PT1069185E

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

Abstract

This record has no abstract on file.

Term

Term ended

Projected expiry passed 2 April 2019, 7.5 years ago.

7 claims: 6 independent, 1 dependent

1
REIVINDICAÇÕES 1. Processo de preparação de um anticorpo humanizado natural que possui regiões determinantes de complementaridade (CDR) derivadas de anticorpos não humanos e uma região estrutural (FR) derivada de um anticorpo humano natural e que possui uma imunogenicidade reduzida, compreendendo o referido método os passos de:(1) preparação de um anticorpo monoclonal não humano responsivo a um antigénio de interesse;
2
(2) preparação de uma pluralidade de anticorpos humanos possuindo elevada homologia com as sequências de aminoácidos das FR nos anticorpos monoclonais do (1) acima;
3
(3) substituição das quatro FR do anticorpo monoclonal não humano do (1) acima com as correspondentes FR do anticorpo humano no (2) acima para gerar um primeiro anticorpo humanizado;
4
(4) determinação da capacidade do anticorpo humanizado gerado no (3) acima para se ligar ao antigénio ou para neutralizar uma actividade biológica do antigénio;
5
(5) substituição de uma a três FR do anticorpo humanizado gerado no (3) acima com a correspondente FR do anticorpo humano que é diferente da utilizada no (3) entre os anticorpos humanos preparados em (2) para gerar o segundo anticorpo humanizado;
6
(6) comparação da capacidade do segundo anticorpo humanizado gerado no (5) acima com o primeiro anticorpo humanizado gerado no (3) acima quanto à ou para capacidade para se ligar ao antigénio neutralizar a actividade biológica do antigénio para deste modo seleccionar um anticorpo humanizado que tenha uma actividade favorável;
7
(7) realização dos passos acima de (3) a (6) para o anticorpo humanizado seleccionado no (6) acima; e (8) repetição dos passos acima de (3) a (6) até ser obtido um anticorpo humanizado possuindo uma actividade equivalente à do anticorpo monoclonal não humano no (1) acima. 2. Processo de acordo com a reivindicação 1, no qual o referido antigénio de interesse é o factor tecidual humano (TF) . 3. Processo para produção de um anticorpo humanizado natural de acordo com qualquer uma das reivindicações 1 a 2 compreendendo os passos de:(1) obtenção de um anticorpo humanizado natural através de um processo de acordo com qualquer uma das reivindicações 1 a 2;(2) introdução de um ácido nucleico codificando o anticorpo humanizado natural numa célula hospedeira;e (3) cultura da célula hospedeira.

Independent claims7

2,932 paragraphs in 16 sections, as filed

DESCRIPTION

HUMANIZED ANTIBODY AGAINST HUMAN TISSUE FACTOR (TF) AND PROCESS FOR CONSTRUCTION OF A HUMANIZED ANTIBODY

Technical Field

The present disclosure relates to a human / mouse chimeric antibody comprising the variable region (V region) of a mouse monoclonal antibody against human tissue factor (TF) and the constant region (region C) of a human antibody; a humanized antibody in which the complementarity determining regions (CDR) of the light chain v region (L chain) and the heavy chain V region (H chain) of a mouse monoclonal antibody against human TF were grafted to a human antibody; the L chain and H chain of said antibody; and a fragment of the V region constituting the L chain or H chain of said antibody. The present disclosure also relates to a process for producing a humanized antibody against human TF.

The present disclosure also relates to DNA encoding the above antibody, specifically a fragment of the V region thereof, and DNA encoding an L chain or H chain containing a V region. The present description also relates to a recombinant vector comprising said DNA, and a host transformed with said vector.

The present disclosure also relates to a process for preparing a chimeric antibody and a humanized antibody against human TF. The present disclosure also relates to a pharmaceutical composition and a therapeutic agent for disseminated intravascular coagulation syndrome (DIC) comprising as active ingredient a humanized antibody against human TF.

Background Art Tissue factor (TF), a cell surface-expressed clotting factor VII receptor, plays an indispensable role in activating clotting factors IX and X by forming a complex with clotting factor VII, and was defined as a practical initiating factor for blood clotting reactions.

TF is known to be expressed in fibroblasts, smooth muscle cells, etc. which constitute the blood vessel, and have a hemostatic function by activating the coagulation system at the time of blood vessel injury.

DIC is a disease in which activation of the coagulation system in a blood vessel leads to a multiple systemic occurrence of blood clots, primarily in the microvasculature. It is not uncommon for platelet reduction and clotting factors due to consumption to lead to bleeding which is the opposite phenomenon to blood clotting. Multiple microthrombi can cause impaired microcirculation in the major organs, which, once developed, leads to irreversible functional impairment and poor prognosis of IHD, and in this sense IHD is considered an important disease.

The estimated underlying disease incidence from the Ministry of Health and Welfare's 1990 and 1992 research reports, Specified Diseases Blood Coagulation Disorders Survey and Study Group is: hematologic malignancies, about 30%; solid tumors, about 20%; infections, about 15%; obstetric diseases, about 10%; liver disease, about 6%; shocks, about 5%; and cardiovascular disease, about 3%. The incidence of IHD is as high as about 15% in leukemia and about 6 to 7% in malignant lymphoma, and about 3% in solid tumors.

IHD develops accompanied by the various diseases mentioned above, but the causative agent of these is the same, which is TF. Thus, the mechanism of onset of IHD is believed to be: abnormally high formation and / or expression of TF in cancer cells in acute leukemia, malignant lymphoma, and solid tumors; increased formation and / or expression of TF in monocytes and / or endothelial cells in infections (in particular, sepsis caused by gram-negative bacilli); TF influx into the blood from necrotic liver tissue in fulminant hepatitis; TF expression in blood vessel lumen in aortic aneurysm, cardiac aneurysm, and giant hemangioma; and also TF influx into the blood in obstetric diseases (amniotic fluid embolism and premature placental detachment) and surgeries, injuries and burns.

The treatment of the original (underlying) diseases is of the utmost concern, which, however, is not easy in practical terms.

As a current method of treating IHD, anticoagulant therapy and replacement therapy are in use. Heparin preparations (fractionated heparin, low molecular weight heparin) are mainly used for anticoagulant therapy. Synthetic protease inhibitors (gabexate mesylate, naphthamostat mesylate) and concentrated plasma (anti-thrombin III and activated protein C preparations) are also used. As replacement therapy, there are platelet concentrates, fresh frozen plasma (fibrinogen supply), washed red blood cells, and the like.

However, current therapeutic agents are unsatisfactory in terms of efficacy and side effects, and in most cases complete disappearance of IHD is impossible. Therefore, there is a need for the use of drugs having high therapeutic effects and low side effects.

On the other hand, further attempts at DIC treatments may include preparations of thrombomodulin, hirudin, and anti-PAF, tissue factor pathway inhibitor (TFPI) agents. Selective FXa inhibitors are attracting attention as orally administrable anticoagulant and / or antithrombotic agents. Also as an agent that neutralizes TF activity, WO 88/07543 discloses a mouse anti-human TF monoclonal antibody, and WO 96/40921 discloses a humanized anti-human TF antibody.

Mouse anti-human TF monoclonal antibodies are expected to be a safe and effective therapeutic agent because they do not exhibit a bleeding symptom associated with major efficacy in IHD. However, mouse monoclonal antibodies are highly immunogenic (sometimes referred to as antigenic), and thus the therapeutic medical value of mouse antibodies in humans is limited. For example, the half-life of mouse antibodies in humans is relatively short and therefore cannot fully exhibit their anticipated effects. In addition, antibody responds to reactions in human mice.

human anti-mouse (HAMA) that develops in to the administered mouse antibody causes immunological that are unfavorable and dangerous patients. Thus, monoclonal antibodies to cannot be repeatedly administered to

To address these problems, methods have been developed to reduce the immunogenicity of non-human derived antibodies, such as (monoclonal antibodies derived from) mice. One is a method of producing a chimeric antibody in which a variable region (V region) of the antibody is derived from a mouse monoclonal antibody, and its constant region (C region) is derived from a suitable human antibody.

Since the chimeric antibody obtained contains the variable region of a full-length original mouse antibody, it is expected to bind to an antigen with the same affinity as that of the original mouse antibody. Furthermore, in the chimeric antibody the ratio of non-human derived amino acid sequences is substantially reduced, and is thus expected to have reduced immunogenicity compared to the original mouse antibody. However, it is still possible that an immune response to the mouse variable region may arise (LoBuglio, AF et al., Proc. Natl. Acad. Sci. USA, 86: 4220-4224, 1989).

Although much more complicated, a second method of reducing mouse antibody immunogenicity is expected to drastically reduce the potential immunogenicity of mouse antibody. In this method, the complementarity determining region (CDR) alone of a mouse antibody is grafted onto a human variable region to produce a remodeled human variable region. As desired, however, some CDR-bearing structural region (FR) amino acid sequences can be grafted from the variable region of a mouse antibody into the human variable region to obtain the closest possible approximation to the structure of the original mouse antibody. . Then the humanized remodeled human variable region is linked to the human constant region. In the finally remodeled humanized antibody, the portions derived from non-human amino acid sequences are only the CDRs and a small portion of the FRs. CDRs comprise hypervariable amino acid sequences and do not have species specific sequences.

For humanized antibody, see also Riechmann, L. et al., Nature 332: 323-327, 1988; Verhoeye, M. et al., Science 239: 1534-1536, 1998; Kettleborough, CA et al., Protein Eng., 4: 773-783, 1991; Maeda, H., Human Antibodies and Hybridoma, 2: 124-134, 1991; Gorman, SD et al., Proc. Natl. Acad. Know. USA, 88: 4181-4185, 1991; Tempest, PR, Bio / Technology, 9: 226-271, 1991; Co, MS et al. , Proc. Natl. Acad. Know. USA 88: 2869-2873, 1991; Cater, P. et al. , Proc. Natl. Acad. Know. USA, 89: 4285-4289, 1992; Co, MS et al. , J. Immunol., 148: 1149-1154, 1992; and Safo, K. et al., Cancer Res., 53: 851-856, 1993.

In conventional humanization technology, part of the structural region (FR) includes an amino acid sequence that has been grafted from the variable region of a mouse antibody to the human variable region. Thus, when it is administered as a therapeutic agent in humans, there is a risk of antibodies being formed against a site having an amino acid sequence not present in humans, although it is merely one to a few amino acids. To circumvent the risk, it was considered a third humanization technology. Thus, the method involves, for the 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 to the mouse antibody FR present in the database using one FR as unit. In this case, several FR of human antibodies present in the database are selected which are sequentially shuffled to prepare a humanized antibody having high activity.

By doing so, it is possible to construct humanized antibodies in which all RF except CDRs in the variable region have amino acid sequences derived from a human antibody. Thus, the humanized antibody having mouse CDR should no longer have more potent immunogenicity than a human antibody containing human CDR.

While the humanized antibody is expected to be useful for the purpose of treatment as mentioned above, there is no fixed process present that is universally applicable to any antibody in the method of producing a humanized antibody, and thus a number of devices are required for construct a humanized antibody that exhibits sufficient binding activity and neutralizing activity for 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 disclosure to provide a human / mouse chimeric antibody comprising the variable region (V region) of a mouse monoclonal antibody against human tissue factor (TF) and the constant region (C region) of a human antibody, a humanized antibody in which the complementarity determining regions (CDR) of the light chain V region (L chain) and the heavy chain V region (H chain) of a mouse monoclonal antibody against human TF were grafted into a human antibody, constituting the L chain and H chain of said antibody; and a fragment of the V region, L chain or H chain of said antibody. It is another object of the present disclosure to provide a process for producing a humanized antibody against human TF.

It is another object of the present disclosure to provide DNA encoding the above antibody, specifically a fragment of the V region thereof, and DNA encoding an L chain or H chain containing a V region. It is another object of the present disclosure to provide a recombinant DNA vector comprising said DNA, and a host transformed with said vector. It is another object of the present disclosure to provide a pharmaceutical composition and a therapeutic agent for disseminated intravascular coagulation syndrome (DIC) comprising as a humanized antibody against human TF as active ingredient.

After intensive study to solve the above problems, the inventors of the present disclosure have successfully obtained an antibody in which the human immunogenicity of mouse monoclonal antibody against human TF is reduced, and have also developed a process for producing a novel humanized antibody, and have thus completed the present description.

Thus, the present disclosure relates to a chimeric H chain comprising the H chain C region of a human antibody and a H chain V region fragment of a mouse monoclonal antibody against human TF. As the H chain V region, one may be mentioned which comprises an amino acid sequence as set forth in SEQ ID NO: 9, and as region C one may be mentioned which is derived from the 0γ4 region.

