Toyo Boseki) using the adjuvanted buffer under the condition of containing 5 μl each of 1 μM FR replacement primers F3EPS and F3EPA, or F3VHS and F3VHA, 0.2 mM dNTPs, 1.0 mM MgCl2 and 2.5 units of KOD DNA polymerase in 100 μl of the reaction mixture, for 5 cycles at a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 74 ° C for 1 minute. After the addition of 100 pmol of exogenous initiators F3PrS and F3para, a polymerase chain reaction was carried out for 25 cycles with the same temperature cycle. DNA fragments amplified by the polymerase chain reaction method were separated by agarose gel electrophoresis using Nu Sieve agarose from 2% GTG (FMC BioProducts). Agarose strips containing DNA fragments approximately 424 base pairs long were removed, 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the fragments of DNA. After precipitation of the purified DNA with ethanol, one third of the volume was dissolved in 14 μl. The obtained polymerase chain reaction mixture was digested with Ball and Ncol and introduced into the plasmid hATR5HVA / CVIDEC (Ball / Ncol) prepared by means of the digestion with Ball and Ncol, and the nucleotide sequence was determined. Plasmids that have the correct sequence were designated hATR5Hvd / CVIDEC and hATR5Hve / CVIDEC. The nucleotide sequence and the corresponding amino acid sequence of the "d" version of humanized H chain contained in plasmid hATR5Hvd / CVIDEC appear in SEQ ID NO: 49, and the amino acid sequence of version "d" also appears in SEQ ID NO: 50. The nucleotide sequence and the corresponding amino acid sequence of the "e" version of humanized H chain contained in plasmid hATR5Hve / CVIDEC appear in SEQ ID NO: 51, and the amino acid sequence of version "e" is also shown in SEQ ID NO: 52. (iv) Construction of versions "f" and "g" of humanized chain H The versions "f" and "g" were generated by the replacement human antibody using the FR replacement method. In order to replace the FR3 in the "f" version with a derivative of human antibody L04345 (DDBJ, Hillson JL et al., J. Exp. Med., 178: 331-336, 1993) and to replace the FR3 in the "g" version with a human antibody derivative S78322 (DDBJ, Bejcek BE., et al., Cancer Res., 55: 2346-2351, 1995), two primers were synthesized each encoding the FR3. The replacement primer of FR F3SSS (SEQ ID NO: 53) of version "f" has a DNA sequence of sense and F3SSA (SEQ ID NO: 54) has an antisense DNA sequence, and the 3 'end of the primers have a complementary sequence of 18 base pairs. F3CDS (SEQ ID NO: 55) version "g" has a sense DNA sequence and F3CDA (SEQ ID NO: 56) has an anti-sense DNA sequence, and the 3 'end of the primers has a sequence complementary to 18 base pairs. F3SSS, F3SSA, F3CDS, and F3CDA were synthesized and purified by Pharmacia Biotech. A polymerase chain reaction was carried out using the KOD DNA polymerase (Toyo Boseki) using the attached buffer provided it contains 5 μl each of 1 μM FR replacement initiators F3SSS and F3SSA, or F3CDS and F3CDA , 0.2 mM dNTPs, 1.0 mM MgCl2, and 2.5 units of KOD DNA polymerase in 100 μl of the reaction mixture, for 5 cycles in a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 74 ° C for 1 minute. After the addition of 100 pmol of exogenous initiators F3Prs and F3PrA, a polymerase chain reaction was carried out for 25 cycles with the same temperature cycle. DNA fragments amplified by the polymerase chain reaction method were separated by agarose gel electrophoresis using NuSieve GTG 2% agarose (FMC BioProducts). Agarose strips containing DNA fragments approximately 424 base pairs long were removed, 3 volumes of (ml / g) TE were added, and then extracted with phenol, phenol-chloroform, and chloroform to purify the fragments. of DNA. After precipitation of the purified DNA with ethanol, one third of the volume was dissolved in 14 μl of water. The obtained polymerase chain reaction mixture was digested with Ball and Ncol, and was introduced into the plasmid hATR5Hva / CVIDEC (Ball / Ncol) prepared by digestion with Ball and Ncol, and the nucleotide sequence was determined. Plasmids having the correct sequence were designated hATR5Hvf / CVIDEC and hATR5Hvg / CVIDEC. The nucleotide sequence and the corresponding amino acid sequence of the "f" version of humanized H chain contained in plasmid hATR5Hvf / CVIDEC, and the amino acid sequence of version "f" are shown in SEQ ID NO: 57 and 58 The corresponding nucleotide sequence and amino acid sequence of the "g" version of humanized H chain contained in plasmid hATR5Hvg / CVIDEC, and the amino acid sequence of version "g" appear in SEQ ID NO: 59 and 60. (v) Construction of the "h" version of the humanized H chain The "h" version was generated by replacing the FR3 of the "a" version with the FR3 derived from another human antibody using the FR replacement method. replacing FR3 in the "h" version with a human antibody derivative Z26827 (DDBJ, van Der Stoep et al., J. Exp. Med., 177: 99-107, 1993), two primers were synthesized each encoding the FR3 The FR replacement F3ADS initiator (SEQ ID NO: 61) of the version " h "has a sense DNA sequence and F3ADA (SEQ ID NO: 62) has an anti-sense DNA sequence, and the 3 'end of the primers has a complementary sequence of 18 base pairs. F3ADS and F3ADA were synthesized and purified by Pharmacia Biotech. A polymerase chain reaction was performed using the KOD DNA polymerase (Toyo Boseki) using the adjuvanted buffer provided it contained 5 μl each of 1 μM of FR replacement primers F3ADS and F3ADA, 0.2 mM dNTPS, 1.0 mM MgCl2 and 2.5 units KOD DNA polymerase in 100 μl of the reaction mixture, for 5 cycles in a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 74 ° C for 1 minute. After the addition of 100 pmol of exogenous initiators F3PrS and F3PrA, a polymerase chain reaction was carried out for 25 cycles with the same temperature cycle. DNA fragments amplified by the polymerase chain reaction method were separated by agarose gel electrophoresis using a NuSive GTG 2% agarose (FMC BioProducts). Agarose strips containing DNA fragments approximately 424 base pairs long were removed, 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the fragments of DNA After precipitation of the purified DNA with ethanol, one third of the volume was dissolved in 14 μl of water. The obtained polymerase chain reaction mixture was digested with Ball and Ncol, and was introduced into the plasmid hATR5Hva / CVIDEC (Ball / Ncol) prepared by digestion with Ball and Ncol, and the nucleotide sequence was determined. Plasmids that have the correct sequence were designated hATR5Hvf / CVIDEC. The corresponding nucleotide sequence and amino acid sequence of the humanized H chain "h" version contained in the hATR5Hvf / CVIDEC plasmid, and the amino acid sequence of the "h" version are shown in SEQ ID NO: 63. The sequence of amino acids from version "h" is shown in SEQ ID NO: 64. (vi) Construction of versions "i" and "j" of humanized chain H The versions "i" and "j" were generated by replacing the FR3 version "a" with FR3 derived from another human antibody using the FR replacement method. In order to replace FR3 in version "i" with a derivative of human antibody U95239 (DDBJ, Manheimer-Lory AAJ., Unpublished), and to replace FR3 in version "j" with a derivative of human antibody L03147 (DDBJ, Collect TA., Et al., Proc. Nati. Acad. Sci. USA, 89: 10026-10030, 1992), two primers were synthesized each encoding the FR3. The FR F3MMS replacement primer (SEQ ID NO: 65) of the "i" version has a sense DNA sequence and F3MMA (SEQ ID NO: 66) has an anti-sense DNA sequence, and the 3 'of the initiators has a complementary sequence of 18 base pairs. F3BMS (SEQ ID NO: 67) of version "j" has a DNA sequence of sense and F3BMA (SEQ ID NO: 68) has an anti-sense DNA sequence, and the 3 'end of the primers has a sequence complementary to 18 base pairs. F3MMS, F3MMA, F3BMS, and F3BMA were synthesized and purified by Pharmacia Biotech. A polymerase chain reaction was performed using Ampli Taq Gold (Perkin-Elmer) using the adjuvanted buffer in the condition of containing 5 μl each of 1 μM of FR F3MMS and F3MMA replacement primers, or F3BMS and F3BMA, 0.2 mM dNTPS, 1.0 mM MgCl2 and 2.5 units of Taq Gold Ampli in 100 μl of the reaction mixture, for 5 cycles in a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute , and 74 ° C for 1 minute. After the addition of 100 pmol of exogenous initiators F3PrS and F3PrA, a polymerase chain reaction was carried out for 25 cycles with the same temperature cycle. DNA fragments amplified by the polymerase chain reaction method were separated by agarose gel electrophoresis using a NuSive GTG 2% agarose (FMC BioProducts). Agarose strips containing DNA fragments approximately 424 base pairs long were removed, 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the fragments of DNA. After precipitation of the purified DNA with ethanol, one third of the volume was dissolved in 14 μl. The obtained polymerase chain reaction mixture was digested with Ball and Ncol and introduced into the plasmid hATR5Hva / CVIDEC (Ball / Ncol) prepared by means of digestion with Ball and Ncol, and the nucleotide sequence was determined. Plasmids that have the correct sequence were designated hATR5Hvi / CVIDEC and hATR5Hvj / CVIDEC. The corresponding nucleotide sequence and amino acid sequence of the "i" version of humanized H chain contained in plasmid hATR5Hvi / CVIDEC and the amino acid sequence of version "i" are shown SEQ ID NO: 69 and 70. The sequence of nucleotides and the corresponding amino acid sequence of the "j" version of humanized H chain contained in plasmid hATR5Hvj / CVIDEC and the amino acid sequence of version "j" are shown in SEQ ID NO: 71 and 72. (vil) Construction of versions "bl" and "di" of chain H humanizada The versions "bl" and "di" were generated by replacing the FR2 of versions "b" and "d" with the FR2 derived from another human antibody using the method FR replacement. In order to replace FR2 with a derivative of the human antibody P01742 (SWISS-PROT, Cunningham BA, et al., Biochemistry, 9: 3161-3170, 1970), two DNA primers encoding FR2 were synthesized. The FR F2MPS replacement vector (SEQ ID NO: 73) has a sense DNA sequence and F2MPA (SEQ ID NO: 74) has an anti-sense DNA sequence. They also have a complementary sequence between them, and have recognition sequences of EcoT221 and Ball at both ends. F2MPS and F2MPA were synthesized and purified by Pharmacia Biotech. F2MPS and F2MPA were fused and digested with EcoT22I and Ball. They were introduced in the hATR5Hvb / CVIDEC plasmids (EcoT22I / BalI) and hATR5Hvd / CVIDEC (EcoT22I / BalI) prepared by digestion with EcoT22I and Ball, and the nucleotide sequence was determined. Plasmids that have the correct sequence were designated hATR5Hvbl / CVIDEC and hATR5Hvdl / CVIDEC. The nucleotide sequence and the corresponding amino acid sequence of the "bl" version of humanized H chain contained in the plasmid hATR5Hvbl / CVIDEC, and the amino acid sequence of the "bl" version is shown in SEQ ID NO: 75 and 76. The corresponding nucleotide sequence and amino acid sequence of the "di" version of humanized H chain contained in the plasmid hATR5Hvdl / CVIDEC , and the amino acid sequence of version "di" appear in SEQ ID NO: 77 and 78. (viii) Construction of versions "b3" and "d3" of humanized chain H The versions "b3 '* d3' were generated by the Replacement of the FR2 of versions with the FR2 derived from another human antibody using the FR replacement method In order to replace the FR2 with a derivative of the human antibody Z80844 (DDDJ, Thomsett AR et al., unpublished) , two DNA primers that encode FR2 were synthesized The FR F2VHS replacement vector (SEQ ID NO: 79) has a sense DNA sequence and F2VHA (SEQ ID NO: 80) has an anti DNA sequence. They also have a complementary sequence between them, and they have sequences of Knowledge of EcoT221 and Ball at both ends. F2VHS and F2VHA were synthesized and purified by Pharmacia Biotech F2VHS and F2VHA were fused and digested with EcoT22I and Ball. They were introduced into the plasmids hATR5Hvb / CVIDEC (EcoT22I / BalI) and hATR5Hvd / CVIDEC (EcoT22I / BalI) prepared by digestion with "EcoT22I and Ball, and the nucleotide sequence was determined.The plasmids having the correct sequence were designated as hATR5Hvb3 / CVIDEC and hATR5Hvd3 / CVIDEC The nucleotide sequence and corresponding amino acid sequence of the humanized H chain "b3" version contained in the hATR5Hvb3 / CVIDEC plasmid, and the amino acid sequence of the "b3" version is shown in SEQ ID NO: 81 and 82. The corresponding nucleotide sequence and amino acid sequence of the "d3" version of humanized H chain contained in plasmid hATR5Hvd3 / CVIDEC, and the amino acid sequence of version "d3" appear in SEQ ID. NO: 83 and 84. (2) Construction of a humanized antibody L chain V region (i) "a" version The L chain V region of humanized ATR-5 antibody was generated by the CDR graft used the polymerase chain reaction method. For the generation of an L chain of humanized antibody (version "a") having structure regions derived from human antibody Z37332 (DDBJ, Welschof M. et al., J. Immunol. Methods, 179: 203-214, 1995) , seven polymerase chain reaction primers were used. The CDR graft primers h5LvlS (SEQ ID NO: 85) and h5Lv4S (SEQ ID NO: 86) have a sense DNA sequence, two CDR graft initiators heLv2A (SEQ ID NO: 87), h5Lv3A (SEQ ID NO: 88) and h5Lv5A (SEQ ID NO: 89) have an anti-sense DNA sequence, and each primer has complementary sequences of 20 base pairs at both ends. Exogenous initiators h5LvS (SEQ ID NO: 90) and h5LvA (SEQ ID NO: 91) have a homology with CDR graft initiators h5LvlS and h5Lv5A. The synthesis and purification of CDR graft primers h5LvlS, h5Lv4S, h5Lv2A, h5Lv3A, h5Lv5A, and h5LvA were referred to Pharmacia Biotech. The polymerase chain reaction solutions contain, in 100 μl, 120 mM Tris-HCl (pH 8.0), 10mM KCl, 6mM (NH4) 2S04, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 mM MgCl2, 2.5 KOD units DNA polymerase (Toyo Boseki), 50 pmol of the CDR graft primers h5LvlS, h5Lv2A, h5Lv3A, h5Lv4S, and h5Lv5A.