Further, the present disclosure relates to a chimeric L chain comprising the L chain C region of a human antibody and a L chain V region fragment of a mouse monoclonal antibody against human TF. As the L chain V region, one may be mentioned which comprises an amino acid sequence as set forth in SEQ ID NO: 15, and as the L chain C region, one may be mentioned which is derived from the Ck region.

Further, the present disclosure relates to a human / mouse chimeric monoclonal antibody against human TF, said antibody comprising the chimeric H chain and the above chimeric L chain.

The present disclosure also relates to a H chain V region fragment of a humanized antibody, said fragment comprising the H chain V region regions (FR) 1-4 of a human antibody and complementarity determining regions (CDR) 1-3 of the H chain V region of a mouse monoclonal antibody against human TF. As CDR 1-3, one may be mentioned which includes the amino acid sequence as set forth in SEQ ID NO: 133-135, respectively. As the H chain V region FR1 of a human antibody, the FR1 of a human antibody having a 40% or greater homology to the H chain V region FR1 of a mouse antibody can be mentioned; as FR2, FR2 of a human antibody that has a 40% or greater homology to H chain V-region FR2 of a mouse antibody can be mentioned; as FR3, FR3 of a human antibody having 40% or more homology to the H chain V-region FR3 of a mouse antibody can be mentioned; and as FR4, FR4 of a human antibody having 40% or more homology to the H chain V region FR4 of a mouse antibody can be mentioned.

Preferably, as the H chain V region FR1 of a human antibody, the FR1 of a human antibody having 50% or greater homology to the H chain V region FR1 of a mouse antibody may be mentioned; as FR2, FR2 of a human antibody having 70% or greater homology to the H chain V-region FR2 of a mouse antibody may be mentioned; as FR3, FR3 of a human antibody having a 65% or greater homology to the H chain V-region FR3 of a mouse antibody can be mentioned; and as FR4, FR4 of a human antibody having 80% or greater homology to the H chain V-region FR4 of a mouse antibody can be mentioned. As specific examples, as FR1 of the H chain V region of a human antibody, human antibody L39130 may be mentioned; as FR2, human antibody L39130, human antibody P01742, and human antibody Z80844 may be mentioned; FR3, human antibody L39130, Z34963 of

<td>antibody</td><td>human,</td><td>P01825</td><td>in</td><td>antibody</td><td>human,</td><td>M62723</td><td>in</td>

<td>antibody</td><td>human,</td><td>Z80844</td><td>in</td><td>antibody</td><td>human,</td><td>L04345</td><td>in</td>

<td>antibody</td><td>human,</td><td>S78322</td><td>in</td><td>antibody</td><td>human,</td><td>Z26827</td><td>in</td>

<td>antibody</td><td>human,</td><td>U95239</td><td>in</td><td>antibody)</td><td>human and</td><td>L03147</td><td>in</td>

<td>antibody</td><td>human;</td><td>It is like</td><td>FR4,</td><td>. Can be</td><td colspan="3">mentioned L39130</td>

of human antibody.

As preferred examples, as FR1 of the H chain V region of a human antibody, human antibody L39130 may be mentioned; as FR2, human antibody L39130 and human antibody Z80844 may be mentioned; as FR3, human antibody Z34963, human antibody M62723, and human antibody U95239 may be mentioned; and as FR4, human antibody L39130 may be mentioned. As more preferred examples, as FR1 of the H chain V region of a human antibody, human antibody L39130 may be mentioned; as FR2, human antibody L39130 may be mentioned; as FR3, human antibody Z34963 and human antibody U95239 may be mentioned; and as FR4, human antibody L39130 may be mentioned.

In addition, as used herein, numbers in the structural regions are based on the definition of Kabat (Kabat, EA et al., US Dept. Health and Services, US Government Printing Offices, 1991).

The present disclosure also relates to a H chain V region fragment of a humanized antibody, said fragment comprising one of the amino acid sequences as set forth in SEQ ID NO: 30, 40, 42, 50, 52, 58, 60, 64, 70, 72, 76, 78, 82, and 84.

The present disclosure also relates to a L-chain V-region fragment of a humanized antibody, said fragment comprising the L-chain V-region FR 1-4 of a human antibody and the V-region CDR 1-3 of the human antibody. L-chain of a mouse monoclonal antibody against human TF. As CDR 1-3, one may be mentioned which includes the amino acid sequence as set forth in SEQ ID NO: 136-138, respectively. As the L-chain V-region FR1 of a human antibody, one can be mentioned that has a 40% or greater homology to the L-chain V-region FR1 of a mouse antibody; as FR2, FR2 of a human antibody having 40% or greater homology to the L-chain V-region FR2 of a mouse antibody may be mentioned; as FR3, FR3 of a human antibody having 40% or greater homology to the L-chain V-region FR3 of a mouse antibody may be mentioned; and as FR4, FR4 of a human antibody having 40% or greater homology to L-chain V-region FR4 of a mouse antibody can be mentioned.

Preferably, as the L chain V region FR1 of a human antibody, the FR1 of a human antibody having 75% or greater homology to the L chain V region FR1 of a mouse antibody may be mentioned; as FR2, FR2 of a human antibody having 80% or greater homology to L-chain V-region FR2 of a mouse antibody may be mentioned; as FR3, FR3 of a human antibody having 70% or greater homology to L-chain V-region FR3 of a mouse antibody may be mentioned; and as FR4, FR4 of a human antibody having 80% or greater homology to L-chain V-region FR4 of a mouse antibody can be mentioned. As specific examples, as FR1 of the L chain V region of a human antibody, human antibody Z37332 may be mentioned; as FR2, human antibody Z37332 and human antibody X93625 may be mentioned; as FR.3, human antibody Z37332, human antibody S68699, and human antibody P01607 may be mentioned; and as FR4, human antibody Z37332 may be mentioned. As more preferred examples, as FR1 of the L chain V region of a human antibody, human antibody Z37332 may be mentioned; as FR2, human antibody X93625 may be mentioned; as FR3, human antibody S68699 may be mentioned; and as FR4, human antibody Z37332 may be mentioned.

In addition, as used herein, numbers in the structural regions are based on the definition of Kabat (Kabat, A.A. et al., US Dept. Health and Services, US Government Printing Offices, 1991).

The present disclosure also relates to a L chain V region fragment of a humanized antibody, said fragment comprising amino acid sequences as set forth in SEC ID NO: 93, 99, 101, 107, and 109.

The present disclosure also relates to the H chain of a humanized human TF antibody, said chain comprising a fragment of the H chain V region of the above humanized antibody and a fragment of the H chain C region of a human antibody. The Cy4 region may be mentioned as region C; such as FR 1-4 derived from a human antibody, those which are each derived from human antibody L39130 (FR1), human antibody L39130 (FR2), human antibody Z34963 or FR3 can be mentioned. Human antibody U95239 (FR3), human antibody L39130 (FR4); and as CDR 1-3, those which are each derived from the amino acid sequence as set forth in SEQ ID NO: 133-135, respectively, may be mentioned.

The present disclosure also relates to the L-chain of a humanized human TF antibody, said chain comprising a fragment of the L-chain V region of the above humanized antibody and a fragment of the L-chain C region of a human antibody. Region Ck may be mentioned as region C; as FR 1-4 derived from a human antibody, mention may be made of those which are each derived from human antibody Z37332 (FR1), human antibody X93625 (FR2), human antibody S68699 , and human antibody Z37332 (FR4); and as CDR 1-3, those which are each derived from the amino acid sequence as set forth in SEQ ID NO: 136-138, respectively, may be mentioned.

The present disclosure also relates to a humanized antibody against human TF, said antibody comprising the L chain and H chain of the above humanized antibody.

The present disclosure also relates to a process for producing a humanized antibody against human TF. The humanization process refers to the method of selecting FR 1-4 by supporting the structure of CDR 1-3 which are the antigen recognition site for the H chain or L chain. Thus, the present disclosure relates to the method of selecting some of the FRs of a human antibody having a high homology to the FR of a mouse antibody with each FR as a unit, and generating a humanized antibody having the desired activity by a sequential shuffling of RF.

More specifically, the claimed invention relates to a process of preparing a natural humanized antibody having a non-human derived complementarity determining rule (CDR) a naturally occurring antibody derived structural region (FR) has reduced immunogenicity. said method comprising the steps of:

(1) preparing a non-human monoclonal antibody responsive to the antigen of interest;

(2) preparing some human antibodies having a high homology to the FR amino acid sequence in the monoclonal antibody 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 the first humanized antibody;

(4) determining the ability of the humanized antibody generated in (3) above to bind antigen or neutralize antigen biological activity;

(5) replacing one to three FR of the humanized antibody generated in (3) above with the corresponding FRs of a human antibody that is different from that used in (3) among the human antibodies prepared in (2) to generate the second humanized antibody. ;

(6) comparing the ability of the second humanized antibody generated in (5) above to the first humanized antibody generated in (3) above for the ability to bind antigen or neutralize antigen biological activity to thereby select a humanized antibody that have a favorable activity;

(7) performing steps (3) to (6) above for the humanized antibody selected in (6) above; and

<td>(8) repetition</td><td>of the steps</td><td>in</td><td>(3) to (6)</td><td>up to</td><td>to be</td>

<td>obtained a</td><td>antibody</td><td colspan="2">humanized</td><td>owning</td><td>an</td>

<td colspan="2">activity equivalent to</td><td>of</td><td>antibody</td><td>monoclonal</td><td>no</td>

<td>human at (1)</td><td>above.</td><td></td><td></td><td></td><td></td>

The claimed invention also relates to the process according to the above paragraph, wherein said antigen of interest is human tissue factor (TF).

The claimed invention also relates to a process for producing a natural humanized antibody according to the preceding two paragraphs comprising the steps of:

(1) obtaining a natural humanized antibody by a process according to either of the preceding two paragraphs;

(2) introducing a nucleic acid encoding the natural humanized antibody into a host cell; and (3) host cell culture.

Once a humanized antibody having a certain degree of human TF neutralizing activity is obtained, further homology screening for a specific FR in the H chain and L chain V region is performed so that a human antibody having a high homology can be selected. By adding the human antibody thus obtained to a group of some human antibodies in step (2) above and further repeating steps (3) to (6), a humanized antibody having the desired activity can be obtained.

The present disclosure also relates to DNA encoding a H chain V region fragment or a H region fragment

L chain V of a mouse monoclonal antibody against human TF. As an amino acid sequence and DNA encoding the H chain V region fragment or region fragment

V of the L chain, one may be mentioned which includes the nucleotide sequence as set forth in SEQ ID NO: 9 or 15, respectively.

The present disclosure also relates to DNA encoding the chimeric H chain or the chimeric L chain above. As DNA encoding said H chain, one may include one which includes the nucleotide sequence as set forth in SEQ ID NO: 9, and as DNA encoding said L chain, one may include the nucleotide sequence as set forth in SEQ. ID NO: 15.

The present disclosure also relates to DNA encoding a H chain V region fragment or L chain V region fragment of the above humanized antibody. As DNA encoding the H-chain V region fragment, one may be mentioned that includes one of the nucleotide sequences as set forth in SEQ ID NO: 29, 39, 41, 49, 51, 57, 59, 63, 69, 71, 75, 77, 81, or 83, and as DNA encoding the L-chain V region fragment, one may be mentioned that includes one of the nucleotide sequences as set forth in SEQ ID NO: 92, 98, 100, 106, or 108. .

The present disclosure also relates to DNA encoding the H chain of a humanized antibody.

The present disclosure also relates to the H chain DNA of a humanized antibody, said DNA comprising DNA encoding one of the amino acid sequences as set forth in SEQ ID NO: 30, 40, 42, 50, 52, 58, 60, 64, 70, 72, 76, 78, 82, or 84. As said DNA, one may be mentioned which includes one of the nucleotide sequences as set forth in SEQ ID NO: 29, 39, 41, 49, 51, 57, 59 , 63, 69, 71, 75, 77, 81, or 83.

The present disclosure also relates to DNA encoding the L chain of the above humanized antibody.

The present disclosure is also the L chain DNA of a humanized antibody, said DNA comprising DNA encoding amino acid sequences as set forth in SEQ ID NO: 93, 99, 101, 107, or 109. As said DNA, it may be mentioned is one that includes one of the nucleotide sequences as set forth in SEQ ID NO: 92, 98, 100,

106, or 108.

The present disclosure also relates to a recombinant DNA vector containing one of the DNAs described above.

The present disclosure also relates to a transformant transformed with a recombinant DNA vector described above.

The present disclosure also relates to a process of generating a chimeric antibody or humanized human TF antibody, said method comprising culturing the above transformant and obtaining a chimeric antibody or humanized human TF antibody from the above. harvested crop.

The present disclosure also relates to a pharmaceutical composition and a therapeutic agent for DIC comprising the above humanized antibody as active ingredient.

Brief Description of the Drawings

Figure 1 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a humanized version a / chimeric L chain antibody, and a chimeric H chain / human L chain antibody version a.

Figure 2 is a graph comparing the neutralizing activity against human TF of a chimeric H chain / chimeric L chain antibody, a humanized version a / chimeric L chain antibody, and a chimeric H chain / L chain antibody humanized version a.

Figure 3 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody and a humanized version a / chimeric L chain H antibody.

Figure 4 is a graph comparing neutralizing activity against human TF of an ATR-5 mouse anti-TF monoclonal antibody, a chimeric H chain / L chain antibody, and a humanized version A / chain H chain antibody. L humanized version a.

Figure 5 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a version b / chimeric humanized H chain antibody, and a version b / chain humanized H chain antibody L humanized version a.

Figure 6 is a graph comparing antigen binding activity of a chimeric H-chain / chimeric L-chain antibody, a chimeric L-chain version humanized H-chain antibody, and a version d / chain humanized H-chain antibody L chimeric.

Figure 7 is a graph comparing the human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version b / chimeric humanized H chain antibody, a version c / humanized H chain antibody Chimeric L, and a humanized H chain antibody version d / chimeric L chain.

Figure 8 is a graph comparing the human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody and a version b humanized H chain antibody / human version L chain antibody.

Figure 9 is a graph comparing antigen binding activity of a chimeric H-chain / chimeric L-chain antibody, humanized chimeric H-chain / L-chain antibody version b, and a humanized version of chimeric H-chain / L-chain antibody ç.

Figure 10 is a graph comparing human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a humanized version b chimeric H chain / L chain antibody, and a chimeric H chain / L chain antibody humanized version c.

Figure 11 is a graph comparing antigen binding activity of a chimeric H-chain / chimeric L-chain antibody, a version b humanized H-chain antibody / version b humanized H-chain antibody, and a version b humanized H-chain antibody / humanized L chain version c.

Figure 12 is a graph comparing human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version b humanized H chain antibody / version b human chain antibody, and a version humanized H chain antibody. b / humanized L chain version c.

Figure 13 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a version b humanized H chain antibody / version b human chain antibody, and a version d humanized H chain antibody. / humanized L chain version b.

Figure 14 is a graph comparing the human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version b humanized H chain antibody / version b human chain antibody, and a version humanized H chain antibody. d / humanized L chain version b.

Figure 15 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a chimeric version e / chain humanized H chain antibody, and a version e / chain chimeric humanized H chain antibody L humanized version b.

<td>Figure 16 is a graph</td><td>which compares the activity</td><td>in</td>

<td>human TF neutralization</td><td>of a chain antibody</td><td>H</td>

<td>chimeric / chimeric L chain humanized version and / L chain</td><td>and a chain antibody chimeric.</td><td>H</td>

<td>Figure 17 is a graph</td><td>which compares the activity</td><td>in</td>

<td>antigen binding</td><td>a chain antibody</td><td>H</td>

<td>chimeric / chimeric L chain humanized version g / L chain</td><td>and a chain antibody humanized version b.</td><td>H</td>

<td>Figure 18 is a graph</td><td>which compares the activity</td><td>in</td>

<td>human TF neutralization</td><td>of a chain antibody</td><td>H</td>

chimeric / L chain chimeric and a humanized H chain antibody version g / humanized L chain version b.

Figure 19 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a version b3 humanized H chain antibody / version b human chain antibody, and a version d3 humanized H chain antibody / humanized L chain version b.

Figure 20 is a graph comparing human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version b3 humanized H chain antibody / version b human chain antibody, and a version humanized H chain antibody. d3 / humanized L chain version b.

Figure 21 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a version I / chimeric human chain H chain antibody, and a version j / chain humanized H chain antibody. L chimeric.

Figure 22 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a version i humanized H chain antibody / version b human chain antibody, and a version j humanized H chain antibody. / humanized L chain version b.

Figure 23 is a graph comparing human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version I / chimeric humanized H chain antibody, and a version j / humanized H chain antibody. chimeric L chain.

Figure 24 is a graph comparing human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version b humanized H chain antibody / version b humanized H chain antibody, a version i humanized H chain antibody humanized L-chain version b, and a humanized H-chain antibody version j / humanized L-chain version b.

Figure 25 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody, a humanized version chimeric H chain / L chain antibody, and a humanized chimeric H chain / L chain antibody b2 version.

Figure 26 is a graph comparing the human TF neutralizing activity of a chimeric H chain / L chain antibody chimeric H chain antibody / human version L chain, and a chimeric H chain / L chain antibody Humanized version B2.

Figure 27 is a graph comparing antigen binding activity of a chimeric H chain / chimeric L chain antibody and a version b humanized H chain / version b2 human chain antibody.

Figure 28 is a graph comparing the human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version b humanized H chain antibody / version b human chain antibody, a version b humanized H chain antibody / humanized L-chain version b, and a humanized H-chain antibody version b / humanized L-chain version b2.

Figure 29 is a graph comparing antigen binding activity of a chimeric H-chain / chimeric L-chain antibody, a version I humanized H-chain antibody / version bl humanized H-chain antibody, and a version I humanized H-chain antibody / humanized L chain version b2.

Figure 30 is a graph comparing the human TF neutralizing activity of a chimeric H chain / chimeric L chain antibody, a version i humanized H chain antibody / version b human chain H antibody, a version i humanized H chain antibody humanized L-chain version bl, and a humanized H-chain antibody version i / humanized L-chain version b2.

Figure 31 is a graph comparing antigen binding activity of a chimeric H-chain / chimeric L-chain antibody, a version b humanized H-chain antibody / version b humanized H-chain antibody, a version I / humanized H-chain antibody humanized L-chain version b, and a humanized H-chain antibody version i / humanized L-chain version b2.

Figure 32 is a graph comparing human TF neutralizing activity (the activity to inhibit Factor Xa production by TF) of a chimeric H chain / chimeric L chain antibody, a humanized version b / humanized L-chain version b, a humanized H-chain antibody version i / humanized L-chain version b, and a humanized H-chain antibody version i / humanized L-chain version b2.

Figure 33 is a graph comparing human TF neutralizing activity (the activity to inhibit Factor X binding) of a chimeric H chain / chimeric L chain antibody, a humanized version b / human chain L chain antibody version b, a version b humanized H-chain antibody / version L humanized version b, and a version b humanized version i / H chain humanized antibody version b2.

Figure 34 is a graph comparing human TF neutralizing activity (the activity to inhibit plasma coagulation through TF) of a chimeric H chain / chimeric L chain antibody, a humanized version b / chain humanized H chain antibody. Humanized L-version b, a humanized version I-chain H / l-chain antibody version b, and a humanized version-I-chain H / human L-version version b2 antibody.

Figure 35 is a figure comparing reactivity with human TF treated under various conditions of a chimeric H chain / chimeric L chain antibody, a version b humanized H chain antibody / version b humanized H chain antibody, a humanized H chain antibody humanized version i / L chain version b, and a humanized version i / H chain humanized antibody b2 version.

Best Mode for Realizing Description

The present description will now be explained in more detail.

1. Preparation of a mouse monoclonal antibody against human TF

A mouse monoclonal antibody against TF can be generated by generating a hybridoma by fusing antibody-producing cells obtained from animals immunized with the antigen with myeloma cells, and then by selecting from the hybridoma obtained from a clone producing the antibody that specifically inhibits TF activity.

Thus, spleen cells from a TF-immunized mouse that was purified from human placenta as antigen were fused to myeloma cells to prepare a hybridoma. For the purpose of hybridoma screening, antibody binding capacity to TF was determined by a Cell ELISA employing the high expression TF J82 cell line, and its TF neutralizing activity was determined in an assay system. which used as index the inhibiting activity of clotting factor X activation (Factor X: FX). As a result, hybridomas that produce 6 antibodies that strongly inhibit FX activation of the TF / VIIa complex have been successfully established.

(1) Antigen preparation

As TF for animal immunization, a peptide may be mentioned which is part of the TF amino acid sequence generated by recombinant DNA technology or chemical synthesis, or human placental-derived TF. For example, human placental purified TF according to the method of Ito et al. (Ito T. et al., J. Biochem. 114: 691-696, 1993) may be used as antigen.

TF obtained is mixed with an adjuvant and then the mixture is used as antigen. As an adjuvant, Freund's complete adjuvant or Freund's incomplete adjuvant can be mentioned, any of which may be mixed.

(2) Immunization and harvesting of antigen-producing antibody cells obtained as above may be administered to non-human mammals, for example mammals such as mice, rats, horses, monkeys, rabbits, goats, sheep, and the like. Immunization may be performed using any of the existing methods, and is primarily performed by intravenous, subcutaneous, and intraperitoneal injection and the like. 0 The immunization period is, but is not limited to, an interval of a few days to a few weeks, preferably an interval of 4-21 days.

Antibody-producing cells may be harvested 2-3 days after the last day of immunization. As antibody producing cells, spleen cells, lymphatic cells, and peripheral blood cells may be mentioned, and spleen cells are generally used. The amount of antigen to be used for each immunization is 0.1-100 pg per mouse.

(3) Determination of antibody titer

To confirm the immune response level of the immunized animal and select the desired hybridoma from the cells after cell fusion treatment, the antibody titer in the immunized animal's blood and the antibody-producing cell culture supernatant is determined.

As antibody detection methods, known methods such as radioimmunoassay enzyme immunoassay (EIA), (RIA), enzyme linked immunosorbent assay (ELISA), and the like may be mentioned.

(4) Cell Fusion

As myeloma cells fused to antibody-producing cells, cell lines that are derived from mice, rats, humans, etc. are used. and which are generally available to one skilled in the art. As cell lines to be used may be mentioned those which have the properties of being drug resistant, unable to survive in the selection medium (e.g. HAT medium) in the unfused state, and capable of only in the molten state. Resistant strains are generally used surviving cell lines do not have

-azaguanine, and these hypoxanthine-guanine28 phosphoribosyl transferase and therefore cannot survive in the medium containing hypoxanthine, aminopterin and thymidine (HAT).

As myeloma cells, a number of known cell lines such as P3 (P3X63Ag8.653) (J. Immunol. 123: 1548-1550, 1979), P3X63Ag8.1 (Current Topics in Microbiology and Immunology 81: 1-7) are preferably used. , 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 (Shulman, M. et al., Nature 276: 269-270, 1978), F0 (from St. Groth, SF et al., J. Immunol. Methods 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.

Antibody producing cells may be obtained from spleen cells, lymphatic cells, and the like. Thus, from the above animals, spleens, lymph nodes, etc. are removed or harvested, and these tissues are dispersed. The dispersed cells are suspended in a medium or buffer such as PBS, DMEM and RPMI1640, filtered with a stainless steel mesh etc., and centrifuged to prepare the desired antibody-producing cells.

The above myeloma cells and antibody producing cells are subjected to cell fusion.

Cell fusion may be performed by contacting the myeloma cells with the antibody producing cells at 30-37 ° C for 1-15 minutes in the presence of a fusion accelerator at a mixing ratio of 1: 1 to 1:10. in an animal cell culture medium such as MEM, DMEM,

RPMI1640, and the like. To accelerate cell fusion, a cell fusion accelerator or a fusion virus such as polyethylene glycol (PEG) of a molecular weight 1,000-6,000, polyvinyl alcohol, or Sendai virus (HVJ) may be used. In addition, commercially available cell fusion instruments (e.g. electroporation) using electrical stimulation can be used to fuse antibody producing cells with myeloma cells.

(5) Selection and cloning of a hybridoma

The desired hybridoma may be selected from the fused cells. As an example, methods may be mentioned which use selective cell growth in a selection medium. Thus, after diluting a cell suspension with a suitable medium, it is plated on a microtiter plate and a selection medium (such as HAT medium) is added to it, which is grown with appropriate replacement of the selection medium. As a result, growing cells can be obtained as hybridoma.

Screening for a hybridoma is performed by the limiting dilution method, the fluorescence activated cell sorting method, and the like, and finally a monoclonal antibody producing hybridoma can be obtained.

Selection of a hybridoma that produces a monoclonal antibody can be accomplished by combining various assay systems. For example, an antigen recognition system such as Cell ELISA, a TF neutralization activity assay system that uses Factor Xa activity as an index, and a neutralization activity assay system such as the assay system. which measure plasma coagulation inhibiting activity are combined to obtain a monoclonal antibody producing hybridoma that has the desired activity. In doing so, a monoclonal antibody producing hybridoma such as ATR-2, ATR-3, ATR-4, ATR-5, ATR-7, and ATR-8 can be obtained.

(6) Monoclonal Antibody Collection

As a method of collecting monoclonal antibody from the obtained hybridoma, a conventional cell culture method, an ascites formation method, and the like may be mentioned.