A polymerase chain reaction was performed using the DNA thermal cycler 480 (Perkin-Elmer) for 5 cycles at a temperature of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute to assemble 5 CDR graft initiators. After the addition of 100 pmol of exogenous initiators h5LvS and h5LvA to the reaction mixture, a polymerase chain reaction was carried out for 30 cycles with a temperature cycle of 94 ° C for 30 seconds, 52 ° C for 1 minute, and 72 ° C for 1 minute to amplify the assembled DNA fragments. The polymerase chain reaction mixture was separated by agarose gel electrophoresis using Nu Sieve agarose of 3% GTG (FMC BioProducts), and the agarose strips containing DNA fragments of approximately 400 base pairs long were removed. . The agarose strips were extracted with phenol and chloroform, the DNA fragments were recovered by precipitation with ethanol. The recovered DNA fragments were digested with the restriction enzymes Spll (Takara Shuzo) and Bgll (Takara Shuzo) at a temperature of 37 ° C for 4 hours. The digestion mixture was extracted with phenol and chloroform, and after the ethanol precipitation of the DNA fragments, they were dissolved in 10 μl of TE. The SplI-BglII DNA fragment prepared as mentioned above coding for the humanized L chain V region and the CVIDEC vector prepared by digestion with Spll and BglII were ligated using the DNA ligation kit ver. 2 (Takara Shuzo) by reaction at a temperature of 16 ° C for one hour in accordance with the instructions enclosed with the kit. The ligation mixture was added to 100 μl of competent E. Coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. After, 300 μl of Hi-Competence Broth (Nippongene) was added, incubated at a temperature of 37 ° C for 1 hour, and then E. coli was plated on LBA agar medium and incubated overnight at a temperature of 37 ° C to obtain an E. coli transformant. The transformant was grown overnight in 3 ml of the LBA medium and from the cell fractions, plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the cDNA coding region in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer) by using the DNA sequencer 373A (Perkin-Elmer). As the sequencing primer, the M13 M4 primer (Takara Shuzo) and the M13 Rv primer (Takara Shuzo) were used, and the sequence was determined by confirmation of the nucleotide sequences in both directions. The plasmid containing the gel encoding the L chain V region of humanized antibody and having a BglII recognition sequence and the Kozak sequence at the 5 'end, and a Spll recognition sequence at the 3' end was designated as hATR5L / CVIDEC. The nucleotide sequence (including the corresponding amino acid sequence) of the "a" version of humanized L chain is shown in SEQ ID NO: 92. The amino acid sequence of the "a" version is also shown in SEQ ID NO: 93 (ii) Versions "b" and "c" Versions "b" and "c" were generated by the replacement (replacement of FR) of FR3 of version "a". For the "b" version, the FR3 derived from the human antibody S68699 (DDBJ, Hougs L. Et al., Exp. Clin Immunogen et., 10: 141-151, 1993) was used, and for the "c" version, the FR3 derived from the human antibody P01607 (SWISS-) was used. PROT, Epp O et al., Biochemistry, 14: 4943-4952, 1975), respectively. The primers F3SS (SEQ ID NO: 94) and F3SA (SEQ ID NO: 95) encoding the FR3 version "b" or the primers F3RS (SEQ ID NO: 96) and F3RA (SEQ ID NO: 97) that encodes the FR3 of the "c" version have a complementary sequence between them, and have the recognition sequences of the restriction enzymes Kpnl and PstI at both ends. The synthesis and purification of F3SS, F3SA, F3RS, and F3RA refer to Pharmacia Biotech. 100 pmol of each of F3SS and F3SA or F3RS and F3RA were fused by treatment at a temperature of 96 ° C for 2 minutes and at a temperature of 50 ° C for two minutes and the double stranded DNA fragments were generated. These double-stranded DNA fragments were digested with the restriction enzyme Kpnl (Takara Shuzo) at a temperature of 37 ° C for one hour and then with the restriction enzyme PstI (Takara Shuzo) at a temperature of 37 ° C for 1 hour. hour. The digestion mixture was extracted with phenol and chloroform and after its precipitation with ethanol, it was dissolved in TE. Plasmid hATR5Lva / CVIDEC was digested with the restriction enzyme Kpnl (Takara Shuzo) at a temperature of 37 ° C for one hour, and then with the restriction enzyme PstI (Takara Shuzo) at a temperature of 37 ° C for 1 hour . The digestion mixture was separated by agarose gel electrophoresis using NuSieve GTG 1.5"agarose (FMC BioProducts), and the agarose strips having DNA fragments of approximately 3000 base pairs in length were extracted. with phenol and chloroform, and after the precipitation of the DNA fragments with ethanol, were dissolved in TE The Kpnl-Pstl DNA fragment prepared according to the aforementioned which encodes the FR3 of the "b" or "c" versions "and the vector hATR5Lva / CVIDEC in which the FR3 was removed by digestion with Kpnl and PstI were ligated using the DNA ligation kit ver.2 (Takara Shuzo) by reaction at a temperature of 16 ° C for 1 hour in accordance with Instructions attached to the kit The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. 300 μl of the Hi-Competence Broth (Nippongene), were incubated at a temperature of 37 ° C for 1 hour, and then E. coli was placed on LBA agar medium plates and incubated during incubation. overnight at a temperature of 37 ° C to obtain an E. coli transformant. The transformant was grown overnight in 3 ml of the LBA medium and from the cell fractions, plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the cDNA coding region in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer) by using the DNA sequencer 373a (Perkin-Elmer). As the sequencing primer, the M13 M4 primer (Takara Shuzo) and the M13 RV primer (Takara Shuzo) were used, and the sequence was determined by confirmation of the nucleotide sequence in both directions.

Plasmids containing the gene encoding version "b" or version "c" where the FR3 of version "a" L chain of humanized antibody was replaced were designated hATR5Lvb / CVIDEC or hATR5Lvc / CVIDEC, respectively . The nucleotide sequence and the corresponding amino acid sequence of the "b" version of humanized L chain contained in plasmid hATR5Lvb / CVIDECH and the amino acid sequence of version "b" are shown in SEQ ID NO: 98 and 99. The nucleotide sequence and the corresponding amino acid sequence of the "c" version of humanized L chain contained in plasmid hATR5Lvc / CVIDEC and the amino acid sequence of version "c" are shown in SEQ ID NO: 100 and 101. ( iii) Versions "bl" and "b2" The versions "bl" and "b2" were generated by replacing the FR2 of version "b". For the "bl" version, the FR2 derived from human antibody S65921 (DDBJ, Tonge DW et al., Year Immunol., 7: 56-62, 1993) was used, and for the "b2" version the FR2 derived from human antibody X93625 (DDBJ) was used. , Cox Jpet al., Eur. J. Immunol., 24: 827-836, 1994) respectively. The primers F2SS (SEQ ID NO: 102) and F2SA (SEQ ID NO: 103) encoding the FR2 of the "bl" version, or the primers F2Xs (SEQ ID NO: 104) and F2XA (SEQ ID NO: 105 ) that encode the FR2 of the "b2" version have a complementary sequence between them, and have the recognition sequences of the restriction enzymes AflII and Spel at both ends. Pharmacia BioTech synthesized F2SS, F2SA, F2XS and F2XA. 100 pmol of each F2SS and F2SA or F2XS and F2XA were fused by treatment at 96 ° C for 2 minutes and at 50 ° C for 2 minutes and double stranded DNA fragments were generated. These double-stranded DNA fragments were digested with the restriction enzymes AflIII (Takara Shuzo) and Spel (Takara Shuzo) at a temperature of 37 ° C for 1 hour. The digestion mixture was extracted with phenol and chloroform, and after precipitating the DNA fragments with ethanol, said fragments were dissolved in TE. Plasmid hATR5LvB / CVIDEC was digested with the restriction enzymes AflII (Takara Shuzo) and Spel (Takara Shuzo) at a temperature of 37 ° C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using a NuSieve GTG 1.5% agarose (FMC BioProducts) and agarose strips containing DNA fragments approximately 3000 base pairs long were extracted. The agarose strip was extracted with phenol and chloroform, and the DNA fragments were precipitated with ethanol, which were then dissolved in TE. The fragment of AflII-Spel prepared according to the aforementioned which codes the FR2 of version "bl" or "b2" and the vector hATR5LvB / CVIDEC where the FR2 was removed by digestion with AflII and Spel were ligated using the kit of DNA ligation see. 2 (Takara Shuzo) by reaction at a temperature of 16 ° C for 1 hour in accordance with the instructions enclosed with the kit. The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. Then, 300 μl of the HiCompetence Broth (Nippongene) was added, incubated at a temperature of 37 ° C for 1 hour, and then E. coli was placed on an LBA agar medium and incubated overnight at a temperature of 37 ° C to obtain an E. coli transformant. The transformant was cultured overnight at a temperature of 37 ° C in 4 ml of the LBA medium, and from the cell fractions, plasmid DNA was prepared using the QIAPrep Spin Plasmid Kit (QIAGEN). The nucleotide sequence of the region encoding cDNA in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-Elmer) through the use of the DNA sequencer 373A (Perkin-Elmer). As the sequencing primer, the M13 M4 primer (Takara Shuzo) and the M13 RV primer (Takara Shuzo) were used, and the sequence was determined by confirmation of the nucleotide sequence in both directions. The plasmids containing the gene coding for the "bl" or "b2" version where the FR2 of the "b" version of the humanized antibody L chain were designated hATR5Lvl / CVIDEC and hATR5Lv2 / CVIDEC respectively. The corresponding nucleotide sequence and amino acid sequence of the humanized L-chain "bl" version contained in plasmid hATR5Lvl / CVIDEC and the amino acid sequence of version "bl" are shown in SEQ ID NO: 106 and 107. nucleotide sequences and the corresponding amino acid sequence of the "b2" version of humanized L chain contained in plasmid hATR5Lvl / CVIDEC and the amino acid sequence of version "b2" appear in SEQ ID NO: 108 and 109. (3) Construction of the humanized antibody expression vector. (i) Combination of humanized H chain and chimeric L chain Plasmid hATR5Lvl / CVIDEC containing a V chain region HH was digested with Nhel and Sali, and a cDNA fragment from the humanized H chain V region was recovered and introduced into hATR5Lvl / CVIDEC (SalI / Nhel) prepared by digestion of chATR5 / N5KG4P, a chATR-5 antibody expression plasmid vector, with Nhel and Salí. The plasmid generated in this way was designated as hHvb-chlv / N5KG4P. Plasmids hATR5Hvc / CVIDEC, hATR5Hvd / CVIDEC, and hATR5Hve / CVIDEC containing an H chain V region were digested with Nhel Y Sali, and a fragment of humanized H chain V region cDNA were recovered and introduced into chATR5 / N5KG4P (Sall / Nehl) prepared by digestion of chATR5 / N5KG4P, a vector of chATR-5 antibody expression plasmids, with Nhel and SalI. The plasmids generated in this way were designated as hHvc-chLv / N5KG4P, hHvd-chLv / N5KG4P, and hHve-chLv / N5KG4P. The hATR5Hvf / CVIDEC and hATR5Hvh / CVIDEC plasmids containing an H chain V region were digested with Nhel Y Sali, and cDNA fragments from the human H chain V region were recovered and introduced into chATR5 / N5KG4P (Sall / Nehl) preparations by digestion of chATR5 / N5KG4P, a vector of antibody expression plasmids of chATR-5, with Nhel and SalI. The plasmids generated in this way were designated as hHvf-chLv / N5KG4P and hHvh-cb.Lv/N5KG4P. The plasmids hATR5Hvi / CVIDEC and hATR5Hvj / CVIDEC containing a V region of H chain were digested with Nhel Y Salí, and cDNA fragments of the humanized H chain V region were recovered and introduced into chATR5 / N5KG4P (Sall / Nehl) preparations by digestion of chATR5 / N5KG4P, a vector of antibody expression plasmids of chATR-5, with Nhel and SalI. The plasmids generated in this way were designated as hHvi-chLv / N5KG4P and hHvj-chLv / N5KG4P.

Plasmids hATR5Hbl / CVIDEC and hATR5Hvdl / CVIDEC containing a V region of H chain were digested with Nhel Y Sali, and cDNA fragments of V chain H region were recovered and introduced into chATR5 / N5KG4P (Sall / Nehl) prepared by digestion of chATR5 / N5KG4P, a vector of chATR-5 antibody expression plasmids, with Nhel and SalI. The plasmids generated in this way were designated as hHvbl-chLv / N5KG4P and hHvdl-chLv / N5KG4P. (ii) Combination of humanized L chain and chimeric chain H Using the antibody expression vector N5KG4P was combined with a chimeric H chain and expressed, and the humanized L chain was evaluated. Plasmids hATR5Lva / CVIDEC, hATR5Lvb / CVIDEC, hATR5Lvc / CVIDEC, hATR5Lvbl / CVIDEC, and hATR5Lvb2 / CVIDEC, were digested with the restriction enzymes BglII (Takara Shuzo) and Spll (Takara Shuzo) at a temperature of 37 ° C for 2 hours. -3 hours. The digestion mixture was separated by agarose gel electrophoresis using 1.5% or 2% NuSieve GTG agarose (FMC BioProducts), and the agarose strips having DNA fragments of approximately 400 base pairs long were removed. The agarose strips were extracted with phenol and chloroform, and then the DNA fragments were precipitated with ethanol, and were dissolved in TE.

The SplI-BglII DNA fragment containing the gene encoding the humanized L chain V region of each of the three versions and the hATR 5Hv / N5KG4P digested with Spll and BglII were ligated using the DNA ligation kit ver. 2 (Takara Shuzo) by reaction at a temperature of 16 ° C for 1 hour in accordance with the instructions enclosed with the kit. The ligation mixture was added to 100 μl of competent E. coli JM109 cells (Nippongene) and incubated for 30 minutes on ice and for 1 minute at a temperature of 42 ° C. Then, 300 μl of Hi-Competence Broth (Nippongene) was added, incubated at a temperature of 37 ° C for 1 hour, and then E. coli was plated in the LBA agar medium and incubated overnight at a temperature of 37 ° C to obtain an E. coli transformant. The transformant was cultured overnight at a temperature of 37 ° C in 250 ml or 500 ml of the LBA medium, and from the cell fractions, plasmid DNA was prepared using the Plasmid Maxi Kit (QIAGEN). The plasmids into which a gene coding for the chimeric H chain and the humanized L chain were introduced were designated chHv-hLva / N5KG4P, chHv-hLvb / N5KG4P, chHv-hLvc / N5KG4P, chHv-hLvbl / N5KG4P, and chHv-hLvb2 / N5KG4P. (iii) Combination of humanized chain H and humanized chain Plasmid hATR5Hva / CVIDEC containing a V region of H chain was digested with Nhel and SalI, and a fragment of cDNA from the humanized H chain V region was recovered and introduced into hLva / N5KG4P (Sall / Nhel) prepared by the digestion of plasmid chHv-hLva / N5KG4P containing the cDNA sequence of the "a" L-chain version of ATR-5 antibody humanized with Nhel and SalI. The plasmid generated in this way was designated hHva-hLva / N5KG4P. The hATR5Hvb / CVIDEC and hATR5Hvc / CVIDEC plasmids containing an H chain V region were digested with Nhel and SalI, and humanized H chain V region cDNA fragments were recovered and introduced into hLva / N5KG4P (Sall / Nhel) prepared by digestion of the plasmid chHv-hLva / N5KG4P containing the cDNA sequence of the "a" version of L chain of humanized ATR-5 antibody with Nhel and SalI. The plasmids generated in this way were designated as hHvb-hLva / N5KG4P and hHvc-hLva / N5KG4P. Plasmids hATR5Hvb / CVIDEC and hATR5Hvd / CVIDEC and hATR5Hve / CVIDEC containing an H chain V region were digested with Nhel and SalI, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by digestion of the plasmid chHv-hLvb / N5KG4P containing the cDNA sequence of the "B" version of L chain of humanized ATR-5 antibody with Nhel and Sali. The plasmids generated in this way were designated as hHvb-hLvb / N5KG4P and hHvd-hLvb / N5KG4P and hHve-hLvb / N5KG4P. The plasmids' hATR5Hvf / CVIDEC and hATR5Hvg / CVIDEC and hATRSHvh / CVIDEC containing a V region of H chain were digested with Nhel and Sali, and cDNA fragments were recovered from the humanized H chain V region, said fragments were introduced into hLvb / N5KG4P (Sall / Nhel) prepared by the digestion of the chHv-hLvb / N5KG4P plasmid containing the cDNA sequence of the " b "of L chain of humanized ATR-5 antibody with Nhel and Sali. The plasmids generated in this way were designated as hHvf-hLvb / N5KG4P hHvg-hLvb / N5KG4P, and hHvh-hLvb / N5KG4P. The hATR5Hvi / CVIDEC and hATR5Hvj / CVIDEC plasmids containing an H chain V region were digested with Nhel and SalI, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by digestion of the chHv-hLvb / N5KG4P plasmid containing the cDNA sequence of the "b" version of the L chain of humanized ATR-5 antibody with Nhel and SalI. The plasmids generated in this way were designated hHvi-hLvb / N5KG4P and hHvj-hLvb / N5KG4P. The hATR5Hvbl / CVIDEC and hATR5Hvdl / CVIDEC plasmids containing an H chain V region were digested with Nhel and SalI, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by digestion of the plasmid chHv-hLvb / N5KG4P containing the cDNA sequence of the "b" version of L chain of humanized ATR-5 antibody with Nhel and SalI. The plasmids generated in this way were designated as hHvbl-hLvb / N5KG4P and hHvdl-hLvb / N5KG4P. Plasmids hATR5Hvb3 / CVIDEC and hATR5Hvd3 / CVIDEC containing an H chain V region were digested with Nhel and SalI, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by digestion of the plasmid chHv-hLvb / N5KG4P containing the cDNA sequence of the L chain version of humanized ATR-5 antibody with Nhel and Salí. The plasmids generated in this way were designated hHvb3-hLvb / N5KG4P and hHvd3-hLvb / N5KG4P. Plasmid hATR5Hvb / CVIDEC containing an H chain V region was digested with Nhel and SalI, and a humanized H chain V region cDNA fragment was recovered and introduced into hLvbl / N5KG4P (Sall / Nhel) and hLvb2 / N5KG4P (Salí and Nhel) prepared by the digestion of plasmids chHv-hLvbl / N5KG4P and chHv-hLvb2 / N5KG4P containing the cDNA sequence of the "bl" and "b2" versions of L chain of NTH humanized antibody ATR-5 and I left. The plasmids generated in this way were designated as hHvb-hLvbl / N5KG4P and hHvb-hLvb2 / N5KG4P.

The hATR5Hvi / CVIDEC plasmid containing an H chain V region was digested with Nhel and SalI, and a humanized H chain V region cDNA fragment was recovered and introduced into hLvbl / N5KG4P (Sall / Nhel) and hLvb2 / N5KG4P (Salí and Nhel) prepared by the digestion of plasmids chHv-hLvbl / N5KG4P and chHv-hLvb2 / N5KG4P containing the cDNA sequence of the "bl" and "b2" versions of L chain of NTH humanized antibody ATR-5 and I left. The plasmids generated in this way were designated as hHvi-hLvbl / N5KG4P and hHvi-hLvb2 / N5KG4P. (4) Transfection in COS-7 cells In order to evaluate the antigen binding activity and the neutralizing activity of the humanized antibody, the aforementioned antibody was expressed transiently in COS-7 cells. The constructed expression plasmid vector was translucent from COS-7 cells by electroporation using the Gene Pulser instrument (Bio Rad). 50 μg or 20 μg of the plasmid was added to 0.78 ml of COS-7 cells suspended in PBS at a cell concentration of 1 x 10 'cells / ml, which was pulsed at 1500 V and 25 μF capacity. After 10 minutes of the recovery period at room temperature, the cells subjected to electroporation were suspended in a DMEM medium (GIBCO) containing 5% ultra-low IgG fetal bovine serum (GIBCO), and cultured using a culture dish. 10 cm or a 15 cm culture dish in an incubator with 5% CO2. After culturing for 24 hours, the culture supernatant was removed by aspiration, and then serum free HBCHO medium (Irvine Scientific) was added. After further culturing for 72 hours or 96 hours, the culture supernatant was collected and centrifuged to remove cell debris. (5) Purification of antibody For the culture supernatant of COS-7 cells, the antibody was purified using the AffiGel Protein A kit MAPSII (Bio Rad) or rProtein A Sepharose Fast Flow (Pharmacia Biotech). Purification using the Affigel kit Protein A MAPSII was performed in accordance with the instructions attached to the kit. Purification using rProtein A Sepharose Fast Flow was carried out in the following manner: one ml of rProtein A Sepharose Fast Flow was filled into a column and the column was equilibrated by 10 volumes of TBS. The COS-7 cell culture supernatant was applied to the equilibrated column, which was then washed with 10 volumes of TBC. The adsorbed antibody fraction was eluted by 13.5 ml of 2.5 mM HCl (pH 3.0). The eluate was neutralized by the addition of 1.5 ml of 1 M Tris-HCL (pH 8.0). By performing the ultrafiltration two or three times for the fraction of purified antibody using the Centriprep 30 or 100 (Amicon), the solvent was replaced by TBS, and finally reached a concentration of approximately 1.5 ml. EXAMPLE 4. QUANTIFICATION OF ANTIBODIES AND EVALUATION OF ACTIVITY. (1) Measurement of antibody concentration by ELISA ELISA plates for antibody concentration measurement were prepared as follows: each well of a 96-well ELISA plate (Maxisorp, NUNC) was immobilized by 100 μl of antibody from IgG? goat antihuman (BIO SOURCE) prepared at a concentration of 1 μg / ml in the immobilization buffer (0.1 M NaHCO, 0.02 NaN3, pH 9.6) (known below as CB). After blocking with 200 μl of the dilution buffer, (50 mM Tris-HCl, 1 mM MgCl2, 0.1 NaCl, 0.05% 0.05% NaN3 0.02% 1% bovine serum albumin (BSA), pH 8.1) (below as DB); the culture supernatant of the COS-7 cells where the antibody was expressed or purified were serially diluted with DB and then added to each well. After incubation at room temperature for one hour followed by washing with Dulbecco PBS containing 0.05% Tween 20 (hereinafter referred to as RB), 100 μl of IgG? antihuman goat conjugate with alkaline phosphatase (Biosource) diluted 1000 times with DB.

After incubation at room temperature for 1 hour followed by washing with RB, Sigmal04 (p-nitrophenyl phosphate, SIGMA) was added to the substrate buffer (20 mM NaHCO3, 10 mM MgCl2, pH 9.8) to one mg / ml, and then the absorbance at 405/655 nm was measured using the Microplate Reader (microplate reader) (Bio Rad). As standard for the measurement of the concentration, IgG4 was used? (union site). (2) Measurement of antigen binding activity. ELISA plates of cells for measuring antigen binding were prepared as follows. The cells used were J82 human bladder carcinoma cells (ATCC HTB-1). To 60 wells of a 96-well cell culture plate, 1 × 10 5 J82 cells were inoculated. This RPMI1640 medium culture containing 10% fetal bovine serum (GIBCO)) for one day in a C02 incubator to allow the fixation of the cells. After discarding the culture liquid, each well was lacquered 2 times with 300 μl of PBS. 100 μl of PBS containing 4% paraformaldehyde (hereinafter referred to as PFA / PBS) was added to each well, and placed on ice for 10 minutes to immobilize the cells. PFA / PBS was discarded and each well was washed twice with 300 μl of PBS and then blocked with 250 μl of DB. The culture supernatant or the purified antibody was serially diluted with DB 100 μl from which it was added to each well. After incubation at room temperature for two hours followed by washing with RB, 100 μl of IgG? antihuman goat conjugate with alkaline phosphatase (BioSource) diluted 1000 times with DB. After * incubation for 1 hour followed by washing with RB, the substrate solution was added, and then the absorbance was measured at 405/655 nm using the Microplate Reader (Bio-Rad). (3) Measurement of neutralizing activity The neutralizing activity of mouse antibody, chimeric antibody and humanized antibody was measured with the activity of inhibition against the amount of factor Xa production by thromboplastin derived from human placenta, Thromborel S (Boehringer AG), as Index. Thus, 60 μl of the buffer (TBS containing 5 mM CaCl 2 and 0.1% BSA) was added to 10 μl of 1.25 mg / ml Thromborel S and 10 μl of appropriately diluted antibody which was then incubated in a 96-well plate. room temperature for 1 hour. 10 μl each of 3,245 μg / ml of human Factor X was added (Celsus Laboratories) and 82.5 ng / ml Human Factor Vlla (Enzyme Research), and then incubated at room temperature for 1 hour. 10 μl of 0.5 M EDTA was added to suspend the reaction, 50 μl of the chromogenic substrate solution was added and the absorbance at 405/655 nm was determined using the Microplate Reader (Bio Rad). After reacting at room temperature for 1 hour, the absorbance was again determined at 405/655 nm. The neutralizing activity can be determined by calculating the residual activity (%) from each change in absorbency with the change in absorbency per hour without addition of antibody as 100% activity. The chromogenic substrate solution was prepared by dissolving the chromogenic substrate Tetstzyme S-2222 (Chromogenix) in accordance with the enclosed instructions, diluting twice with purified water and mixing with a solution of polybrene (0.6 mg / ml hexadimethylene bromide, SIGMA) at 1: 1. (4) Evaluation of the activity (i) Combination of the "a" version of humanized H chain and a chimeric L chain An antibody (a-ch) that is the "a" version of humanized H chain combined with the chimeric L chain was generated and said antibody was assayed to determine antigen binding activity by the cell ELISA. It was found that the antigen decreased in the high concentration (figure 1). Neutralizing activity against the antigen by inhibition of Fxa production was weak compared to the neutralizing activity of the chimeric positive control antibody (ch-ch) (Figure 2). Therefore, the decision was made to carry out the high version of the humanized H chain by replacement of FR. The chimeric antibody used here was the antibody expressed in COS-7 cells, purified and evaluated. (ii) Combination of the "a" version of humanized L chain and a chimeric H chain An antibody (ch-a) which is the "a" version of humanized L chain combined with a chimeric H chain was generated, and said antibody to determine the activity of binding to the antigen through cell ELISA. It was found that the antigen has the same binding activity or a binding activity greater than the activity of the chimeric antibody (Figure 1). On the other hand, the neutralizing activity against the antigen was weak compared to the activity of the chimeric positive control antibody (Figure 2). Therefore, the decision was made to carry out the elevated version of the humanized L chain by replacement of FR. The chimeric antibody employed herein was the chimeric antibody expressed in COSA cells, purified and evaluated. (iii) Combination of version "a" of humanized chain H and version "a" of humanized chain L An antibody (aa) that is version "a" of humanized chain H combined with version "a" of chain L Humanized was generated, and was assayed to determine antigen binding activity through cell ELISA. It was found that the amount bound to the antigen showed a decrease in the side of the high concentration "figure 3". Neutralizing activity against the antigen by inhibition of FXa production was weak compared to the neutralizing activity of the chimeric positive control antibody (Figure 4). Accordingly, the decision was made to carry out the elevated version of the humanized H chain and humanized L chain by replacement of FR. The chimeric antibody used here was the chimeric antibody expressed in COSA cells, purified and