In the cell culture method, a hybridoma is cultured in an animal cell culture culture medium such as RPMI1640 medium supplemented with 1020% fetal calf serum, DMEM medium, serum free medium or the like under a culture condition. (e.g. 37 ° C, 5% CO 2 concentration) for 2-14 days, and then antibody is obtained from the culture supernatant.

In the ascites formation method, a hybridoma is given intraperitoneally to a mammal-like animal species from which myeloma cells are derived, and hybridoma proliferates in large quantities. Then ascites or serum is collected 1-4 weeks later.

Where antibody purification is required in the above antibody method, purification is performed by selecting, as appropriate, a known method such as ammonium sulfate fractionation, ion exchange chromatography and affinity chromatography, or by your combination.

2. DNA cloning encoding the V region of a monoclonal antibody against human TF (i) Preparation of mRNA

To clone DNA encoding the H chain and L chain V region of a mouse monoclonal antibody against human TF, total RNA is isolated from the hybridoma collected by a known method such as the guanidinaultracentrifugation 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 through the centrifugation oligo (dT) -cellulose column attached to 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) cDNA preparation and amplification

From the mRNA obtained in (i) above, L chain V and H chain V region cDNA, respectively, is synthesized using an inverse transcriptase. Synthesis of cDNA can be performed using an Oligo-dT primer or a suitable primer (for example, the synthetic cDNA primer attached to the kit) which hybridizes to the L chain C region or the H chain C region.

CDNA amplification can be performed by PCR based on the 5'-RACE method (Frohman, MA et al., Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988; Belyavsky, A. et al. , Nucleic Acids Res. 17: 2919-2932, 1989) using the 5'-Ample FINDER RACE kit (CLONETECH) together with the L chain and the H chain. Thus, a cDNA adapter is attached to the ends of a double stranded cDNA synthesized above, and then a polymerase chain reaction (PCR) is performed for the DNAs encoding the H chain V region and the L chain V region ( DNA encoding an L chain V region fragment is hereinafter abbreviated as L chain V region DNA or L chain V region DNA; the same is true for H chain V region, etc.).

As a primer for DNA amplification of a região chain V region, Adapter primer 1 can be used for the 5 'end primer, and mouse antibody H chain constant region MHC-G1 primer (SEQ ID NO: 1) (ATR-2, ATR-3, ATR-4, and ATR-5) (ογΐ region) or the MHC-G2a primer (SEQ ID NO: 2) (ATR-7 and ATR-8) (CY2a region) (ST Jones et al., Biotechnology, 9: 88, 1991). For example, for the 5 'end primer, the adapter Primer 1 attached to the kit may be used, and for the 3' end primer the L chain κ chain constant region primer (Ck region) of a mouse antibody may be used. (such as the MKC primer having the nucleotide sequence as set forth in SEQ ID NO: 3).

(iii) DNA purification and nucleotide sequence determination

PCR products are subjected to agarose gel electrophoresis according to a known method. After excision of the desired DNA fragment, the DNA is recovered and purified, and then ligated into a vector DNA.

DNA may be purified by phenol and chloroform extraction (J. Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989) or using a commercially available kit (e.g., GENECLEAN II; BIO101). A vector DNA used to retain DNA fragments may be any known (e.g., pUC19 and Bluescript, etc.).

Above DNA and a vector DNA are ligated using a known binding kit (made by Takara Shuzo) to obtain a recombinant vector. After the resulting recombinant DNA vector is introduced into a competent Escherichia coli JM109 (Nippongene) etc. cell, ampicillin resistant colonies are selected, and then a vector DNA based on a known method is prepared (J. Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). After the above vector is digested with a restriction enzyme, the DNA nucleotide sequence of interest can be determined by a known method (e.g. the dideoxy method) (J. Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press , 1989). According to the present description, an automated nucleotide sequence determination instrument (DNA Sequencer 373A, Perkin-Elmer) may be used.

(iv) Complementarity Determining Region (CDR)

The H chain V region and the L chain V region form an antigen binding site, and the overall structure is similar to each other. Thus, every four hypervariable structural regions (FR) are linked through three regions ie complementarity determining regions (CDR). The amino acid sequences of the

RF are well conserved, while CDR amino acid sequences are very highly variable (Kabat, EA et al., Sequence of Proteins of Immunologic Interest, US Dept. Health and Human Services, 1983).

Many regions of the top four FR take the form of a β-leaf structure, with the result of the CDR forming a loop. CDR can sometimes form parts of the β sheet structure. Thus, the three CDRs are kept in close proximity to each other three-dimensionally, and the FRs constitute an antigen binding site along with the three CDRs.

Based on such facts, by adjusting the amino acid sequence of a mouse monoclonal antibody against human TF in the Kabat generated antibody amino acid sequence database (US Sequence of Proteins of Immunologic Interest, US Dept. Health and Human Services, 1983). ), their homology can be examined and thus CDRs can be found.

The sequence of a CDR altered by insertion, substitution, or deletion may be included in the present disclosure provided that it retains the binding to or neutralization activity of human TF when a humanized antibody using it is generated. For example, those having a 90-100% homology with each of the CDRs for SEQ ID NO: 133-138 or with each CDR in the V region of SEQ ID NO: 139-141, 143-144, 145 may be mentioned. -147, and 149-150. Preferably, sequences having a homology of 95-100% may be mentioned. More preferably, sequences having a 98-100% homology may be mentioned.

3 Preparation of an Chimeric Antibody Expression Vector

Once cloned DNA fragments encoding mouse L chain (antibody L chain or H chain may hereinafter be referred to as mouse L chain for human antibody and human H chain for human antibody H chain). ) and mouse H chain V region, DNA encoding mouse V region is ligated to DNA encoding the constant region of a human antibody and expressed to obtain a chimeric anti-human TF antibody.

Basic methods for generating a chimeric antibody include ligating a mouse leader sequence and a V region sequence in the cloned cDNA to a sequence encoding the human antibody constant region already in a mammalian cell expression vector. Alternatively, they comprise ligating a mouse leader sequence and a V region sequence in the cloned cDNA to a sequence encoding the human antibody C region, and then binding it to a mammalian cell expression vector.

Fragments of the human antibody C regions may be those of the H chain C region and the L chain C region of any human antibody. For example, ογΐ, ογ2, ογ3, or ογ4 may be mentioned for those in the human H chain, and CÃ or Ck for the L chain, 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 is prepared under the control of an expression regulatory region such as a stimulator / promoter system, and a single expression vector (see, for example, WO 94/11523) containing DNA encoding a mouse L chain V region and a human L chain C region under the control of a region such as a system the expression vector is regulator of enhancer / promoter expression. It is then used to co-transform a host cell 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 IgG1K-like antibody expression vector N5KG1 (V) and an IgG4K-like antibody expression vector N5KG4P can be used.

(i) Construction of the H chain of a chimeric antibody

An expression vector for an H chain of a chimeric antibody 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 H chain C region, for example, the ΟγΟ, 0γ2, Cy3, or Cy4 region can be mentioned.

As used herein, to introduce cDNAs encoding a mouse H chain V region into an expression vector, a suitable nucleotide sequence may be introduced into said cDNA by the PCR method. For example, such a suitable nucleotide sequence may be introduced into an expression vector by performing PCR using PCR primers designed to have a recognized sequence of a suitable restriction enzyme at the 5 'end of said cDNA, and, for better efficiency of transcription, the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol., 196: 947-950, 1987) just prior to the start codon of said cDNA, and PCR primers designed to have a recognized sequence of a suitable restriction enzyme at the 3 'end of said cDNA.

Following treatment of a cDNA encoding the mouse H chain V region thus constructed with a suitable restriction enzyme, it is inserted into the above expression vector and a chimeric H chain expression vector containing DNA encoding the C region of the DNA is then constructed. H chain (Ογΐ or Cy4).

(ii) Construction of an expression vector containing cDNA encoding the L chain κ chain of a chimeric antibody

An expression vector for an L chain of a 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 region C of the L chain can be mentioned, for example, the region Ck and CÃ.

As used herein, to construct an expression vector containing cDNA encoding a mouse L chain V region, a suitable nucleotide sequence may be introduced into said cDNA by the PCR method. For example, such a suitable nucleotide sequence may be introduced into said cDNA by performing PCR using PCR primers designed to have a recognized sequence of a suitable restriction enzyme at the 5 'end of said cDNA, and for better transcription efficiency. , the Kozak consensus sequence, and PCR primers designed to have a recognized sequence of a suitable restriction enzyme at the 3 'end of said cDNA.

After treatment of a mouse L-chain V-region cDNA so constructed with a suitable restriction enzyme, it is inserted into the above expression vector and then a chimeric L-chain expression vector containing C-region DNA is constructed. L chain (Ck region).

4 Humanized Antibody Preparation (1) Human Antibody Homology Search

To want a humanized antibody in which the CDRs of a mouse monoclonal antibody are grafted to a human antibody, there is preferably a high homology between the FRs of a mouse monoclonal antibody and the FRs of a human antibody. Thus, the H chain and L chain V regions of a mouse anti-human TF monoclonal antibody are compared with the V region of all known antibodies whose structure was published using a Database. At the same time, they are compared to the human antibody subgroups (HSG: human subgroup) (Kabat, EA et al., US Dep. Health and Human Services, US Government Printinq Offices, 1991) classified by Kabat et al. based on RF length and amino acid homology.

Based on the HSG classification of Kabat et al., The H chain V regions can be broken down into HSGI in III; for example, the H chain region V of the mouse anti-human ATR-5 monoclonal antibody has a 67.8% homology to the HSGI consensus sequence. On the other hand, the regions

V from the κ chain of the human L chain can be grouped into HSGI in IV; for example, the L-chain κ chain V region of the mouse anti-human ATR-5 monoclonal antibody has a 72.3% homology to the HSGI consensus sequence.

When a mouse antibody is humanized by conventional technology, the amino acid sequence of some of the V region FR of a CDR-bearing mouse antibody can be grafted into the human V region FR as desired so that the structure of the CDR from a humanized V region may more closely resemble that of the original mouse antibody. However, there are no fixed rules as to which V region FR amino acids of a mouse antibody to graft in the V region FR of a human antibody. Accordingly, much effort is required to specify the amino acids that are essential for maintaining the structure of CDR.

There is also a risk that a human antibody against the grafted amino acid sequence in the human V region of the V region of a mouse antibody could be formed by the FR. In accordance with the present disclosure, to change all amino acid sequences except for CDR in the humanized antibody to the amino acid sequences derived from a human antibody, the FRs of a human antibody having high homology to the FR of mouse antibodies present in the antibody were screened. database, with one FR as unit, for the four FRs (FR1-4) that are required to maintain the three-dimensional structure of the CDR. 0 The following is a result of the database homology search for each H chain V region and L chain V region FR of an ATR-5 monoclonal antibody.

Table 1

<td>At the.</td><td>gives</td><td>FR</td><td></td><td>No. of</td><td>Homology</td><td>with each FR</td><td>gives</td><td>SEQ</td><td>ID</td>

<td></td><td></td><td></td><td></td><td>Access</td><td>region V</td><td>from chain H</td><td>of</td><td>AT THE:</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>antibody</td><td>of mouse (%)</td><td></td><td></td><td></td>

<td>FRl</td><td>gives</td><td>jail</td><td>H</td><td>L39130</td><td> 53, 0</td><td></td><td></td><td> 110</td><td></td>

<td>FR2</td><td>gives</td><td>jail</td><td>H</td><td>L39130</td><td> 92, 9</td><td></td><td></td><td> 111</td><td></td>

<td>FR3</td><td>gives</td><td>jail</td><td>H</td><td>L39130</td><td> 62,5</td><td></td><td></td><td> 114</td><td></td>

<td>FR4</td><td>gives</td><td>jail</td><td>H</td><td>L39130</td><td> 90, 9</td><td></td><td></td><td> 124</td><td></td>

<td colspan="10">Table 2</td>

<td>At the.</td><td>gives</td><td>FR</td><td>At the. Access</td><td>of Homology region V antibody</td><td>with each of the chain mouse</td><td>FR L %)</td><td>gives of</td><td>SEQ AT THE:</td><td>ID</td>

<td>FRl</td><td>gives</td><td>L chain</td><td>Z37332</td><td> 78,3</td><td></td><td></td><td></td><td> 125</td><td></td>

<td>FR2</td><td>gives</td><td>L chain</td><td>Z37332</td><td> 80,0</td><td></td><td></td><td></td><td> 126</td><td></td>

<td>FR3</td><td>gives</td><td>L chain</td><td>Z37332</td><td> 71, 9</td><td></td><td></td><td></td><td> 129</td><td></td>

<td>FR4</td><td>gives</td><td>L chain</td><td>Z37332</td><td> 90,0</td><td></td><td></td><td></td><td> 132</td><td></td>

(2) Designing DNA encoding a humanized antibody V region The first step in designing DNA encoding a humanized antibody V region is to select each RF from a V region of a human antibody on which the design is based. In RF shuffling, it is necessary to select for each FR a highly varied FR from the V region of the human antibody.