evaluated, (iv) Combination of versions "b", "c" and "d" of humanized chain H and chimeric chain L Antibodies ("b-ch" , "c-ch", and "d-ch", respectively) which are the humanized H chain submitted to high version by replacement of FR combined with a chimeric L chain were generated, and were assayed to determine the binding activity with the antigen through cell ELISA, "d-ch" presented a binding activity equal to the binding activity of the chimeric antibody, and "b-ch" and "c-ch" presented slightly lower binding activity (Figures 5 and 6). On the other hand, the neutralizing activity against the antigen compared to the neutralizing activity of the chimeric positive control antibody was almost equal in "b-ch", and slightly weak in "d-ch". In the "c-ch" version, it was significantly lower than that of the chimeric antibody (figure 7). Accordingly, the "b" and "d" versions of the humanized H chain were considered to be high-activity versions of the humanized H chain. (v) Combination of version "b" of humanized chain H and version "a" of humanized chain L An antibody (ba) which is version "b" of humanized chain H subjected to elevation by replacement of FR combined with the "a" version of humanized L chain was generated, and was assayed to determine antigen binding activity through cell ELISA. It was found that the quantity bound to the antigen decreased in the high concentration "figure 5". On the other hand, the neutralizing activity against the antigen was significantly weak compared to the neutralizing activity of the chimeric positive control antibody (Figure 8). Therefore, "b-a" and "a-a" were those that show high activity. The chimeric antibody used here was the antibody expressed in COS-7 cells, purified and evaluated. (vi) Combination of the "b" and "c" versions of the humanized L chain and a chimeric H chain. Antibodies ("ch-b" and "ch-c", respectively) which are versions "b" and "c" of humanized chain L combined with a chimeric chain H were generated, and it was found that both had a binding activity with the antigen and a neutralizing activity against the antigen equal to the chimeric antibody (figures 9 and 10). Accordingly, versions "b" and "c" were selected as a candidate for an L chain of humanized antibody. Version "b" derived from mouse antibody having a minor amino acid in the number of amino acid residues is considered superior to version "c" in terms of antigenicity. The chimeric antibody employed herein was the antibody expressed in purified and washed DG44 CHO cells. In the evaluation that we present below, the antibody was used as a positive control. (vii) Combination of version "b" of humanized chain H and version "b" and version "c" of chain L immobilized Antibodies ("bb" and "bc", respectively) which are the "b" versions of the humanized H chain combined with the humanized L-chain "b" and "c" versions were generated and tested to determine the binding activity with the antigen and the neutralizing activity against the antigen. Both had slightly lower activity than the chimeric antibody activity in both the binding activity and the neutralization activity (Figures 11 and 12). (viii) Combination of versions "b" and "d" of humanized chain H and version "b" of chain humanized Antibodies ("bb" and "db", respectively) which are the humanized chain H subjected to a version increased by replacement of FR combined with the humanized L chain version "b" were generated, and said antibodies were assayed for antigen binding activity by means of cell ELISA, "db" showed a binding activity equal to the binding activity of the chimeric antibody, and "bb" exhibited a slightly lower binding activity at the high concentration (Figure 13). On the other hand, the neutralizing activity against the antigen in comparison with the neutralizing activity against the chimeric positive control antibody was slightly on "b-b", and significantly weak on "d-b" (Figure 14). Accordingly, it was shown that "b-b" is a version of high neutralizing activity, whereas "d-b" is a version of high binding activity. (ix) Combination of the "e" version of the humanized H chain, and a chimeric L chain and the "b" version of humanized L chain Antibodies ("e-ch" and "e-b", respectively) which are the "e" version of the humanized L chain combined with a chimeric L chain and the humanized "b" version were generated, "e-ch" presented an antigen-binding activity equal to the linkage activity of the chimeric antibody, but in "eb" the amount of antibody that was expressed was very small and most of the binding activity was lost (Figure 15). The neutralizing activity against the "e-ch" antigen was significantly lower compared to the activity of the chimeric antibody (Figure 16). Consequently, it was concluded that the "e" version of the H chain combined with the "d" version of the L chain did not work well, (x) Combination of the "f", "g" and "h" versions humanized chain H and version "b" humanized chain L Antibodies ("fb", "gb" and "hb", respectively) which are combined versions "f", "g" and "h" humanized chain H with version "b" of chain humanized L were generated. In antibodies "f-b" and "h-b", the amount of antibody expressed was small. In the case of versions "f" and "h", antibodies combined with the chimeric L chain were generated, but were not expressed. "g-b" reached saturation at a low concentration, and had a weaker binding activity than the binding activity of the chimeric antibody (FIG. 17). The neutralizing activity against the "g-b" antigen was significantly weak compared to the neutralizing activity of the chimeric antibody (Figure 18). (xi) Combination of the "bl" and "di" versions of the humanized H chain, and the "b" version of the humanized L chain Antibodies ("bl-b" and "dl-b", respectively) which are the versions " bl "and" di "of humanized chain H combined with version" b "of humanized chain L were generated. Almost no antibody was expressed in them. For these, antibodies combined with a chimeric L chain were generated, but not expressed. (xii) Combination of versions "b3" and "d3" of humanized chain H, and version "b" of humanized chain L Antibodies ("b3-b" and "d3-b", respectively) which are the versions " b3"and" d3"of humanized chain H combined with version" b "of humanized chain L were generated. The antigen binding activity of "d3-b" was slightly lower than the binding activity of the chimeric antibody, and the binding activity of "b3-b" was much lower (Figure 19). The neutralizing activity against the antigen of "b3-b" was greater than that of "b-b", but was less than that of the chimeric antibody, and "d3-b" and "b-b" remained the same as activity (Figure 20). (xiii) Combination of the "i" and "j" versions of the humanized H chain, and the chimeric L chain and the "b" version of humanized L chain Antibodies ("i-ch" and "j-ch", respectively) which are the "i" and "j" versions of the humanized H chain combined with the chimeric L chain, and antibodies ("ib" and "jb", respectively) combined with the "b" version of the humanized L chain, and said antibodies were assayed to determine the binding activity with the antigen and the neutralizing activity against the antigen. The binding activity of any of the antibodies was almost equal to the binding activity of the chimeric antibody (Figures 21 and 22). "i-ch" exhibited a neutralizing activity greater than the neutralizing activity of the chimeric antibody, and the "j-ch" neutralizing activity was significantly less than that of the chimeric antibody (Figure 23). "i-b" exhibited a neutralizing activity equal to the neutralizing activity of the chimeric antibody, and "j-b" exhibited a significantly weaker neutralizing activity than the neutralizing activity of the chimeric antibody (Figure 24). Versions "bl" and "b2" of chain L humanized When the antibodies ("ch-bl" and "ch-b2", respectively) which are the "bl" and "b2" versions of humanized L chain, combined with a chain Chimeric H were generated, both had an antigen binding activity equal to the binding activity of the chimeric antibody (Figure 25). For the neutralizing activity against the antigen, "ch-bl" presented a binding activity equal to the binding activity of the chimeric antibody, while "ch-b2" presented a slightly higher activity than the chimeric antibody activity in the high concentration (figure 26). The "bl" and "b2" versions may be candidates for the humanized antibody L chain, but "b2" is superior in that it has a higher activity. (xv) Combination of version "b" of humanized chain H and version "b2" of humanized chain L An antibody ("b-b2") that is version "b" of chain humanized combined with version "b2" "Humanized chain was generated, and was tested to determine antigen binding activity and neutralizing activity against the antigen. The binding activity was slightly less than the binding activity of the chimeric antibody (Figure 27). The neutralizing activity, even though it was slightly greater than the neutralizing activity of "b-b", was less than the neutralizing activity of "i-b" (Figure 28). (xvi) Combination of version "i" of humanized H chain and "bl" version or "b2" version of humanized L chain. Antibodies were generated ("i-bl" and "i-b2", respectively) that are version "i" of humanized chain H combined with version "bl" or version "b2" of humanized chain L, and said antibodies were tested to determine antigen binding activity and neutralizing activity against the antigen. The binding activity of "i-b2" was almost equal to the binding activity of the chimeric antibody, and the binding activity of "i-bl" was slightly less than the binding activity of the chimeric antibody (figure 29). The neutralizing activity of "i-bl 'li-b2' was slightly greater than the neutralizing activity of the chimeric antibody and" ib ", which was in a decreasing order of" i-b2">" i-bl "(FIG. EXAMPLE 5. PREPARATION OF HUMANIZED ANTIBODY THAT PRODUCES CHO CELLS AND EVALUATION OF ITS ACTIVITY (1) Establishment of a cell line that produces CHO stably, in order to establish cell lines that stably produce a humanized antibody (bb, ib, and i-b2), an antibody expression gene vector was introduced into CHO cells (DG44) acclimated to a serum-free medium.

Plasmid DNA, hHvb-hLvb / N5KG4P, hHvi-hLvb / N5KG4P, and hHvi-hLvb2 / N5KG4P were digested with the restriction enzyme SspI (Takara Shuzo) and linearized, extracted with phenol and chloroform, and purified by ethanol precipitation. The linearized expression gene vector was introduced into the DG44 cells using the electroporation instrument (Gene Pulser, Bio Rad). The DG44 cells were suspended in PBS at a cell concentration of ixl07 cells / ml, and at approximately 8 ml of this suspension 10 or 50 μg of the DNA was added, which was subjected to pulses of 1,500 V and capacity of 25 μF. After 10 of the recovery period at room temperature, the treated cells were suspended in a CHO-S-SFMII medium (GIBCO) containing hypoxanthine / thymidine (GIBCO) (hereinafter referred to as HT), which was inoculated in two plates of 96 wells (Falcon) at a rate of 100 μl / well, and cultured in a C02 incubator. Eight to nine hours after the start of the culture, 100 μl / well of the CHO-S-SFMII medium containing HT and 1 mg / ml of GENETICIN (GIBCO) were added to change to 500 μg / ml of the GENETICIN selection medium and the cells in which the antibody gene had been introduced were selected. The medium was changed with a fresh medium once every 3-4 days with 1/2 volume. At the point of time approximately 2 weeks after switching to the selection medium, an aliquot of the culture supernatant was recovered from the well in which a favorable cell growth was observed 4-5 days later. The concentration of the antibody expressed in the culture supernatant was measured by ELISA in accordance with what was described above to measure the concentration of antibodies, and cells having a high yield of antibody production were selected. (2) Large-scale coding of humanized antibody After the DG44 cell lines selected according to the above-mentioned which produce the humanized antibody ("bb", "ib", and "i-b2") were cultured for a few days in 500 ml / bottle of medium CHO-S-SFMII using a 2 L roller bottle (CORNING), the culture medium was harvested and a fresh CHO-S-SFMII culture medium was added and cultivated again. The culture medium was centrifuged to remove cell debris, and filtered with a 0.22 μm or 0.45 μm filter. Repeating this, a total of about 2 L each of the culture supernatant was obtained. From the obtained culture supernatant, an antibody was purified by the CONSEP LC100 system (Millipore) connected to an affinity column of Protein A (Poros). (3) Measurement of antibody concentration by ELISA ELISA plates for antibody concentration measurement were prepared as follows: each well of a 96-well ELISA plate (Maxisorp, NUNC) was immobilized with 100 μl of IgG? antihuman goat (BioSource) prepared at a concentration of 1 μg / ml with CB. After blocking with 200 μl of DB, the culture supernatant of the CHO cells in which the antibody had been expressed or the purified antibody was serially diluted with DB, and agiregated to each well. After incubation at room temperature for 1 hour and washing with RB, 100 μl of IgG? antihuman goat conjugated with alkaline phosphatase (BioSource) diluted 1000 times with DB. After incubation at room temperature for 1 hour and washing with DB, 100 μl of the substrate solution was added, and then the absorbance at 405/655 nm was measured using the Microplate Reader (BioRad). As a standard for the measurement of the concentration, IgG4 was used? human (The Binding Site). (4) Measurement of antigen binding activity ELISA plates for cells for antigen binding measurement were prepared as follows. The cells used were J82 human bladder carcinoma cells (ATCC HTB-1), which were inoculated in a 96-well cell culture plate at a cell count of 1 × 10 5 cells. This was cultured (RPMI1640 medium containing 10% fetal bovine serum (GIBCO)) for one day in a CO 2 incubator to allow the cells to attach there. After discarding the culture liquid, each well was washed twice with PBS. 100 μl of PFA / PBS was added to each well, and placed on ice for 10 minutes to immobilize the cells. PFA / PBS was discarded, and each well was washed twice with 300 μl of PBS and then blocked with 250 μl of DB. Based on the previous result of the measurement, the purified antibody was serially diluted with DB starting at 10 μg / ml by a factor of 2, 100 μl of which was added to each well. After incubation at room temperature for 2 hours and after washing with RB, 100 μl of IgG? goat antihuman conjugated with alkaline phosphatase (BioSource) diluted 1000 times with DB. After incubation at room temperature for 1 hour and after washing with RB, 100 μl of the substrate solution was added, and the absorbance was measured at 405/655 nm using the Microplate Reader (Bio-Rad) . (5) Measurement of neutralizing activity against TF (factor inhibition activity against Fxa production) The factor Xa production inhibition activity of the humanized antibody was measured with the activity of inhibition against factor Xa production activity by the thromboplastin derived from human placenta, Thromborel S (Boehringer AG), as an index. Thus, 60 μl of the buffer (TBS containing 5 mM CaCl 2 and 0.1% BSA) was added to 10 μl of 5 mg / ml of Thromborel S and 10 ml of the antibody, which was then incubated in a 96-well plate at room temperature for 1 hour. Antibody ~ was serially diluted with the buffer starting at 200 μg / ml by a factor of 5. 10 μl each of 3,245 μg / ml human factor X (Celsus Laboratories) and 82.5 ng / ml human factor Vlla (Enzyme) Research) were added and further incubated at room temperature for 45 minutes. 10 μl of 0.5 M EDTA was added to stop the reaction. 50 μl of the chromogenic substrate solutions were added and the absorbance was determined at 405/655 nm by means of the Microplate Reader (Bio Rad). After reacting at room temperature for 30 minutes, the absorbance was again determined at 405/655 nm. The residual activity (%) was determined from each absorption change by absorption change for 30 minutes without the addition of antibody as a 100% activity. The chromogenic substrate solution was prepared by dissolving the chromogenic substrate Testzyme S-2222 (Chromogenix) in accordance with the accompanying instructions, and with mixing with the polybrene solution (0.6 mg / ml hexadimethylene bromide, SIGMA) at 1: 1. (6) Measurement of neutralizing activity against TF (inhibition activity against FX binding). The inhibition activity against the FX binding of the humanized antibody was measured using placental-derived thromboplastin, Thromborel S (Boehringer AG), where a TF and factor Vlla complex had previously been formed and the inhibitory activity was measured. against the FX link with the factor Xa production activity of the TF-Fvlla complex as an index. A) Yes, 60 μl of the buffer (TBS containing 5 mM CaCl2 and 0.1% BSA) was added to 10 μl of 5 mg / ml of Thromborel S and 10 ml of human Vlla factor (Enzyme Research), which was pre-incubated on a plate of 96 wells at room temperature for 1 hour. 10 μl of the antibody solution was added, incubated at room temperature for 5 minutes and 10 μl of 3,245 μg / ml factor X (Celsus Laboratories) were added and incubated at temperature also for 45 minutes. The antibody was serially diluted with the regulator starting at 200 μg / ml by a factor of 2. 10 μl of 0.5 EDTA was added to suspend the reaction. 50 were added. 50 μl of the chromogenic substrate solution was added and absorbance was determined at 405/655 nm through the Microplate Reader (Bio Rad). After reacting at room temperature for 30 minutes, the absorbance at 405/655 nm was again measured. Residual activity (%) was determined from each absorption change by absorption change for 30 minutes without the addition of antibody as a 100% activity. The. chromogenic substrate solution was prepared by dissolving the chromogenic substrate Testzyme S-2222 (Chromogenix) in accordance with the enclosed instructions, and mixed with a solution of polybrene (0.6 mg / ml hexadimethylene bromide, SIGMA) at 1: 1. (7) Measurement of the neutralizing activity against the activity of inhibition against plasma coagulation. The neutralizing activity against TF (inhibitory activity against plasma coagulation) of humanized antibody was measured using the prothrombin time determined using the thromboplastin derived from human placenta, Thr? Mborel S (Boehringer AG) as an index. Thus, 100 μl of human plasma (Cosmo Bio) were placed in a sample cup to which 50 μl of antibody diluted in various concentrations were added, and heated at a temperature of 37 ° C for 3 minutes. 50 μl of 1.25 mg / ml Thromborel S that had previously been preheated to 37 ° C were added to initiate plasma coagulation. The coagulation time was measured using Amelung KC-10A connected to the Amelung CR-A (both from M.C. Medical). The antibody was serially diluted with TBS containing 0.1% BSA (hereinafter known as BSA-TBS) starting at 80 μg / ml by a factor of 2. with the coagulation time of no antibody addition as a plasma coagulation activity of 100% TF, the residual TF activity was calculated from each coagulation time in addition of antibody based on the standard curve obtained by graphically depicting the Thromborel S concentration and the clotting time. The standard curve was created from various concentrations of Thomborel S and the clotting time was measured. 50 μl of BSA-TBS was added to 50 μl of appropriately diluted Thromborel S, which was heated at a temperature of 37 ° C for 3 minutes, 100 μl of preheated human plasma was added at a temperature of 37 ° C to initiate the coagulation, and the clotting time was determined. It was diluted in series Thromborel with Hank's buffer (GIBCO) containing 25 mM CaCl2, starting at 6.25 mg / ml by a factor of 2. Thromborel concentration was plotted on the abscissa axis and the clotting time in the axis of the ordinates in a log-log paper, which became a standard curve. (8) Activity evaluation All humanized antibodies, "b-b-", and "i-b2" exhibited activity equal to or greater than the activity of the chimeric antibody (Figure 31). For the activity of inhibition against Fxa production, the activity of FX inhibition and the activity of inhibition against plasma coagulation also, the humanized antibodies, "bb", "i-b2" presented an activity equal to or greater than the activity of the chimeric antibody and the activity was, in descending order "i-b2" > "l-b" > "b-b" (figures 32, 33, and 34). EXAMPLE 6. KINETIC ANALYSIS IN TF INTERACTION AND ANTI-TF ANTIBODY USING BIACORE A kinetic analysis of the antigen-antibody reaction was carried out using BIACORE. The recombinant protein G was immobilized on a sensor chip, to which the antibody was coupled. The purified recombinant TF (a soluble TF in which the FLAG peptide was labeled in 1-219) was used as the antigen, and the soluble TFs prepared in various concentrations were used as analytes. From the sensorgram obtained, they were calculated for kinetic meters (constants of dissociation speed Kdiss, and constants of speed of union Kass). For a kinetic analysis reference is made to "Kinetic Analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system" (Karlsson, R. et al., (1991) J. Immunol. Methods 145: 229-240). (1) Immobilization of G protein on the sensor flake The G protein (ZYMED) is immobilized on the CM5 sensor flake (BIACORE). The shock absorber HBS-EP (HEPES) was used as shock absorber 0. 01 M, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate 20 (volume / volume)) (BIACORE) and the flow rate was 5 μl / min. Carboxymethyldextrane carboxyl groups in the CM5 sensor chip were activated by injection of 0.05 M N-hydroxysuccinimide (NHS) / N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride 0.2 M (EDC). Subsequently, 10 μl of 50 μg / ml protein G was injected, and this was repeated three times for immobilization. Protein G was prepared by dissolving in 10 mM Tris-HCl buffer (pH 7.5) at a concentration of 10 mg / ml, and diluting to 50 μg / ml with a buffer of 10 mM sodium acetate (pH 4.0). In addition, 100 μl of 1.0 M ethanolamine hydrochloride (pH 8.5) was injected to block excess active groups. To this, 10 μl of glycine buffer-0.1 M hydrochloric acid (pH 2.5) and 10 μl of 10 mM hydrochloric acid were injected to wash the bound substances non-covalently. By doing this for each flow cell and injecting 10 μl of version "ib2" of humanized anti-TF antibody, which confirmed the binding of approximately 1000 RU. (2) Interaction of immobilized TF-antibody and human TF. Human TF where the FLAG peptide had been bound to a C-terminus of amino acid sequence 1-219 was expressed in CHO cells and was purified. This was used as the soluble TF. As an experiment buffer, the HBS-EP buffer was used. 10 μl of 72 mM antibody solution were injected at a flow rate of 20 μl / min to immobilize the antibody. The antibody was diluted with the HBS-EP buffer. 40 μl of the soluble human TF solution in various concentrations were injected at a flow rate of 30 μl / min. In the analysis, a period of 80 seconds for the injection was established as the binding phase and then changed to the HBS-EP buffer to establish the dissociation phase of 120 seconds. After the dissociation phase was completed, 10 μl of 20 mM hydrochloric acid was injected to reconstitute J_a sensor flake. The binding, dissociation, and reconstitution were established as one cycle, and the sensorgram was obtained for each antibody. The soluble human TF solution was prepared in concentrations of 250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.81 nM, and 3.91 nM using the HBS-EP buffer. As a control, the sensorgram obtained by injection of the HBS-EL buffer used for dilution was used. The aforementioned procedure was carried out for each of the flow cells number 1 to 3. (3) Interaction kinetic analysis The data file of interest was read, and the reaction patterns were compared by superscription using as baseline the sensorgram of the HBS-EP shock absorber. In addition, using a program of analytical application "BIAevaluation 2.1" (Pharmacia) exclusively for BIACORE, which calculates the kinetic parameters (Kass junction speed constant and Kdiss dissociation speed constant) by curve fitting, the analysis was carried out kinetic of the interaction. In order to determine the Kass junction speed constants, the type 4 analysis model was used (BIAevaluation 2.1 Software HandBook, A1-A5). Based on the calculated values for each flow cell, kinetic parameters were obtained for each antibody. The result (average of the values calculated for each flow cell +/- standard deviation) is shown in Table 6. Table 6 Kinetic parameters of chimeric and humanized anti-TF antibody (n = 3) chimeric bb ib i-b2 Kdiss [ xl0 ~ 4l / s] 5.06 +/- 0.12 9.52 +/- 0.22 6.49 +/- 0.17 6.35 +/- 0.15 Kass [xl05l / Ms] 4.65 +/- 0.32 4.15 +/- 0.27 4.67 +/- 0.30 5.44 + / -0.36 KD [xl0"9M] 1.09 +/- 0.09 2.30 +/- 0.15 1.39 +/- 0.13 1.17 +/- 0.11 EXAMPLE 7. MEASUREMENT OF REACTIVITY ANTI-TF ANTI-TF HUMANIZED ANTIBODY FOR HUMAN TF Using the hybridization method point absorption ("Protein Experimental Method of Molecular Biological Research, Revised ", Yodosha, edited by Takenawa Tadao i, page 101), the non-denatured TF reactivity, denatured TF in non-reduced condition, and denatured TF in reduced condition were investigated using TF where the FLAG had been labeled in The extracellular region was expressed in CHO cells and purified (ShTF) STF was diluted with each of three buffers (buffer A: 10 mM Tris-HCl pH 8.0, buffer B: 10 mM Tris-HCl (pH 8.0), urea 8 M, buffer C: 10 mM Tris-HCl pH 8.0, 8 M urea, 5 mM DTT Non-denatured TF was treated with buffer A, while non-reductively denatured TF was treated with buffer B, and TF reductively denatured was treated with buffer C. Each sample was treated for 24 hours at room temperature After the treatment, the sample was applied on a nitrocellulose membrane (BioRad) 0.5 μl, 1 μl, and 2 μl of the sample (3 μg / ml) a the membrane, and the membrane was dried in the air. It was blocked with DB (50 mM Tris-HCl, pH 8.1, 0.15 M NaCl, 1 mM MgCl2, 0.05% (volume / volume) Tween 20, 0.02% (weight / volume) NaN3, 1% (weight / volume) BSA) . The membrane reacted in the DB containing the humanized anti-TF antibody or the DB (control). After washing with PBS containing 0.05% (volume / volume) of Tween 20, it reacted with DB containing anti-human IgG antibody labeled with peroxidase (DAKO). After washing with PBS containing 0.05% (volume / volume) of Tween 20, it was treated with the Western Blot ECL reagent (Amerscham) and exposed to an X-ray film for 30 seconds. As shown in Figure 35, the chimeric anti-TF antibody and the humanized anti-TF antibodies (versions "bb", "ib", and "ib2") reacted to all the non-denatured TF, non-reductively denatured TF, and TF reductively denatured. EXAMPLE 8. CONFIRMATION OF ANTITHROMBOTIC EFFECTS IN THE ACUTE DICRAT RAT MODEL The antithrombotic effects of the anti-TF antibody were confirmed in a model of thromboplastin-induced DIC using rats. Thus, a human thromboplastin solution was injected continuously into the vein of male SD rats at 40 mg / kg for 3 hours to create a model of DIC. The anti-TF antibody (i-b2 chimeric and humanized anti-TF antibody) was administered intravenously at a dose of 0.2 mg / kg five minutes before the start of injection of the thromboplastin solution. Fifteen minutes after completing the continuous injection of the thromboplastin solution, aggregated blood was extracted with citrate from the abdominal aorta, for which the platelet count, activated partial thromboplastin time (aPTT), fibrinogen concentration were measured. (Fib), the concentration of soluble fibrin monomer complex (sFMC) and the concentration of thrombin / antithrombin III complex (TAT). The result shown in Table 7 indicated that continuous thromboplastin injection caused a decrease in platelet count, extended aPTT, decreased fibrinogen concentration, increased concentrations of sFMC and TAT, and an evident hypercoagulated state. In contrast, both chimeric and humanized anti-TF antibodies inhibited these changes equally strongly. The result showed that the humanized anti-TF antibody is useful as an antithrombotic agent.