For the ATR-5 monoclonal antibody according to the present description, three FR region V of human antibody for FR.2 and 10 for FR.3 were selected based on the result of the homology search between the H chain V region of all mouse antibodies and each FR to the H chain. For the L chain, three FR region V of the human antibody FR2 and three FR3 can be selected based on the result of homology screening between the mouse antibody L chain V region and each FR.

For both humanized H-chain and L-chain V regions, L-chain V regions, L39130 and Z37332 can be selected, having a high homology to the H-chain and L-chain V region of mouse antibody ATR-5. respectively. To allow easy RF shuffling in the generation of these humanized antibodies, suitable restriction enzyme recognition sites can be designed at suitable sites on each CDR and FR. By doing so, only one of the RF can easily be replaced.

Examples of such sites include an Ecol221 restriction enzyme recognition site in humanized H chain CDR1, a Bali restriction enzyme recognition site in CDR2, an NcoI restriction enzyme recognition site in

CDR3 is 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 PstI restriction in CDR3, and an AccIII restriction enzyme recognition site in FR3.

Based on the version thus designed, RF shuffling can be performed for each FR to obtain a humanized antibody having the desired activity.

(3) Preparation of a V region fragment of the humanized antibody The humanized antibody of the present invention is such that the C region and V region FRs of said antibody are derived from a human antibody and the V region CDR is derived from an antibody. of mouse. V region fragments of the humanized antibody of the present invention may be generated by a method called CDR grafting by the PCR method, if human antibody DNA fragments are available as a template. Such as

<td>used here graft</td><td>in</td><td>CDR is a</td><td>method</td><td>at the</td><td>what</td><td>is</td>

<td>generated a fragment of</td><td>DNA</td><td colspan="2">coding a</td><td>CDR</td><td>in</td><td>one</td>

<td>mouse antibody,</td><td>what</td><td>is exchanged</td><td>through the</td><td>CDR</td><td>in</td><td>one</td>

human antibody as a template.

When DNA fragments of a human antibody are not available as a template, the nucleotide sequence recorded in the database can be synthesized using a DNA synthesizer, and the V region of the humanized antibody can be generated using the PCR method. In addition, when the amino acid sequence is only recorded in the database, the entire nucleotide sequence can be deduced based on the amino acid sequence based on the frequency of antibody codon use reported by Katat, EA et al. (US Dep. Health and Human Services, US Government Printing Offices, 1991). The nucleotide sequence may be synthesized using a DNA synthesizer, and V region fragments of the humanized antibody may be generated using the PCR method.

(i) DNA construct and an expression vector encoding the humanized H chain V region

According to the present disclosure, DNA encoding the humanized H chain V region can be constructed by obtaining the H chain V region coding gene of a human antibody to be used as a template for the humanized antibody and then synthesizing the entire sequence. nucleotide DNA encoding the humanized H chain V region using a DNA synthesizer, followed by the PCR method. For example, L39130 having high homology to the H chain V region of the anti-human ATR-5 mouse monoclonal antibody, can be generated as the humanized H chain V region version a. To generate the humanized H chain version V region a, for example, 5 primers as set forth in SEQ ID NOs: 22-26 and 2 exogenous primers as set forth in SEQ ID NOs: 27 and 28 are used separately.

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 graft primers, hR5Hv3A (SEQ ID NO: 25) and hR5Hv5A (SEQ ID NO: 26) have an antisense DNA sequence, each having a complementary 18-35 bp sequence at both ends of the primers. hR5Hv1S is designed to have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol. 196: 947-950, 1987) and a Sall recognition site, and hR5Hv5A is designed to have a Nhel recognition site. The exogenous primers, hR5HvPrS (SEQ ID NO: 27) and hR5HvPrA (SEQ ID NO: 28) also have homology to the CDR graft primers, hR5HvlS and hR5Hv5A.

Using the PCR method, five primers are assembled to synthesize an entire cDNA, and upon addition of an exogenous primer to them the DNA is amplified. Assembly by the PCR method as used herein means that hR5Hv1S, hR5Hv2S, hR5Hv4S, hR5Hv3A, and hR5Hv5A are linked through their complementary sequences and the V-region DNA of the entire humanized H chain is synthesized.

The H chain C region of the human antibody can be any human H chain C region, and for example human H chain Cyl, Cy2, Cy3, or Cy4 can be mentioned.

H chain V region DNA of the humanized antibody constructed as above may be ligated to DNA from any human antibody H chain C region, for example the Cyl or Cy4 human H chain C region. As described in the construction of a chimeric antibody H chain, upon treatment with a suitable restriction enzyme, it is ligated to DNA encoding a human H chain C region under the control of an expression regulatory region such as a stimulator / promoter system. to generate an expression vector containing the humanized H chain V region and human H chain C region DNA.

(ii) DNA construct 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 disclosure, DNA encoding the humanized L chain V region can be constructed by obtaining a L chain V region gene from a human antibody to be used as a template and then synthesizing the entire DNA nucleotide sequence encoding the humanized L-chain V region using a DNA synthesizer, followed by the PCR method. For example,

Ζ37332 having a high homology to the L-chain V region of the mouse anti-human TF monoclonal antibody

<td>ATR-5 can be generated humanized version a.</td><td>how</td><td>region V</td><td>from jail</td><td>L</td>

<td>To generate region V of</td><td>jail</td><td>L humanized</td><td>version a,</td><td>the</td>

<td>graft initiators</td><td>from CDR</td><td>h5LvlS (SEQ</td><td>ID NO: 85)</td><td>and</td>

h5Lv4S (SEQ ID NO: 86) have a sense DNA sequence and the CDR graft primers h5Lv2A (SEQ ID NO: 87), h5Lv3A (SEQ ID NO: 88), and h5Lv5A (SEQ ID NO: 89) have an antisense DNA sequence each having a complementary 20 bp sequence at both ends of the primer. The h5Lv1S primer is designed to have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol. 196: 947-950, 1987) and a restriction enzyme recognition site BgIII, and h5Lv5A is also designed to have a restriction enzyme recognition site Spl1. The exogenous primers h5LvS (SEQ ID NO: 90) and h5LvA (SEQ ID NO: 91) also have homology to the h5LvlS and h5Lv5A CDR graft primers.

As for the humanized H chain V region, using the PCR method, five primers are assembled to synthesize an entire cDNA, and after addition of an exogenous primer to them the DNA can be amplified.

The human antibody L-chain C region can be any human L-chain C region, and for example the human L-chain C1 and Ck may be mentioned.

The L chain V region DNA of the humanized antibody constructed as above may be ligated to the DNA of any human L chain C region, for example one derived from the human L chain C k or C k region. After treatment with a suitable restriction enzyme, it is ligated to DNA encoding a human L chain C region under the control of an expression regulatory region such as a stimulator / promoter system to generate an expression vector containing DNA encoding the V region of the human. humanized L chain and the C region of the human L chain κ chain.

Even if a V region fragment of a humanized antibody is generated as mentioned above, it is not always clear whether said V region fragment has antibody activity (ie antigen binding, antigen neutralizing activity, etc.). Thus, it is necessary to investigate the presence of activity by combining it with a humanized H chain and expressing it in an animal cell such as COS-7.

(iii) H-chain V and L-chain FR shuffling of the humanized antibody

The present inventors performed transient expression of a humanized antibody containing a humanized H chain and L chain V region in an animal cell such as COS-7 to investigate antigen binding activity and neutralizing activity, and found that the antibody has antigen binding and neutralizing activity but that the activity is not adequate compared to the chimeric antibody.

The present inventors can solve this problem by sequentially shuffling each FR of the H chain V region and the humanized L chain. The antibody used in RF shuffling can be selected from existing databases. The RF of the selected human antibody can be synthesized based on the nucleotide sequence demonstrated in the databases using a DNA synthesizer. At this point, as mentioned above, by addition of the designed restriction enzyme recognition sequence to the CDR or FR, it can be easily shuffled with the H chain V and L chain V FR of the humanized antibody generated above. By investigating the activity of the humanized antibody thus generated, a humanized antibody having antigen binding and neutralizing activity can be obtained.

For example, the H chain V-region FR3 of the humanized antibody may be shuffled with the human antibody-derived FR3 Z34963 (GenbBank, Borrentzen M. et al., Proc. Natl. Acad. Sci. USA, 91: 12917-12921 , 1994).

FR shuffle primers 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 antisense DNA sequence. FR shuffling primers F3RFFS, F3RF3S, F3RFFA, and F3RFBA can be synthesized using a DNA synthesizer.

F3RFFS and F3RFFA, and F3RFBS and F3RFBA were ligated, which were digested with Bali and Xhol, and Ncol and Xhol, respectively. By introduction into plasmid hATR5Hva / CVIDEC (Bali / Ncol) prepared by digestion with Bali and Ncol, and confirming the nucleotide sequence thereof, a plasmid having the correct sequence can be obtained. 0 The plasmid thus obtained containing the humanized antibody H chain was designated as hATR5Hvb / CVIDEC, and the humanized H chain contained in hATR5Hvb / CVIDEC was designated version b. The nucleotide sequence and corresponding amino acid sequence are shown in SEQ ID NO: 39, and the amino acid sequence of version b is shown in SEQ ID NO: 40.

Similarly, FR derived from the H chain and L chain V region of another human antibody selected from the database can also be shuffled with the H chain V and L chain V region FR of a humanized antibody. .

To select a more preferred human antibody for shuffling of the H chain V region and the L chain V region of a humanized antibody, the following can be performed. Thus, a combination of the humanized version b antibody H chain with a chimeric antibody L chain has a neutralizing activity equal to that of a chimeric antibody or a mouse antibody. However, the combination of the H chain of the humanized version b antibody with the L chain of the humanized version a antibody has a lower neutralizing activity than that of a chimeric antibody or a mouse antibody.

In such cases, to select a human antibody to be a candidate for RF shuffling, homology screening can be performed, for example, for FR3 (Accession No. Z34963: SEQ ID NO: 115) of the humanized antibody In version b, a human antibody having a high homology to this sequence can be obtained. For example, as FR3 of the H chain V region of the human antibody thus selected, U95239 (SEQ ID NO: 122) and L03147 (SEQ ID NO: 123) may be mentioned.

The H chain V region amino acid sequences of the humanized antibody thus generated are shown in Table 3 and 4, and the L chain V region amino acid sequence of the humanized antibody is shown in Table 5.

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12345678901234567899123 45673901234 567890123456789 0123456 Z37332 (a) DIQ.MTQSPSSLSASVGDRVT1TC KASQDIKSFLS WYQQKPGKAPKLLIY YATSLAD

S68699 (b) -------------------------------------------------- --------- P0'607 (c) ----------------------------------- -------------------- S65921 (bl) -------------------------- --------- F ------ ST -------- X93625 (b2) --------------------- ------------------- B ---- S --------- FR3 CDR3 FR4

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78901234567890123456789012345678 901234567 8901234567 Z37332 (a) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQHGESPYT FGGGTKVBIK

S68699 (b) Y ------------------------------------ P01607 (c) ----- --------- Y ----------- í ---------------------—

S65921 (bl) -------- Y ------------------------------------ X93625 (b2) -------------- Y -------------------------------- ---- Each version of the H chain and L chain V region of the humanized antibody thus constructed may be ligated to the DNA of any human H chain C region or L chain C region, for example human H chain Cy4 regions. and human L chain Ck, respectively. Following treatment with a suitable restriction enzyme, it is ligated to a DNA encoding the human H chain Cy4 region and the human L chain Ck region under the control of an expression regulatory region such as a stimulator / promoter system, and a vector is generated. of expression containing a DNA encoding each version of the humanized H-chain V-chain and the human H-chain C-region and human L-chain Ck 0γ4 region.

A DNA encoding the humanized antibody H chain V region 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 introduced into a single vector (see , e.g. WO 94/11523), and then said vector is used to transform a host cell. Then, the transformed host may be cultured in vivo or in vitro to produce the desired humanized antibody.

5 Production of chimeric antibody and humanized antibody

To produce a chimeric antibody or humanized antibody, a DNA encoding an H chain V region and an H chain C region and a DNA encoding an L chain V region and an L chain C region can be linked to a single vector, which is transformed into a suitable host cell to produce the antibody. Thus, for expression of the 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 V region mouse L chain and a human H chain C region are introduced into a single expression vector (see, for example, WO 94/11523) under the control of an expression regulatory region such as a stimulator / promoter system.