Table 7 Measurement element normal group without control group administering with administration of thromboplastinization of solvent platelet count 115.5 ± 11.8 82.9 ± 14.3 (xlOVmm3) aPTTC (sec) 20.1 ± 1.1 36.2 ± 13.9 Concentration of fibri100.0 ± 4.2 64.8 ± 20.0 nanogen (normal group = 100%) Concentration sFMC 74.2 ± 5.5 3517 ± 3645 (μg / ml) Concentration TAT 3.4 ± 0.6 29.6 ± '31.0 (ng / ml) Element of measurement group with group with administration humanized chimeric antibody antibody administration platelet count 100.7 ± 12.9 96.1 ± 13.3 (xlOVmm3) aPTTC (sec) 22.3 ± 0.7a) 21.8 ± 1.3a) Fibri- concentration 101.0 ± 6.6a) 98.9 ± 5.7a) Ndgen (normal group = 100%) Concentration sFMC 129.9 ± 46.8a: 66.5 ± 23.0a) (μg / ml) Concentration TAT 3.8 ± or 4.2 ± 0.9 (ng / ml) __ (Mean ± standard deviation) Significance of difference in relation to the control group with solvent administration: a): p < 0.01, b): p < 0.05. REFERENCE EXAMPLE 1. PREPARATION OF MONOCLONAL ANTIBODY ANTI-TF 1. Purification of human TF The purification of TF from human placenta was carried out according to the Ito method (Ito, T. et al., J. Biol. Chem., 114: 691-696, 1993). Thus, the human placenta was homogenized in a Tris-regulated saline solution (TBS, pH 7.5) containing 1.0 mM benzamidine hydrochloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM diisopropyl fluorophosphate, and 0.02% sodium azide, and then the precipitate was degreased with cold acetone. The degreased powder obtained was suspended in the aforementioned buffer containing 2% Triton X-100 to solubilize the TF. The supernatant was subjected to affinity chromatography using a column Concanavalin A-Sepharose 4B (Pharmacia) and column Sepharose 4B (Pharmacia) bound to anti-TF antibody, and the purified TF was obtained. It was concentrated with an ultrafiltration membrane (PM-10, Amicon) and stored as the purified sample at a temperature of 4 ° C). The content of TF in the purified sample was quantified by Sandwich ELISA which combined a commercially available anti-TF monoclonal antibody (American Diagnostica) and polyclonal antibody (American Diagnostica) with recombinant TF as a standard. The purity in the purified sample was confirmed by subjecting the sample to SDS-PAGE using a polyacrylamide gel with a density gradient of 4 to 20% and staining the product with silver. 2. Immunization and Hybridoma Preparation After mixing the purified human TF (approximately 70 μg / ml) with an equal volume of Freund's complete adjuvant (Difco), he was immunized subcutaneously in the abdomen of male Balb / c mice of 5 weeks old. age (Nippon Charles River) at 10 μg TF / mouse. On days 12, 18, and 25, a subcutaneous reinforcement of TF mixed with Freund's incomplete adjuvant, 5 μg / mouse TF was applied, and as final immunization, the TF solution diluted with PBS was administered intraperitoneally at a rate of 5 μg / mouse on day 32. Three days after the final immunization, the spleen cells were prepared from four mice, and said cells were fused on the mouse myeloma cell line P3U1 at 1/5 cell counts by the method of polyethylene glycol. The fused cells were suspended in RPMI-1640 medium (mentioned below as RPMI medium) (Lifetech Oriental) containing 10% fetal bovine serum, which was inoculated into 400 wells per mouse (approximately 400 cells / well) of a plate of 96 wells. On days 1, 2, 3, and 5 after fusion, half the volume of the medium was exchanged with the RPMI medium (hereinafter known as HAT medium) containing HAT (Dainippon Seiyaku) and Hl (Boehringer Mannheim GmbH) for carry out the HAT selection of hybridoma. The hybridomas selected by the screening method described below were cloned by carrying out a limiting dilution twice. For limiting dilution, 0.8 cells were inoculated per well in the two 96-well plates. For wells in which a single colony was confirmed by microscopic examination, clones were selected by the following measurement of TF binding activity and neutralizing activity against TF. The obtained clones were acclimated from the HAT medium to the RPMI medium. After confirming the absence of reduction of the antibody production capacity due to acclimation, a limiting dilution was again carried out for complete cloning. By the above procedure, hybridomas were established that produce 6 antibodies (ATR-2, 3, 4, 5, 7, and 8) that strongly inhibit the binding of the TF / factor Vlla complex and factor X. 3. Formation of ascites and purification of antibodies The formation of ascites of the established hybridomas was carried out according to the standard method. In this way, 106 hybridomas that were subcultured in vitro were grafted intraperitoneally in male BALB / c mice that had previously received two intravenous administrations of mineral oil. The ascites was collected from the mice that had a swollen abdomen 1-2 weeks after grafting. The purification of the ascites antibody was carried out using the ConSepLClOO system (Millipore) equipped with the Protein A column (Nippon Gaishi). 4. ELISA for cells J82 human bladder carcinoma cells (Fair DS et al., J. Biol. Chem., 262: 11692-11698, 1987) of which are known to express TF at a high level were obtained from of ATCC and subcultured and maintained in the RPMI medium under the conditions of 37 ° C, 5% C02, and 100% humidity. ELISA plates for cells were prepared by inoculating J82 cells to a 96-well plate at a rate of 10 5 cells / well, culturing for one day under the above-mentioned conditions, stirring the medium and then washing twice with a buffered saline solution. with phosphate (PBS), adding a solution of 4% paraformaldehyde (PFA), and allowing to stand on ice for 10 minutes for immobilization. After removal of PFA, the plate was washed with PBS, the Tris buffer (blocking buffer) containing 1% BSA and 0.02% sodium azide was added, and the plate was stored at a temperature of 4 ° C. until its use. ELISA was carried out for cells in the following manner. The blocking buffer was removed from the plate prepared in accordance with the aforementioned, to which was added an anti-TF antibody solution or a hybridoma culture supernatant and reacted at room temperature for 1.5 hours. After washing with PBS containing 0.05% Tween , goat anti-mouse IgG conjugated with alkaline phosphatase (H + L) (Zymed) reacted for 1 hour. After washing, 1 mg / ml disodium p-nitrophenyl phosphate (Sigma) was added, and one hour later the absorbance at 405/655 nm was measured to determine the amount of anti-TF antibody that bound J82 cells. 5. Neutralizing assay assay system against TF with factor Xa activity as Index To 50 μl of Tris-regulated saline (TBS: pH 7. 6) containing 5 mM CaCl2 and 0.1% bovine serum albumin, 10 μl of a thromboplastin solution derived from human placenta (5 mg / ml) (Thromborel S) was added (Boehring) and 10 μl of a factor Vlla solution (82.5 ng / ml) (American Diagnostic), and reacted at room temperature for 1 hour to allow the formation of the TF / Factor Vlla complex. Then, 10 μl of a predetermined concentration of a diluted anti-TF antibody solution or hybridoma culture supernatant and 10 μl of a Factor X solution (3.245 μg / ml) (Celsus Laboratories) were added and reacted for 45 minutes, and 10 μl of 0.5 M EDTA was added to suspend the reaction. Fifty μl of a 2mM S-2222 solution (Daiichi Kagaku Yakuhin) were added, and changes in absorbance were measured at 405/655 nm in 30 minutes and established as the activity that produced TF factor X. In this method, the antibody activity that inhibits the binding of the TF / factor Vlla complex and factor X can be determined. 6. Activity assay system and inhibition against plasma coagulation Fifty μl of an appropriately diluted anti-TF antibody solution was mixed with 100 μl of a commercially available normal human plasma (Kojin Bio) and reacted at a temperature of 37 ° C for 3 minutes. Then 50 μl of a thromboplastin solution derived with human placenta (1.25 mg / ml) was added, and the time to plasma coagulation was measured using the plasma coagulation measuring instrument (CR-A: Amelung). 7. Determination of antibody isotype The culture supernatant of the hybridoma and the purified antibody, the mouse monoclonal antibody isotyping kit (manufactured by Amersham) was used to confirm the isotype of the antibody. The results appear below. Table 8 Immunoglobulin Isotype of Anti-TF Monoclonal Antibody ATR-2 IgGl, k ATR-3 IgGl, k ATR-4 IgGl, k ATR-5 IgGl, k ATR-7 IgG2a, k ATR-8 IgG2a, k REFERENCE EXAMPLE 2. METHOD FOR PREPARING HUMAN SOLUBLE TF - Soluble human TF (shTF) was prepared in the following manner. The gene encoding the region of human TF penetration where amino acids at position 220 and later had been replaced by the FLAG M2 peptide was inserted into a mammalian cell expression vector (containing the neomycin resistance gene). and the DHFR gene), and was introduced into CHO cells. For the human TF cDNA sequence, reference is made to an article by James H. Morrissey et al. (Cell (1987) 50: 129-135). The sequence of genes and the amino acid sequence of this soluble human TF appear in SEQ ID NO: 151. After the pharmacological selection with G418, the expressed cells were selected, which were subjected to expression amplification with methotrexate, and were established the cells that express shTF. The cells were cultured in the serum-free CHO-S-SFMII medium (GIBCO) to obtain a culture supernatant containing shTF. It was diluted twice with an equal volume of a 40 mM Tris-HCl buffer (pH 8.5), which was added to the Q-Sepharose Fast Flow column (100 ml, Pharmacia Biotech) equilibrated with a 20 mM Tris-HCl buffer ( pH 8.5). After washing with the same buffer containing 0.1 M NaCl, the NaCl concentration was changed to 0.3 M, and shTF was eluted from the column. To the fraction of shTF obtained, ammonium sulfate was added to a final concentration of 2.5 M, and centrifuged (10,000 revolutions per minute, 20 minutes) to precipitate contaminating proteins. The TOYOPEARL butyl supernatant (30 ml, TOSO) was added, and then washed with a 50 mM Tris-HCl buffer (pH 6.8) containing 2.5 M ammonium sulfate. In the 50 mM Tris-HCl buffer (pH 6.8) , the concentration of ammonium sulfate was reduced linearly from 2.5 M to 0 M to allow shTF elution. The peak fractions containing shTF were concentrated by Centri-Prep 10 (Amicon). The concentrate was added to the TSKgel G3000S WG column (21.5 x 600 mm, TOSO) equilibrated with a 20 mM Tris-HCl buffer (pH 7.0) containing 150 mM NaCl, and the peak fraction of shTF was collected. It was filter sterilized with a 0.22 μm membrane filter and the product was placed in the soluble human TF (shTF). The concentration of the sample was calculated considering that the molar extinction coefficient of the sample epsilon = 40,130 and molecular weight = 43,210. FREE TEXT FROM THE SEQUENCE LIST The content of the sequence list <; 223 > It is the following: SEQ ID NO: 1 Initiator MHC-G1 SEQ ID NO: 2 Initiator MHC-G2a SEQ ID NO: 3 Initiator MKC SEQ ID NO: 4 Initiator M13 M4 SSEEQQ IIDD NNOO :: 55: Initiator M13 RV SEQ ID NO: 6: Sequence of amino acids of the V region of the H chain of the mouse anti-TF monoclonal antibody ATR-2 and the nucleotide sequence encoding it SEQ ID NO: 7: Amino acid sequence of the V region of the H chain of the anti-monoclonal antibody -TF of mouse ATR-3 and the sequence of nucleotides that encode it SEQ ID NO: 8: Amino acid sequence of the V region of the H chain of the monoclonal antibody anti-TF of mouse ATR-4 and the sequence of nucleotides that encode SEQ ID NO: 9: Amino acid sequence of the V region of the H chain of the mouse anti-TF monoclonal antibody ATR-5 and the nucleotide sequence encoding it SEQ ID NO: 10: Amino acid sequence of the V region of the H chain of the mouse anti-TF monoclonal antibody ATR-7 and the nucleotide sequence that or encode SEQ ID NO: 11: Amino acid sequence of the V region of the H chain of the mouse anti-TF monoclonal antibody ATR-8 and the nucleotide sequence encoding it SEQ ID NO: 12: Amino acid sequence of the region V of the L chain of the mouse anti-TF monoclonal antibody ATR-2 and the nucleotide sequence encoding it SEQ ID NO: 13: Amino acid sequence of the V region of the L chain of the mouse anti-TF monoclonal antibody ATR -3 and the nucleotide sequence encoding it SEQ ID NO: 14: Amino acid sequence of the V region of the L chain of the mouse anti-TF monoclonal antibody ATR-4 and the nucleotide sequence encoding it SEQ ID NO: 15: Amino acid sequence of the V region of the L chain of the mouse anti-TF monoclonal antibody ATR-5 and the nucleotide sequence encoding it SEQ ID NO: 16: Amino acid sequence of the V region of the L chain of the mouse anti-TF monoclonal antibody ATR-7 and the sequence of nucleotides encoding it SEQ ID NO: 17: Amino acid sequence of the V region of the L chain of the mouse anti-TF monoclonal antibody ATR-8 and the nucleotide