For expression of the humanized antibody, a

DNA encoding a humanized H chain V region and a human H chain C region and DNA encoding a humanized 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 an expression regulatory region such as a stimulator / promoter system. These vectors are used to transform host cells. Then, the transformed host cell can be cultured in vivo or in vitro, and thus the chimeric antibody or humanized antibody can be produced.

Two expression vectors can also be generated, each containing an H chain V region and an L chain V region. Thus, for the chimeric antibody an expression vector containing a DNA encoding a region is generated.

Mouse H chain V and a human H chain C region under the control of a stimulator / promoter system and an expression vector containing a DNA encoding a region

Mouse L chain V and a human L chain C region under the control of a stimulator / promoter system, and for the humanized antibody an expression vector containing a DNA encoding a humanized H chain V region and a C region is generated of the human H chain under the control of a stimulator / promoter system and an expression vector containing a DNA encoding a humanized L chain V region and a human L chain C region under the control of a stimulator / 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 C region of the mouse. human L-chain under the control of an expression regulatory region such as a stimulator / promoter system, and for the humanized antibody an expression vector is generated that contains a DNA encoding a humanized H chain V region 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 a stimulator / promoter system.

These expression vectors are then used to co-transform host cells such as mammalian cells, and transformed cells are cultured in vitro or in vivo to produce a chimeric antibody or a humanized antibody (see, for example, WO 91/16928).

As stated hereinabove, a transformant transformed with a gene encoding the desired chimeric antibody or humanized antibody is cultured, and the chimeric antibody or humanized antibody produced can be separated from inside or outside the cell and purified to homogeneity.

Isolation and / or purification of the chimeric antibody or humanized antibody, or desired protein of the present disclosure, may be accomplished using a Protein A-sepharose column. Other methods include, but are not limited to, the separation and / or purification methods used for common proteins. By way of example, the chimeric antibody or humanized antibody may be isolated and / or purified by combining, as appropriate, various chromatographic, ultracentrifugation, desalting, dialysis and the like.

To produce the chimeric antibody or humanized antibody of the present disclosure against human TF, any expression system may be used. For example, when eukaryotic cells are used, animal cells (e.g., established mammalian cell lines), fungal cells or yeast cells may be used, and when prokaryotic cells are used, bacterial cells (such as Escherichia cells) may be used. coli). Preferably, the chimeric antibody or humanized antibody of the present invention is expressed in mammalian cells such as COS cells or CHO cells.

In these cases, useful common promoters may be used for expression in mammalian cells. For example, the immediate human cytomegalovirus immediate promoter (HCMV) is preferably used. Examples of expression vectors containing the HCMV promoter include HCMV-VH-HCyI, HCMVVL-HCk, and the like, and those derived from pSV2neo (WO 92-19759).

Other promoters for mammalian cell gene expression that may be used herein include viral promoters such as retrovirus promoters, polyoma virus, adenovirus, and simian virus 40 (SV40), and promoters derived from mammalian cells such as factor human polypeptide chain elongation chain (HEFla). For example, expression can be easily achieved by the method of Mulligan et al. (Nature (1979) 277: 108) when the

SV40, or by the method of Mizushima et al. (Nucleic

Acids Res. (1990) 18: 5322) when the HEFla promoter is used.

As a source of replication, SV40 derivatives, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like may be used. In addition, for gene copy number amplification in the host cell system, expression vectors may include as selectable markers the phosphotransferase gene APH (3 ') II or I (neo), the thymidine kinase gene ( TK), the xanthine-guanine phosphoribosyl transferase gene from E. coli (Ecogpt), the dihydrofolate reductase (dhfr) gene and the like.

6 Evaluation of antigen binding activity and neutralizing 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 antibody concentration measurement can be prepared as follows: Each well of a 96-well ELISA plate (eg, Maxisorp, NUNC) is immobilized with 100 µl of prepared human goat antiIgGy antibody (BioSource). to a concentration of 1 pg / mL.

After blocking with 200 µL dilution buffer (hereinafter referred to as DB; 50 mM Tris-HCl, 1 mM MgCl 2, 0.15 M NaCl, 0.05% Tween 20, NaN3 0.02%, 1% bovine serum albumin (BSA), pH 7.2), culture supernatants of COS-7 cells or CHO cells in which the chimeric antibody or humanized antibody was expressed, or the chimeric antibody or Purified humanized antibodies are serially diluted and then added to each well. Then 100 µl of alkaline phosphatase-conjugated goat anti-human IgG antibody is added, 100 µl of 1 mg / ml substrate solution (Sigmal04, p-nitrophenyl phosphate, SIGMA) is added, and then the absorbance at 405 µg / ml is measured. 655 nm using the Microplate Reader (Bio Rad). As a standard for concentration measurement, human IgG4K (The Binding Site) may be used.

(2) Measurement of antigen binding activity

Cell ELISA plates for measuring antigen binding activity are prepared as follows: J82 Human Bladder Carcinoma Cells (ATCC HTB-1) are inoculated into 60 wells of a 96-well cell culture plate at a counting. 1χ10 cells5 cells These are grown (RPMI1640 medium containing 10% fetal bovine serum (GIBCO)) for one day in a CO incubator.2 to allow cells to adhere to it. After discarding the culture liquid, each well is washed twice with 300 µl PBS. 100 µl PBS containing 4% paraformaldehyde (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 PBS, and then blocked with 250 µl DB. 100 µl of culture supernatants containing a chimeric antibody or a humanized antibody, or a chimeric antibody or purified humanized antibodies are serially diluted, then added to each well, and incubated at room temperature for 2 hours. After washing with rinse buffer (hereinafter referred to as RB PBS containing 0.05% Tween 20), 100 µl alkaline phosphatase-conjugated goat anti-human IgGY antibody (BioSource) diluted 1000 times with DB is added. After incubation at room temperature for 1 hour and washing with RB, the substrate solution is added, and then the absorbance at 405/655 nm is measured using the Microplate Reader (Bio-Rad).

(3) Measurement of neutralizing activity

Neutralizing activity of mouse antibody, chimeric antibody, and humanized antibodies can be measured with inhibitory activity against Factor Xa production by thromboplastin derived from human placenta Thromborel S (Boehringer AG) as an index. Thus, 60 pL of buffer (TBS containing 5 mM CaCl2 and 0.1% BSA) is added to 10 pL of 1.25 mg / mL Thromborel S and 10 pL of an appropriately diluted antibody, which is then incubated in a plate. 96 wells at room temperature for 1 hour.

10 µl each of 3.245 pg / mL human Factor X (Celsus Laboratories) and 82.5 ng / mL human Factor Vila (Enzyme Research) are added, and then incubated at room temperature for another hour. After 10 µl of 0.5 M EDTA is added to stop the reaction, 50 µl of the chromogenic substrate solution is added and the absorbance at 405/655 nm is determined. After reaction at room temperature for 1 hour, the absorbance at 405/655 nm is again determined. Neutralizing activity can be determined by calculating the residual activity (%) of each change in absorbance with the change in absorbance in the absence of antibody addition as 100% activity.

The chromogenic substrate solution is prepared by dissolving the Testzyme S-2222 (Chromogenix) chromogenic substrate according to the accompanying instructions, diluting 2 times with purified water and then mixing with a polybrene solution (0.6 mg / mL bromide). hexadimethylene, SIGMA) at 1: 1.

7 Interaction kinetic analysis of humanized antibody and soluble TF

The kinetic parameters, ie, dissociation constants (KD), dissociation rate constants (kdiss), and binding rate constants (kass) of the anti-TF antibody of this disclosure can be determined by BIACORE.

Recombinant G protein is immobilized on a sensor chip to which the antibody is bound, and purified recombinant TF (a soluble TF 1-219 on which the FLAG peptide has been labeled)

<td colspan="2">(hereafter referred to</td><td colspan="2">as a TF</td><td>soluble) is</td><td>used</td>

<td>as antigen</td><td>While</td><td>TF</td><td>soluble</td><td>prepared</td><td>to several</td>

<td>concentrations is</td><td>used</td><td>how</td><td>object</td><td>of analysis</td><td>. Starting</td>

<td>of the sensogram</td><td>obtained,</td><td>are</td><td colspan="2">calculated</td><td>parameters</td>

kinetics (dissociation rate constant kdiss, and binding rate constant kass), 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 (Karlsson, R. et al., (1991) J. Immunol. Methods 145: 229-240).

It is preferred that the anti-TF antibody of the present disclosure has a lower dissociation constants (KD) value as it will have a higher neutralizing activity. In the anti-TF antibody of the present disclosure, KD values are preferably not greater than 2.30> <10 "8 [1 / M], preferably not exceeding 2.30χ10-9 [1 / M], and most preferably not exceeding 1,17χ10 ~9 [1 / M].

In addition, KD values are determined from two parameters, the dissociation rate constant (kdiss) and the binding rate constant (kass) (KD = kdiss / kass). Thus, it is evident that when a kdiss is low and a kass is high, then the value of KD becomes low.

Specifically, in the case of the anti-TF antibody of the present disclosure, kdiss values may be no greater than 9.52x10-3 [1 / sec] Preferably the kdiss values are no greater than 9.52χ10 ~4 [1 / sec], most preferably not exceeding 6.35χ10 “4 [1 / sec]

On the other hand, kass values may be no less than 4.15x104 [1 / M'sec]. Preferably, the kass values are not less than 4.15χ105 [1 / M'seg], and most preferably not less than 4.65χ105 [1 / M'sec].

In addition, preferably anti-TF antibodies have a kdiss value of not more than 9.52χ10 ~3 [1 / sec] and a kass value of not less than 4.15x104 [1 / M * sec]

More specifically, for the anti-TF antibody of the present disclosure, the KD values are in the range of 1.9 χ 110

2.30χ10 “8 [1 / M], preferably 1.09χ10 “9 - 2.30χ10 “9 [1 / M], and most preferably 1.09χ10 ~9- 1.39χ10 ~9 [1 / M].

<td colspan="2">In addition, the values of kdiss</td><td colspan="2">are in the range of</td>

<td>6.0x10-4</td><td>9.52x10 “3 [1 / sec] of</td><td>preference</td><td>5.06x10-4 -</td>

<td>9.52x10-4</td><td>[1 / sec], and higher</td><td>preference</td><td>5.06x10-4 -</td>

And, the kass values are in the range of 4.15> <104 5.44χ105 [1 / M'seg], preferably 4.15χ105 - 5.44x105 [1 / M'seg], and most preferably 4.65χ105 - 5.44χ105 [1 / Μ · sec]

Although these KD values, kdiss values, and kass values can be obtained by, in addition to BIACORE, Scatchard analysis, and the like, it is preferred to use BIACORE.

8 Measurement of the reactivity of the humanized antibody to human TF dot-blot hybridization method can be used to investigate the reactivity of undenatured TF, denatured TF under non-reducing conditions and denatured TF under reducing conditions.

TF may be one that has been purified from human tissue, or has been expressed in mammalian cells such as CHO cells and purified, and may be used for research. As denaturing agent, guanidine hydrochloride or SDS, etc. can be used instead of urea. As a reducing agent, an SH reducing agent such as 2-mercaptoethanol may be used instead of DTT. For detection of humanized antibody, multi-substance labeled human IgG antibody may be used. As used herein, the labeling agents may be radioisotopes, biotin, fluorogenic substances such as

FITC, enzymes such as peroxidase and alkaline phosphatase, and the like. The anti-TF antibody of this disclosure reacts with either undenatured TF, denatured TF under non-reducing conditions, and denatured TF under reducing conditions.

9 Pharmaceutical compositions and therapeutic agents for DIC comprising a humanized antibody as active ingredient

To confirm the therapeutic effect of a humanized antibody on human TF, the humanized anti-human TF antibody is administered to an animal having a strong symptom of IHD, and then IHD indices are measured to confirm therapeutic effects.

The antibody as used herein is a humanized antibody to human TF. The antibody neutralizes human TF activity by binding to human TF, and preferably a humanized ATR5 antibody may be mentioned. The method of generating the humanized antibody ATR5 is described in the Examples.

The antibody as used herein can be purified to high purity by combining common purification methods such as desalting, gel filtration method such as HPLC, affinity chromatography using a Protein A column, and the like. The antibody thus purified can be confirmed to recognize human TF with high precision using common immunological means such as radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), or an immunofluorescent antibody method (Immunofluorescence Analysis) and the like.