sequence encoding it SEQ ID NO: 18: Initiator ch5HS SEQ ID NO: 19: Initiator ch5HA SEQ ID NO: 20: Initiator ch5LS SEQ ID NO: 21: Initiator ch5LA SEQ ID NO: 22: CDR graft initiator hR5HvlS SEQ ID NO: 23: CDR graft initiator hR5Hv28 SEQ ID NO: 24: CDR graft initiator hR5Hv4S SEQ ID NO: 25: CDR graft initiator hR5Hv3A SEQ ID NO: 26: CDR graft initiator hR5hv5A SEQ ID NO: 27: Initiator hR5HvPrS SEQ ID NO: 28: Initiator hR5HvPrA SEQ ID NO: 29 : Amino acid sequence of the humanized H chain V region, "a" version and the nucleotide sequence encoding it SEQ ID NO: 30: Amino acid sequence of the humanized H chain V region, version "a" SEQ ID NO : 31: Mixing initiator FR F3RFFS SEQ ID NO: 32: Mixing initiator FR F3RFBS SEQ ID NR: 33 : Mixing initiator FR F3RFFA SEQ ID NO: 34: Mixing initiator FR F3RFBA SEQ ID NO: 35: Mixing initiator FR F3NMFS 'SEQ ID NO: 36: Mixing initiator FR F3NMBS SEQ ID. NO: 37: FR mixing primer F3NMFA SEQ ID NO: 38: FR mixing primer F3NMBA SEQ ID NO: 39: Amino acid sequence of the humanized H chain V region, version "b" and the nucleotide sequence encoding it SEQ ID NO: 40: Amino acid sequence of the humanized H chain V region, version "b" SEQ ID NO: 41: Amino acid sequence of the humanized H chain V region, version "c" and the nucleotide sequence that it encodes SEQ ID NO: 42: Amino acid sequence of the humanized H chain V region, version "c" SEQ ID NO: 43 FR mixing primer F3EPS SEQ ID NO: 44 FR mixing primer F3EPA SEQ ID NO: 45 Initiator F3PrS SEQ ID NO: 46 Initiator F3PrA SEQ ID NO: 47 Mixture initiator FR F3VHS SEQ ID NO: 48 Mixture initiator FR F3VHA SEQ ID NO: 49: Amino acid sequence of the humanized H chain V region, version "d" and the nucleotide sequence encoding it SEQ ID NO: 50: Amino acid sequence of the humanized "H" chain V region, "d" version SEQ ID NO: 51: Amino acid sequence of the V region of humanized H chain, "e" version and the nucleotide sequence encoding it SEQ ID NO: 52: Amino acid sequence of the humanized H chain V region, "e" version SEQ ID NO: 53 FR mixing primer F3SSS SEQ ID NO: 54 FR Mixing Initiator F3SSA SEQ ID NO: 55 FR Mixing Initiator F3CDS SEQ ID NO: 56 FR Mixing Initiator F3CDA SEQ ID NO: 57: Amino Acid sequence of humanized H chain V region, "f" version and the nucleotide sequence encoding it SEQ ID NO: 58: Amino acid sequence of the humanized H chain V region, version "f" SEQ ID NO: 59: Amino acid sequence of the humanized H chain V region, version " g "and the nucleotide sequence encoding it SEQ ID NO: 60: Amino acid sequence of the a humanized H chain region V, version "g" SEQ ID NO: 61: FR mixing primer F3ADS SEQ ID NO: 62: Mixing primer FR F3ADA SEQ ID NO: 63: Amino acid sequence of the H chain V region humanized version "h" and the nucleotide sequence encoding it SEQ ID NO: 64: Amino acid sequence of the humanized H chain V region, "h" version SEQ ID NO: 65: FR F3MMS mixing primer SEQ ID NO : 66: Mixing initiator FR F3MMA SEQ ID NO: 67: Mixing initiator FR F3BMS SEQ ID NO: 68: Mixing initiator FR F3BMA SEQ ID NO: 69: Amino acid sequence of the humanized H chain V region, version " i "and the nucleotide sequence encoding it SEQ ID NO: 70: Amino acid sequence of the humanized H chain V region, version" i "SEQ ID NO: 71: Amino acid sequence of the humanized H chain V region, "j" version and the nucleotide sequence that encodes it SEQ ID NO: 72: Amino acid sequence of the V region of humanized H chain, version "j" SEQ ID NO: 73: Mixing initiator FR F2MPS SEQ ID NO: 74: Mixing initiator FR F2MPA SEQ ID NO: 75: Amino acid sequence of the humanized H chain V region, version " bl "and the nucleotide sequence encoding it SEQ ID NO: 76: Amino acid sequence of the humanized H chain V region," bl "version SEQ ID NO: 77: Amino acid sequence of the humanized H chain V region, "di" version and the nucleotide sequence that encodes it SEQ ID NO: 78: Amino acid sequence of the humanized H chain V region, "di" version SEQ ID NO: 79: Mixing initiator FR F2VHS SEQ ID NO: 80 : FR Mixing Initiator F2VHA SEQ ID NO: 81: Amino acid sequence of the humanized H chain V region, "b3" version and the nucleotide sequence encoding it SEQ ID NO: 82: Amino acid sequence of the V region of humanized H chain, version "b3" SEQ ID NO: 83: Amino acid sequence of the V region of ac dena H humanized, version "d3" and the nucleotide sequence encoding it SEQ ID NO: 84: Amino acid sequence of the humanized H chain V region, version "d3" SEQ ID NO: 85: FR mixing vector h5LvlS SEQ ID NO: 86: mixing vector FR h5Lv4S SEQ ID NO: 87: mixing vector FR h5Lv2A SEQ ID NO: 88: mixing vector FR h5Lv3A SEQ ID NO: 89: mixing vector FR h5Lv5A SEQ ID NO: 90: Initiator h5LvS SEQ ID NO: 91: Initiator h5LvA SEQ ID NO: 92: Amino acid sequence of humanized L chain V region, "a" version and the nucleotide sequence encoding it SEQ ID NO: 93: Amino acid sequence of the Humanized L chain region V, version "a" SEQ ID NO: 94: FR mixing primer F3SS SEQ ID NO: 95: FR mixing primer F3SA SEQ ID NO: 96: FR F3RS mixing primer SEQ ID NO: 97: Mixer initiator FR F3RA SEQ ID NO: 98: Amino acid sequence of humanized L chain V region, "b" version and nucleotide sequence etids encoded by it SEQ ID NO: 99: Amino acid sequence of the humanized L chain V region, version "b" SEQ ID NO: 100: Amino acid sequence of the humanized L chain V region, "c" version and the nucleotide sequence encoding it SEQ ID NO: 101: Amino acid sequence of the V chain region L humanized, version "c" SEQ ID NO: 102: Mixing initiator FR F2SS SEQ ID NO: 103: Mixing initiator FR F2SA SEQ ID NO: 104: Mixing initiator FR F2XS SEQ ID NO: 105: FR mixing initiator F2XA SEQ ID NO: 106: Amino acid sequence of humanized L chain V region, "bl" version and the nucleotide sequence encoding it SEQ ID NO: 107: Amino acid sequence of humanized L chain V region, version "bl" SEQ ID NO: 108: Amino acid sequence of humanized L chain V region, "b2" version and the nucleotide sequence encoding it SEQ ID NO: 109: Amino acid sequence of humanized L chain V region , version "b2" SEQ ID NO: 110: Amino acid sequence of FRl of the region Humanized H chain V, full version SEQ ID NO: 111: FR2 amino acid sequence of the region Humanized H chain V, versions "a" to "j" SEQ ID NO: 112: FR2 amino acid sequence of the region Humanized H chain V, "bl" and "di" versions SEQ ID NO: 113: FR2 amino acid sequence of the region Humanized H chain V, "b3" and "d3" versions SEQ ID NO: 114: FR3 amino acid sequence of humanized H chain V region, "a" version SEQ ID NO: 115: FR3 amino acid sequence of the region Humanized H chain V, versions "b", "bl", and "b3" SEQ ID NO: 116: FR3 amino acid sequence of the region H humanized chain V, version "c" SEQ ID NO: 117: FR3 amino acid sequence of the region H humanized chain V, versions "d", "di", and "d3" SEQ ID NO: 118: FR3 amino acid sequence of the region Humanized H chain V, version "e" SEQ ID NO: 119: FR3 amino acid sequence of the humanized H chain V region, version "f" SEQ ID NO: 120: FR3 amino acid sequence of the region Humanized H chain V, version "g" SEQ ID NO: 121: FR3 amino acid sequence of the region Humanized H chain V, version "h" SEQ ID NO: 122: FR3 amino acid sequence of the humanized H chain V region, "i" version SEQ ID NO: 123: FR3 amino acid sequence of the region Humanized H chain V, version "j" SEQ ID NO: 124: FR4 amino acid sequence of the humanized H chain V region, all versions SEQ ID NO: 125: FRl amino acid sequence of the region Humanized L chain V, all versions SEQ ID NO: 126: FR2 amino acid sequence of the region Humanized L chain V, versions "a", "b" and "c" SEQ ID NO: 127: FR2 amino acid sequence of the region Humanized L chain V, version "bl" SEQ ID NO: 128: FR2 amino acid sequence of the region Humanized L chain V, version "b2" SEQ ID NO: 129: FR3 amino acid sequence of humanized L chain V region, "a" version SEQ ID NO: 130: FR3 amino acid sequence of the region Humanized L chain V, versions "b", "bl" and "b2" SEQ ID NO: 131: FR3 amino acid sequence of the region Humanized L chain V, version "c" SEQ ID NO: 132: FR4 amino acid sequence of the region Humanized L chain V, all versions SEQ ID NO: 133: CDR1 amino acid sequence of the humanized H chain V region, all versions SEQ ID NO: 134: CDR2 amino acid sequence of the humanized H chain V region, all versions SEQ ID NO: 135: CDR3 amino acid sequence of humanized H chain V region, all versions SEQ ID NO: 136: CDR1 amino acid sequence of humanized L chain V region, all versions SEQ ID NO : 137: CDR2 amino acid sequence of humanized L chain V region, all versions SEQ ID NO: 138: CDR3 amino acid sequence of humanized L chain V region, all versions SEQ ID NO: 139: Amino acid sequence of the V chain H region of the mouse anti-TF monoclonal antibody ATR-2 SEQ ID NO: 140: Amino acid sequence of the H chain V region of the mouse anti-TF monoclonal antibody ATR-3 SEQ ID NO: 141: Amino acid sequence of the H chain V region of the mouse anti-TF monoclonal antibody ATR -4 SEQ ID NO: 142: Amino acid sequence of the V chain H region of the mouse anti-TF monoclonal antibody ATR-5 SEQ ID NO: 143: Amino acid sequence of the V chain H region of the anti-TF monoclonal antibody of mouse ATR-7 SEQ ID NO: 144: Amino acid sequence of the V chain H region of the mouse anti-TF monoclonal antibody ATR-8 SEQ ID NO: 145: Amino acid sequence of the L chain V region of the mouse anti-TF monoclonal antibody ATR-2 SEQ ID NO: 146: Amino acid sequence of the L chain V region of the mouse anti-TF monoclonal antibody ATR -3 SEQ ID NO: 147: Amino acid sequence of the L chain V region of the mouse anti-TF monoclonal antibody ATR-4 SEQ ID NO: 148: Amino acid sequence of the L chain V region of the mouse anti-TF monoclonal antibody ATR-5 SEQ ID NO: 149: Amino acid sequence of the L chain V region of the mouse anti-TF monoclonal antibody ATR-7 SEQ ID NO: 150: Amino acid sequence of the L chain V region of the mouse anti-TF monoclonal antibody ATR-8 SEQ ID NO: 151: Amino acid sequence of soluble human TF and the nucleotide sequence encoding it SEQ ID NO: 152: Amino acid sequence of soluble human TF.

Claims

CLAIMS A heavy chimeric chain (H) comprising a H chain variable region (V) of a mouse monoclonal antibody against human tissue factor (TF) and a constant (H) chain region H of a human antibody, wherein said V chain region H has any of the following amino acid sequences: (1) the amino acid sequence of SEQ ID NO: 139 (ATR-2), (2) the amino acid sequence of SEQ ID NO: 140 (ATR-3), (3) the amino acid sequence of SEQ ID NO: 141 (ATR-4), (4) the amino acid sequence of SEQ ID NO: 142 (ATR-5), (5) the amino acid sequence of SEQ ID NO: 143 (ATR-7), and (6) the amino acid sequence of SEQ ID NO: 144 (ATR-8).
The chimeric H chain in accordance with the claim 1, wherein said V chain region H has the amino acid sequence of SEQ ID NO: 142.
The chimeric H chain according to claim 1 or according to claim 2, wherein said C chain region H is the C region. ? l, C? 2, C? 3 or C? 4.
The chimeric chain H according to any one of claims 1 to 3, wherein said region V of chain H has the amino acid sequence of SEQ ID NO: 142, and said region C of chain H is the region C? 4.
A chimeric light chain (L) comprising the L chain region V of a mouse monoclonal antibody against human TF and the L chain C region of a human antibody, wherein said L chain region V has any of the following sequences of amino acids: (1) the amino acid sequence of SEQ ID NO: 145 (ATR-2), (2) the amino acid sequence of SEQ ID NO: 146. { ATR-3), (3) the amino acid sequence of SEQ ID NO: 147 (ATR-4), (4) the amino acid sequence of SEQ ID NO: 148 (ATR-5), (5) the amino acid sequence of SEQ ID NO: 149 (ATR-7), (6) the amino acid sequence of SEQ ID NO: 150 (ATR-8).
The chimeric L chain according to claim 5, wherein said V chain region L has the amino acid sequence of SEQ ID NO: 148.
The chimeric L chain according to claim 5 or according to claim 6, wherein said region C of chain L is region C? or CK.
The chimeric L chain according to any of claims 5 to 7, wherein said chain region V L has the amino acid sequence of SEQ ID NO: 148, and said region of C of chain L is CK.
A chimeric antibody to human TF comprising a chimeric H chain according to any of claims 1 to 4 and a chimeric L chain according to any of claims 5 to 8.
A chimeric antibody against human TF comprising a chimeric H chain according to claim 4 and a chimeric L chain according to claim 8.
A humanized H chain V region comprising a region of complementarity determination (CDR) of H chain V region of a mouse monoclonal antibody against TF human and a region of structure (FR) of V region of H chain of a human antibody, wherein said CDRs include the following amino acid sequences: H-CDR1: Asp Tyr Tyr Met His (SEQ ID NO: 133), H-CDR2 : Gly Asn Asp Pro Wing Asn Gly HIs Ser Met Tyr Asp Pro Lys Phe Gln Gly (SEQ ID NO: 134), and H-CDR3: Asp Ser Gly Tyr Ala Met Asp Tyr (SEQ ID NO: 135).