Pharmaceutical compositions or therapeutic agents for DIC of the present disclosure comprising as active ingredient the humanized TF antibody may be administered non-perorally systemically or locally. For example, the method of administration may be selected by intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection, and may be selected as appropriate based on the age and condition of the patient. The effective dosage is chosen from the range of 0.01 mg to 1000 mg per kg body weight per administration. Alternatively, a dosage of 10 mg / body, preferably 1 to 1000 mg / body per patient may be chosen.

collagen, alcohol a sodium polymer,

Pharmaceutical compositions and DIC therapeutic agents of the present disclosure containing a humanized human TF antibody as active ingredient may contain pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, polyvinyl, polyvinylpyrrolidone, carboxyvinyl, sodium polyacrylic carboxymethylcellulose, sodium alginate, water-soluble dextran, carboxymethyl starch, pectin, methylcellulose, ethylcellulose, gum gum arabic, barrack, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, pharmaceutically surfactant

Additives used are acceptable lactose, one and the like sorbitol chosen from, but not limited to, the above or combinations thereof as appropriate depending on the dosage form of the present disclosure.

Effects of the invention

According to the present disclosure, there is provided a chimeric antibody and a humanized antibody against human TF, and a claimed method of generating a humanized antibody. These antibodies are used as therapeutic agents due to their low antigenicity.

Examples

The present invention will now be explained in more detail with reference to the following Examples.

Example 1. Cloning of DNA encoding the V region of a mouse monoclonal antibody against human TF (1) Preparation of mRNA

MRNA was prepared from the hybridomas ATR-2, ATR-3, ATR-4, ATR-5 (IgG1K), ATR-7, and ATR-8 (IgG2aK) using the QuickPrep mRNA Purification Kit (Pharmacia Biotech). Each hybridoma cell was completely homogenized in the extraction buffer according to the instructions attached to the kit, and then mRNA was purified through the oligo (dT) -cellulose centrifugation column, followed by ethanol precipitation. The mRNA precipitate was dissolved in the elution buffer.

(2) Preparation and amplification of gene cDNA encoding a V region of a mouse antibody (i) Cloning of H chain V region cDNA

Cloning of the gene encoding the H chain V region of a mouse monoclonal antibody against human TF was performed using the 5'-RACE method (Frohman, MA et al., Proc. Natl. 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 performed according to the instructions attached to the kit.

Using about 1 pg of mRNA prepared as above as a template, the attached cDNA synthesis primer was added to the kit, which was reacted with a reverse transcriptase at 42 ° C for 60 minutes to reverse transcribe to cDNA. This was reacted with DNA polymerase I, DNA ligase, and RNAseH at 16 ° C for 1.5 hours, and with T4 DNA polymerase at 16 ° C for 45 minutes to thereby synthesize a double stranded cDNA. The double stranded cDNA was extracted with phenol and chloroform, and recovered by precipitation in ethanol.

By overnight reaction with T4 DNA ligase at 16 ° C, a cDNA adapter was ligated to both ends of the double stranded cDNA. The reaction mixture was diluted 50-fold with 10 mM Tricine-KOH (pH 8.5) containing 0.1 mM EDTA. Using this as a template, the gene encoding the H chain V region was amplified by PCR. The adapter primer 1 attached to the kit was used for the 5 'end primer, and for the 3' end primer was used the MHCG1 primer (SEQ ID NO: 1) (ATR-2, ATR-3, ATR-4, and ATR-5) or the primer MHC-G2a (SEQ ID NO: 2) (ATR-7 and ATR-8) (S. T. Jones, et al., Biotechnology, 9: 88-89, 1991).

The H-chain V region PCR solutions of the ATR-2, 3, 4, and 5 antibody contained, in 100 µl, 120 mM Tris-HCl (pH 8.0), 10 mM KCl (NH4)2ONLY4 6 mM, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), MgCl2 1 mM, 2.5 units KOD DNA polymerase (Toyo Boseki), 30-50 pmole of adapter primer 1 as well as MHCG1 primer, and 1-5 µl of a cDNA reaction mixture to which the CDNA.

All PCRs were performed using Thermal Cycler 480 DNA (Perkin-Elmer), and PCR was performed for thirty cycles at a temperature cycle of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 74 ° C for 1 minute.

(ii) Cloning of L chain V region cDNA

Cloning of the L-chain V-encoding gene of a mouse monoclonal antibody against human TF was performed using the 5'-RACE method (Frohman, MA et al., Proc. Natl. 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 performed according to the instructions attached to the kit. Using about 1 pg of mRNA prepared as above as a template, the cDNA synthesis primer was added and reacted with a reverse transcriptase at 42 ° C for 60 minutes to reverse transcribe to cDNA.

This was reacted with DNA polymerase I, DNA ligase, and RNAseH at 16 ° C for 1.5 hours, and with T4 DNA polymerase at 16 ° C for 45 minutes to thereby synthesize a double stranded cDNA. The double stranded cDNA was extracted with phenol and chloroform, and recovered by precipitation in ethanol. By overnight reaction with T4 DNA ligase at 16 ° C, a cDNA adapter was ligated to both ends of the double stranded cDNA. The reaction mixture was diluted 50-fold with 10 mM Tricine-KOH (pH 8.5) containing 0.1 mM EDTA. Using this as a template, the gene encoding the H chain V region was amplified by PCR. Adapter primer 1 was used for the 5 'end primer, and for the 3' end primer the MKC primer (SEQ ID NO: 3) (ST Jones, et al., Blotecnology, 9: 88-89, 1991).

The PCR solutions contained, in 100 µl, 120 mM Tris-HCl (pH 8.0), 10 mM KCl (NH4)2ONLY4 6 mM, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), MgCl2 1 mM, 2.5 units KOD DNA polymerase (Toyo Boseki), 3050 pmole of adapter primer 1 as well as MKC primer, and 1 µl of a cDNA reaction mixture to which the cDNA adapter was attached.

(3) Purification and fragmentation of PCR products

The above PCR reaction mixture was extracted with phenol and chloroform, and the amplified DNA fragments were recovered by ethanol precipitation. DNA fragments were digested with restriction enzyme Xmal (New England Biolabs) at 37 ° 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 about 500 bp DNA fragments as the V region of the chain. H and DNA fragments of about 500 bp in length as the L chain V region were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol, which were then dissolved in 10 µl 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA (hereinafter referred to as TE)

Xmal digested DNA fragments prepared as above containing genes encoding a mouse H chain V region and a mouse L chain V region and the plasmid vector pUC19 prepared by Xmal digestion were ligated using the DNA ligation kit see .2 (Takara Shuzo) reacting at 16 ° C for 1 hour according to the instructions attached to the kit.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C.

Then 300 Âµl of Hi-Competence Broth (Nippongene), incubated at 37Â ° C for 1 hour, was added. Escherichia coli was then plated on an LB-agar medium (Molecular Cloning: A Laboratory Manual, Sambrook, et al., Cold Spring Harbor Laboratory Press, 1989) containing 100 pg / ml ampicillin (hereinafter referred to as LBA- agar), and incubated overnight at 37 ° C to obtain an E. coli transformant.

The transformant was grown overnight in 3 mL or 4 mL of an LB medium containing 50 pg / ml ampicillin (hereinafter referred to as LBA medium) at 37 ° C, and DNA fractions were prepared from the cell fractions. using the QIAprep Spin Plasmid Kit (QIAGEN), and then the nucleotide sequence was determined.

(4) Determination of nucleotide sequence of gene encoding mouse antibody V region

The nucleotide sequence of the cDNA coding region in the above plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin-

<td colspan="3">Elmer) through the DNA Sequencer</td><td rowspan="2">373A were</td><td colspan="2">(Perkin-Elmer)</td>

<td>As initiator</td><td>in</td><td>sequencing,</td><td>used</td><td> 0</td>

<td>M4 Initiator</td><td>M13</td><td>(Takara Shuzo)</td><td>(SEQ</td><td>ID NO: 4) and</td><td>O</td>

<td>RV Initiator</td><td>M13</td><td>(Takara Shuzo)</td><td>(SEQ</td><td>ID NO: 5), and</td><td>The</td>

<td>sequence was</td><td colspan="2">determined through</td><td>in</td><td>confirmation</td><td>gives</td>

nucleotide sequence in both directions.

Thus obtained plasmids containing the gene encoding the hybridoma-derived mouse H chain V region

ATR-2, ATR-3, ATR-4, ATR-5, ATR-7, and ATR-8 were designated as ATR-xHv / pUCl 9 (x = 2, 3, 4, 5, 7, or 8), and thus obtained plasmids containing the gene encoding a mouse L-chain V region 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 plasmid ATRxHv / pUC19 (x = 2, 3, 4, 5, 7, or 8) (including corresponding amino acid sequences) are shown in SEQ ID NO: 6th to 11, respectively, and the nucleotide sequences of the genes encoding the L chain V region of each mouse antibody contained in plasmid ATRxLv / pUC19 (x = 2, 3, 4, 5, 7, or 8) (including corresponding sequences of amino acids) are shown in SEQ ID NO: 12 to 17, respectively.

Example 2. Construction of Chimeric Antibody

An ATR-5 chimeric antibody was generated in which the V region of the mouse ATR-5 antibody was bound to the C region of a human antibody. A chimeric antibody expression vector was constructed by ligating the gene encoding the ATR-5 antibody V region with an expression vector encoding the human antibody C region.

(1) Construction of a chimeric antibody H chain V region

The H chain V region of the ATR-5 antibody was modified by the PCR method to bind it to an expression vector encoding the human antibody H chain C region. The 5'-end primer ch5HS (SEQ ID NO: 18) was designed to hybridize to the 5 'end of the V-region encoding DNA and to have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol 196: 947-950, 1987) and a restriction enzyme recognition sequence SalI. The 3 'end primer ch5HA (SEQ ID NO: 19) is designed to hybridize to the 3' end of DNA encoding the J region and to have an Nhel restriction enzyme recognition sequence.

The PCR solutions contained, in 100 µl, 120 mM Tris-HCl (pH 8.0), 10 mM KOI, (NH4)2ONLY4 6 mM, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), MgCl2 1 mM, 2.5 units KOD DNA polymerase (Toyo Boseki), 50 pmole of ch5HS primer and ch5HA primer, as well as 1 µl of plasmid ATR5Hv / pUC19 as template DNA. For PCR, DNA Thermal Cycler 480 (Perkin-Elmer) was used, and PCR was performed for thirty cycles at a temperature cycle of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 74 ° C for 1 minute.

The PCR reaction mixture was extracted with phenol and chloroform, and the amplified DNA fragments were recovered by ethanol precipitation. The DNA fragments were digested with Nhel restriction enzyme (Takara Shuzo) at 37 ° C for 1 hour, and then with Sall restriction enzyme (Takara Shuzo) at 37 ° C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using a 3% NuSieve GTG agarose (FMC BioProducts), and the agarose strips containing about 450 bp long DNA fragments were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol and then dissolved in 20 µl TE.

As a cloning vector, an altered promoter vector (hereinafter referred to as CVIDEC) was used in which the restriction enzyme recognition sequences Nhel, Sall, and Spll, Bglll were introduced. The gene fragment prepared as above encoding the mouse H chain V region and the CVIDEC vector prepared by digestion with Nhel and Sall were ligated using the ver.2 DNA binding kit (Takara Shuzo) by reacting them. 16 ° C for 1 hour according to the instructions attached to the kit.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated on LBA-agar medium and incubated overnight at 37 ° C to obtain an E. coli transformant. The transformant was grown overnight at 37 ° 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 plasmid cDNA coding region was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (PerkinElmer) through the 373A DNA Sequencer (Perkin-Elmer). As sequencing primer, the M13 M4 Primer (Takara Shuzo) and M13 RV Primer (Takara Shuzo) were used, and the sequence was determined by confirming the nucleotide sequence in both directions. The plasmid containing the gene encoding the ATR-5 antibody H-chain V region, a SalI recognition sequence and the 5 'end Kozak consensus sequence, and the 3' end Nhel recognition sequence have been designated. as chATR5Hv / CVIDEC.

(2) Construction of an L chain V region of a chimeric antibody

The ATR-5 antibody L chain V region was modified by the PCR method to bind it to an expression vector encoding the human antibody L chain C region. The 5 'end primer ch5LS (SEQ ID NO: 20) was designed to hybridize to the 5' end of DNA encoding the V region and to have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol 196: 947-950, 1987) and a restriction enzyme recognition sequence Bg111. The 3 'end primer ch5LA (SEQ ID NO: 21) is designed to hybridize to the 3' end of DNA encoding the J region and to have a Spl1 restriction enzyme recognition sequence.

The PCR solutions contained, in 100 µl, 120 mM Tris-HCl (pH 8.0), 10 mM KCl (NH4)2ONLY4 6 mM, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), MgCl2 1 mM, 2.5 units KOD DNA polymerase (Toyo Boseki), 50 pmole of ch5LS primer and ch5LA primer, as well as 1 pL of plasmid ATR5Lv / pUC19 as template DNA. Thermal Cycler 480 DNA (Perkin-Elmer) was used for PCR, and PCR was performed for thirty cycles at a temperature cycle of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 74 ° C for 1 minute. .