A humanized H chain V region, where said FRs include the following amino acid sequences: H-FR1: Gln Val Gln Leu Leu Glu Ser Gly Ala Val Leu Ala Arg Pro Gly Thr Ser Val Lys lie Ser Cys Lys Ala Ser Gly Phe Asn lie Lys (SEQ ID NO: 110), H-FR2: any of the following sequences (1) a (3): (1) Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp lie Gly (SEQ ID NO: 111), (2) Trp Val Arg Gln Wing Pro Gly Gln Gly Leu Glu Trp Met Gly (SEQ ID NO : 112), and (3) Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp lie Gly (SEQ ID NO: 113), H-FR3: any of the following sequences (1) to (10): (1) Arg Ala Lys Leu Thr Ala Ala Thr Ser Ala Ser lie Ala Tyr Leu Glu Phe Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg (SEQ ID NO: 114), (2) Arg Val Thr lie Thr Wing Asp Thr Ser Thr Asn Thr Wing Tyr Met Glu Leu Being Law Arg Ser Glu Asp Thr Wing He Tyr Tyr Cys Wing Arg (SEQ ID NO: 115), (3) Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Wing Wing Asp Thr Wing Val Tyr Tyr Cys Wing Arg (SEQ ID NO: 116), (4) Arg Val Thr He Thr Wing Asp Glu Ser Thr Being Thr Wing Tyr Met Glu Leu Being Ser Leu Arg Being Glu Asp Being Wing Val Tyr Phe Cys Wing Arg (SEQ ID NO: 117), (5) Arg Val Ser He Thr Wing Asp Glu Ser Thr Lys He Wing Tyr Met Glu Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Wing Arg (SEQ ID NO: 118), (6) Arg Val Thr He Thr Wing Asp Thr Ser Thr Ser Thr Wing Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr 'Wing Val Tyr Tyr Cys Wing Arg ( SRQ ID NO: 119), (7) Lys Wing Thr Leu Thr Wing Asp Glu Being Ser Thr Wing Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Ser Wing Val Tyr Ser Cys Wing Arg (SEQ ID NO: 120), 18) Arg Val Thr Met Ser Wing Asp Lys Ser Ser Wing Wing Tyr Leu Gln Trp Thr Ser Leu Lys Wing Being Asp Thr Wing He Tyr Phe Cys Wing Arg (SEQ ID NO: 121), (9) Arg Val Thr He Thr Wing Asp Thr Ser Thr Ser Thr Val Phe Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Wing Val Tyr Tyr Cys Wing Arg (SEQ ID NO: 122), and (10) Arg Val Thr Phe Thr Wing Asp Thr Ser Wing Asn Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Wing Asp Thr Ala Val Tyr Tyr Cys Ala Arg (SEQ ID NO: 123), and FR4: Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser (SEQ ID NO: 124).
13. The humanized H chain V region according to claim 11 or according to claim 12, having an amino acid sequence in accordance with that set forth in SEQ ID NO: 30 (version a), SEQ ID NO: 40 (version b), SEQ ID NO: 42 (version c), SEQ ID NO: 50 (version d), SEQ ID NO: 52 (version e), SEQ ID NO: 58 (version f), SEQ ID NO: 60 (version g), SEQ ID NO: 64 (version h), SEQ ID NO: 70 (version i), SEQ ID NO: 72 (version j), SEQ ID NO: 76 (version bl), SEQ ID NO: 78 (version di), SEQ ID NO: 82 (version b3), or SEQ ID NO: 84 (version d3).
14. The humanized H chain V region according to any of claims 11 to 13, having the amino acid sequence of SEQ ID NO: 40 (version b).
The humanized H chain V region according to any of claims 11 to 13, having the amino acid sequence of SEQ ID NO: 70 (version i).
A humanized L chain V region comprising L chain V region CDRs of a mouse monoclonal antibody against human TF and human L chain V region FRs, wherein said CDRs include the following amino acid sequences: L-CDR1: Lys Ala Ser Gln Asp He Lys Ser Phe Leu Ser (SEQ ID NO: 136), L-CDR2: Tyr Ala Thr Ser Leu Ala Asp (SEQ ID NO: 137), and L-CDR3: Leu Gln His Gly Glu Ser Pro Tyr Thr (SEQ ID NO: 138).
The humanized L chain region V according to claim 16, wherein said FRs include the following amino acid sequences: L-FRl: Asp He Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr He Thr Cys (SEQ ID NO: 125), L-FR2: any of the following sequences (1) to (3): (1) Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr (SEQ ID NO: 126), (2) Trp Phe Gln Gln Lys Pro Gly Lys Pro Pro Lys Thr Leu He Tyr (SEQ ID NO: 127), and (3) Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu He Tyr (SEQ ID NO: 128), L-FR3: any of the following sequences (1) to (3): (1) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Be Ser Be Leu Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys (SEQ ID NO: 129), (2) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Be Ser Leu Gln Pro Glu Asp Phe Wing Thr Tyr Tyr Cys (SEQ ID NO: 130), and (3) Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Be Ser Leu Gln Pro Glu Asp He Wing Thr Tyr Tyr Cys ( SEQ ID NO: 131), and L-FR4: Phe Gly Gly Gly Thr Lys Val Glu He Lys (SEQ ID NO: 132).
The humanized L chain region V according to claim 16 or according to claim 17, having an amino acid sequence according to that set forth in SEQ ID NO: 93 (version a), SEQ ID NO: 99 (version b), SEQ ID NO: 101 (version c), SEQ ID NO: 107 (version bl) or SEQ ID NO: 109 (version b2).
The humanized L chain region V according to any of claims 16 to 18, having the amino acid sequence of SEQ ID NO: 99 (version b).
The humanized L chain region V according to any of claims 16 to 18, having the amino acid sequence of SEQ ID NO: 109 (version b2).
A humanized H chain of an antibody to human TF, said chain comprises the humanized H chain V region according to any of claims 11 to 15 and the H chain C region of a human antibody.
A humanized H chain of an antibody to human TF, said chain comprises the humanized H chain V region (version b) according to claim 14 and the H chain C region of a human antibody.
A humanized H chain of an antibody to human TF, said chain comprises the humanized H chain V region (version i) according to claim 15 and the H chain C region of a human antibody.
The humanized H chain according to any of claims 21 to 23, wherein said C chain region H of a human antibody is C? 1, C? 2, C? 3 or C? 4.
A humanized L chain of an antibody to human TF, said chain comprises a humanized L chain V region according to any of claims 16 to 20 and an L chain C region of a human antibody.
A humanized L chain of an antibody to human TF, said chain comprises the humanized L chain region V (version b) according to claim 19 and an L chain C region of a human antibody.
A humanized L chain of an antibody against human TF, said chain comprises the humanized L chain region V (version b2) according to claim 20 and an L chain C region of a human antibody.
The humanized L chain according to any of claims 25 to 27, wherein said C chain region L of a human antibody is C? or CK.
A humanized antibody against human TF, said antibody comprises a humanized H chain according to any of claims 21 to 24, and a humanized L chain according to any of claims 25 to 28.
A humanized antibody against human TF, said antibody comprises a humanized H chain (version b) according to claim 22 and a humanized L chain (version b) according to claim 26. 1.
A humanized antibody against human TF, said antibody comprises a humanized H chain (version i) according to claim 23 and a humanized L chain (version b) according to claim 26.
32. A humanized antibody against human TF, said The antibody comprises the humanized H chain (version i) according to claim 23 and the humanized L chain (version b2) according to claim 27.
33. The DNA encoding a chimeric H chain according to any of claims 1 to 4.
34. A DNA encoding a chimeric H chain according to any one of claims 2 to 4.
35. A DNA encoding a chimeric L chain according to any of claims 5 to 8.
36. A DNA encoding a chimeric L chain according to any one of claims 6 to 8.
37. A DNA encoding a humanized H chain V region according to any of claims 11 to 15.
38. A DNA encoding the humanized H chain V region (version b) according to claim 14.
39. A DNA encoding the humanized H chain V region (version i) according to claim 15.
40. A DNA encoding a humanized L chain region V according to any of claims 16 to 20.
41. A DNA encoding the humanized L chain V region (version b) according to claim 19.
42. A DNA encoding the humanized L chain region V (version b2) according to claim 20.
43. A DNA encoding a humanized H chain according to any of claims 21 to 24.
44. A DNA encoding the humanized H chain ( version b) according to claim 22 or 24.
45. A DNA encoding the humanized H chain (version i) according to claim 23 or 24.
46. A DNA encoding a humanized L chain in accordance with any of the Claims 25 a 28.
47. A DNA encoding the humanized L chain (version b) according to claim 26 or 28.
48. A DNA encoding a humanized L chain (version b2) according to claim 27 or 28.
49. An expression vector comprising DNA encoding a chimeric H chain according to claim 33.
50. An expression vector comprising DNA encoding a chimeric H chain according to claim 34.
51. An expression vector comprising DNA encoding a chimeric L chain according to claim 35.
52. An expression vector comprising DNA encoding a chimeric L chain according to claim 36.
53. An expression vector comprising DNA encoding a chimeric H chain according to claim 33 and DNA encoding a chimeric L chain according to claim 35.
54. An expression vector comprising DNA encoding a chimeric H chain in accordance with claim 1. 34 and DNA encoding a chimeric L chain according to claim 36.
55. An expression vector comprising DNA encoding a humanized H chain according to claim 43.
56. An expression vector comprising DNA encoding a Humanized H chain (version b) according to claim 44.
57. An expression vector comprising DNA encoding a c humanized adena H (version i) according to claim 45.
58. An expression vector comprising DNA encoding a humanized L chain according to claim 46.
59. An expression vector comprising DNA encoding an L chain humanized (version b) according to claim 47.
60. An expression vector comprising DNA encoding a humanized L chain (version b2) according to claim 48.
61. An expression vector comprising DNA encoding a humanized H chain according to claim 43 and a DNA encoding a humanized L chain according to claim 46.
62. An expression vector comprising DNA encoding a humanized H chain (b version) in accordance with claim 44 and a DNA encoding a humanized L chain (h version) according to claim 47.
63. An expression vector comprising DNA encoding a humanized H chain (version i) in accordance with claim 45 and a DNA encoding a humanized L chain (version b) according to claim 47.
64. An expression vector comprising DNA encoding a humanized H chain (version i) according to claim 45 and a DNA encoding a humanized L chain (version b2) according to claim 48.
65. A host transformed with an expression vector comprising DNA encoding a chimeric H chain. a according to claim 49 and by an expression vector comprising a DNA encoding a chimeric L chain according to claim 51.
66. A host transformed with an expression vector comprising DNA encoding a chimeric H chain of according to claim 50 and by an expression vector comprising a DNA encoding a chimeric L chain according to claim 52.
A host transformed with an expression vector according to claim 53.
68. A host transformed with an expression vector according to claim 54
69. A host transformed with an expression vector comprising DNA encoding a humanized H chain according to claim 55, and with an expression vector comprising DNA encoding a humanized L chain according to claim 58.
70. A host transformed with an expression vector comprising DNA encoding a humanized H chain (version b) in accordance with claim 56, and with an expression vector comprising DNA encoding a humanized L chain (version b) according to claim 59.
71. A host transformed with an expression vector comprising DNA encoding a humanized H chain (version i) according to claim 57, and with an expression vector comprising DNA encoding a humanized L chain (version b) according to claim 59.
72. A host transformed with an expression vector comprising DNA encoding a humanized H chain (version i) according to claim 57, and with an expression vector comprising DNA encoding a humanized L chain (version b2) according to claim 6.
73. A host transformed with an expression vector in accordance with claim 61.
74. A host transformed with an expression vector according to claim 62.
75. A host transformed with an expression vector according to claim 63.
76. A host transformed with an expression vector according to claim 64.
77. A process for the preparation of a chimeric antibody against human TF, comprising the culture of a host according to claim 65 and, harvesting the chimeric antibody to from that crop.
78. A process for the preparation of a chimeric antibody against human TF, which comprises culturing a host according to claim 66, and harvesting the chimeric antibody from said culture. .
79. A process for the preparation of a chimeric antibody against human TF, comprising culturing a host according to claim 67, and harvesting the chimeric antibody from said culture.
80. A process for the preparation of a chimeric antibody against human TF, comprising the culture of a host according to claim 68, and harvesting a chimeric antibody from said culture.
81. A process for the preparation of a chimeric antibody against human TF, comprising culturing a host according to claim 69, and harvesting the humanized antibody from said culture. .
A process for the preparation of a humanized antibody against human TF, comprising the culture of a host according to claim 70, and harvesting the humanized antibody from said culture. .
A process for the preparation of a humanized antibody against human TF, comprising the culture of a host according to claim 71, and - harvesting the humanized antibody from said culture. .
A process for the preparation of a humanized antibody against human TF, comprising the culture of a host according to claim 72, and harvesting the humanized antibody from said culture. .
A process for preparing a humanized antibody against human TF, which comprises culturing a host according to claim 73, and harvesting the humanized antibody from said culture. .
A process for the preparation of a humanized antibody against human TF, comprising the culture of a host according to claim 74, and harvesting the humanized antibody from said culture.
A process for the preparation of a humanized antibody against human TF, comprising the culture of a host according to claim 75, and harvesting the humanized antibody from said culture.
A process for the preparation of a humanized antibody against human TF, comprising the culture of a host according to claim 76, and harvesting the humanized antibody from said culture.
A process for the preparation of a natural humanized antibody having regions of complementarity determination (CDRs) derived from non-humans and a structure region (FR) derived from a natural human antibody and having a reduced immunogenicity, said method comprises the steps of: (1) preparing a non-human monoclonal antibody that responds to an antigen of interest; (2) preparing a plurality of human antibodies having a high homology to the amino acid sequence of the FRs in the monoclonal antibodies of (1) above; (3) replacing the four FRs of a human antibody in (2) above with the corresponding FRs of the non-human monoclonal antibody of (1) above to generate a first humanized antibody;(4) determining the ability of the humanized antibody generated in (3) above to bind with the antigen or to neutralize a biological activity of the antigen; (5) replacing one to three FRs of the humanized antibody generated in (3) above with the corresponding FRs of the human antibody which is different from that used in (3) among the human antibodies prepared in (2) to generate the second humanized antibody; (6) compare the capacity of the second humanized antibody generated in (5) above and the first humanized antibody generated in (3) above to determine the binding capacity with the antigen or to neutralize the biological activity of the antigen thus selecting a humanized antibody who has a favorable activity; (7) carrying out steps of (3) to (6) above for the humanized antibody selected in (6) - above; and (8) repeating the above steps of (3) to (6) until a humanized antibody having an activity equivalent to the non-human monoclonal antibody in (1) above is obtained.
The process according to claim 89, wherein said antigen of interest is human tissue factor (TF).
A humanized antibody prepared by the process according to claim 89.
92. A humanized antibody prepared by the process according to claim 90.
93. A therapeutic agent for disseminated intravascular coagulation syndrome (DIC), said agent comprises the humanized antibody according to any of claims 29 to 32 and claim 92.