The PCR reaction mixture was extracted with phenol and chloroform, and the amplified DNA fragments were recovered by ethanol precipitation. The DNA fragments were digested with restriction enzyme Spll (Takara Shuzo) at 37 ° C for 1 hour, and then with restriction enzyme Bglll (Takara Shuzo) at 37 ° C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using a 3% NuSieve GTG agarose (FMC BioProducts), and the agarose strips containing about 400 bp long DNA fragments were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol and then dissolved in 20 µl TE.

gene fragment prepared as above encoding mouse L-chain V region and CVIDEC vector prepared by Spl1 and BglII digestion were ligated using DNA binding kit ver.2 (Takara Shuzo) and reacted at 16 ° C for 1 hour according to the instructions attached to the case.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated in 100 Âµg / ml LBA agar medium and incubated. overnight at 37 ° C to obtain an E. coli transformant. The transformant was grown overnight at 37 ° 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 cDNA coding region in the plasmid was determined using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (PerkinElmer) through the 373A DNA Sequencer (Perkin-Elmer). As sequencing primer, the M13 M4 Primer (Takara Shuzo) and M13 RV Primer (Takara Shuzo) were used, and the sequence was determined by confirming the nucleotide sequence in both directions. The plasmid containing the gene encoding the ATR-5 antibody L-chain V region and having a BglII recognition sequence and the 5 'end Kozak consensus sequence and a 3' end Spl1 recognition sequence 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 by IDEC Pharmaceuticals. As a vector, the H5KG1 (V) IgG1 type antibody expression vector and the N5KG4P IgG4 type antibody expression vector were used. 0 ATR-5 chimeric antibody expression vector was generated by ligation of a gene encoding the ATR-5 H chain V region to the Sall-Nhel site located immediately before the human antibody H chain C region of the N5KG1 expression vector (V) or N5KG4P and the ligation of a gene encoding the ATR-5 L-chain V region to the Bglll-Spl1 site located immediately before the human antibody L-chain C region of the N5KG1 (V) or N5KG4P expression vector.

(i) Introduction of the H-chain V-region plasmid chATR5Hv / CVIDEC was digested with Nhel restriction enzyme (Takara Shuzo) at 37 ° C for 3 hours and with Sall restriction enzyme (Takara Shuzo) at 37 ° C for 3 hours. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose strips containing about 450 bp long DNA fragments were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol and then dissolved in 20 µl TE.

N5KG1 (V) and N5KG4P expression vectors were digested with Nhel restriction enzyme (Takara Shuzo) at 37 ° C for 3 hours, and with Sall restriction enzyme (Takara

Shuzo) at 37 ° C for 3 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 about 9000 bp in length were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol and then dissolved in 20 µl TE.

The Sall-Nhel DNA fragment prepared as above containing the gene encoding the H chain V region and Sall and Nhel digested N5KG1 (V) or N5KG4P were ligated using the ver.2 DNA binding kit (Takara Shuzo) making react at 16 ° C for 1 hour according to the attached instructions.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated in 100 Âµg / ml LBA agar medium and incubated. overnight at 37 ° C to obtain an E. coli transformant. The transformant was grown overnight at 37 ° C in 3 mL of LBA medium, and from the cell fractions plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN). These plasmids containing the ATR-5 chimeric antibody H-chain genes were designated as chATR5Hv / N5KG4 (V) and chATR5Hv / N5KG4P, respectively.

(ii) Introduction of the V-region L-chain plasmid chATR5Lv / CVIDEC was digested with restriction enzymes BglII (Takara Shuzo) and Spll (Takara Shuzo) at 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 about 400 bp long DNA fragments were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol and then dissolved in 20 µl TE.

The chATR5Hv / N5KG1 (V) and chATR5Hv / N5KG4P plasmids were digested with restriction enzymes BglII (Takara Shuzo) and Spll (Takara Shuzo) at 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 about 9400 bp in length were excised. The agarose strips were extracted with phenol and chloroform, the DNA fragments were precipitated with ethanol and then dissolved in 20 µl TE.

The SplI-BglII DNA fragment prepared as above containing the L-chain V region gene encoding Spl1 and BglII digested chATR5Hv / N5KG1 (V) or chATR5Hv / N5KG4P were ligated using the ver.2 DNA binding kit ( Takara Shuzo) by having them react at 16 ° C for 1 hour according to the attached instructions.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated in 100 Âµg / ml LBA agar medium and incubated. overnight at 37 ° C to obtain an E. coli transformant. The transformant was cultured overnight at 37 ° C in 1 L of 2xYT medium containing 50 pg / ml ampicillin and from plasmid fractions plasmid DNA was prepared using the Plasmid Maxi Kit (QIAGEN). These plasmids containing the gene encoding the ATR-5 chimeric antibody were designated as chATR5 / N5KG1 (V) and chATR5 / N5KG4P, respectively.

(4) COS-7 cell transfection

To evaluate antigen binding activity and neutralizing activity of the chimeric antibody, the above expression plasmid was transfected into COS-7 cells and the antibody was transiently expressed.

The chATR5 / N5KG1 (V) or chATR5 / N5KG4P plasmid was transduced to COS-7 cells by electroporation using the Gene Pulser instrument (Bio Rad). 50 µg of plasmid was added to 0.78 mL of COS-7 cells suspended in Dulbecco (-) PBS (hereinafter referred to as PBS) at a cell concentration of 1χ107 cells / mL, which were subjected to 1,500 V pulses and 25 pF capacitance.

After 10 minutes of the recovery period at room temperature, the electroporated cells were suspended in a DMEM medium containing 5% IgG ultra-low fetal bovine serum (GIBCO), and cultured using a 10 cm culture box in a CO incubator.2 at 5%. After culturing for 24 hours, the culture supernatant was aspirated, and then HBCHO serum free medium (Irvine Scientific) was added. After culturing for a further 72 hours, the culture supernatant was harvested and centrifuged to remove cell debris.

(5) Antibody Purification

From the COS-7 cell culture supernatant, the chimeric antibody was purified using rProtein A Sepharose Fast Flow (Pharmacia Biotech) as follows.

1 ml of rProtein A Sepharose Fast Flow was filled into a column and the column was equilibrated by 10 volumes of TBS. COS-7 cell culture supernatant was applied to the equilibrated column, which was then washed with 10 volumes of TBS.

The traction of adsorbed antibody was then eluted through 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 1 M Tris-HCl (pH 8 .0).

By twice ultrafiltration for the traction of purified antibody using Centriprep 100 (Amicon), the solvent was replaced with 50 mM Tris-HCl (pH 7.6) containing 150 mM NaCl (hereinafter referred to as TBS), and was finally concentrated to about 1.5 mL.

(6) Establishment of a stable producing CHO cell line

To establish a cell line stably producing chimeric antibody, the above expression plasmid was introduced into CHO cells (DG44) acclimated to serum free CHOS-SFMII (GIBCO) medium.

The chATR5 / N5KG1 (V) or chATR5 / N5KG4P plasmid was cleaved with the SspI restriction enzyme (Takara Shuzo) to linearize the DNA, and after phenol and chloroform extraction, the DNA was recovered by ethanol precipitation. The linearized plasmid was transduced to DG44 cells by electroporation using the Gene Pulser instrument (Bio Rad). 10 µg of plasmid was added to 0.78 mL of PBS-suspended DG44 cells at a cell concentration of 1χ107 cells / mL, which were subjected to 1,500 V pulses and 25 pF capacitance.

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 CO incubator.2 at 5%. On the day after culture initiation, the medium was changed to a selection medium containing CHO-S-SFMII (GIBCO) medium containing hypoxanthine / thymidine (GIBCO) and 500 pg / mL GENETICIN (G418 Sulphate, GIBCO) to select the cells into which the antibody gene had been introduced. After changing the selection medium, cells were examined under a microscope about two weeks later. After favorable cell growth was observed, the amount of antibody produced was measured by the ELISA described below for determination of antibody concentration, and cells having a high antibody production were selected.

Example 3. Humanized antibody construction (1) Humanized antibody H chain construction (i) Humanized H chain construction version a

The H chain of the humanized ATR-5 antibody was generated using a CDR graft by the PCR method. To generate the H chain of humanized version a antibody having the FR derived from human antibody L39130 (DDBJ, Gao L. et al., Unpublished, 1995), seven PCR primers were used. The hR5Hv1S (SEQ ID NO: 22), hR5Hv2S (SEQ ID NO: 23), and hR5Hv4S (SEQ ID NO: 24) CDR graft primers have a sense DNA sequence, and the hR5Hv3A CDR graft primers ( SEQ ID NO: 25) and hR5Hv5A (SEQ ID NO: 26) have an antisense DNA sequence, each primer having a complementary 18-35 bp sequence at both ends thereof.

hR5Hv1S was designed to have the Kozak consensus sequence (Kozak, M. et al., J. Mol. Biol. 196: 947-950, 1987) and a Sall recognition site, and hR5Hv5A was designed to have a site of consensus. of Nhel recognition. The exogenous primer hR5HvPrS (SEQ ID NO: 27) has homology to the hR5HvlS CDR graft primer, and hR5HvPrA (SEQ ID NO: 28) has homology to the hR5Hv5A CDR graft primer.

The CDR graft primers hR5Hv1S, hR5Hv2S, hR5Hv3A, hR5Hv4S, and hR5Hv5A, and the exogenous primers hR5HvPrS and hR5HvPrA were synthesized and purified by Pharmacia Biotech.

PCR was performed using KOD DNA polymerase (Toyo Boseki) and the attached buffer provided that it contained 120 mM Tris-HCl (pH 8.0), 10 mM KOI, (NH4) 6 mM 2SO 4, 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) pmole of each of the CDR graft primers hR5Hv1S, hR5Hv2S, hR5Hv3A, hR5Hv4S, hR5Hv5A at 98 pL for 5 cycles one cycle and

temperatures of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute. After further addition of 100 pmole of the hRSHvPrS and hR5HvPrA exogenous primers, PCR was performed for 25 cycles in a 100 µL system with the same temperature cycle . DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using a 2% NuSieve GTG agarose (FMC BioProducts).

Agarose strips containing about 430 bp long DNA fragments were excised to which 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the DNA fragments. After precipitation of ethanol-purified DNA, one third of the volume was dissolved in 17 µl of water. The obtained PCR reaction mixture was digested with Nhel and Sall, and was ligated to the plasmid vector CVIDEC prepared by digestion with Nhel and Sall using the ver.2 DNA binding kit (Takara Shuzo) according to the instructions attached to case.

Since mutation and / or deletion was observed before or after the Ecol221 recognition site, each fragment having the correct sequence was ligated and then subcloned into CVIDEC to determine the nucleotide sequence. The plasmid having the correct sequence was designated as hATR5Hva / CVIDEC. The nucleotide sequence and corresponding humanized H chain amino acid sequence version a contained in plasmid hATR5Hva / CVIDEC are shown in SEQ ID NO: 29. The amino acid sequence of version a is also shown in SEQ ID NO: 30.

(ii) Construction of humanized H chain versions b and c

The b and c versions were generated by replacing version 3 FR3 with FR3 derived from another human antibody using the RF shuffling method. To replace FR3 in version b with a derivative of the human antibody Z34963 (DDBJ, Borretzen M. et al., Proc. Natl. Acad. Sci. USA, 91: 12917-12921, 1994), the four DNA primers were generated. encoding the FR3. FR shuffle primers F3RFFS (SEQ ID NO: 31) and F3RFBS (SEQ ID NO: 32) have a sense DNA sequence and F3RFFA (SEQ ID NO: 33) and F3RFBA (SEQ ID NO: 34) have a antisense DNA sequence. F3RFFS and F3RFFA have a complementary sequence to each other, and have Bali and XhoI recognition sequences at both ends. F3RFBS and F3RFBA have a complementary sequence to each other and have XhoI and NcoI recognition sequences at both ends.

To replace FR3 in version c with one derived from human antibody P01825 (SWISS-PROT, Poljak RJ et al., Biochemistry, 16: 3412-3420, 1977), four FR3-encoding DNA primers were generated. FR shuffle primers 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 a antisense DNA sequence.

F3NMFS and F3NMFA have a complementary sequence to each other and have Bali and Xhol recognition sequences at both ends.

F3NMBS and F3NMBA have, and have XhoI and NcoI recognition sequences at both ends.

F3RFFS, F3RFBS, F3RFFA, F3RFBA, F3NMFS, F3NMBS, F3NMFA, and F3NMBA were synthesized by Pharmacia Biotech. F3RFFS and F3RFFA, and F3RFBS and F3RFBA were ligated, and were digested with Bali and Xhol, and Ncol and Xhol, respectively. They were introduced into plasmid hATR5Hva / CVIDEC (Bali / Ncol) prepared by digestion with Bali and Ncol, and the nucleotide sequence was determined. The plasmid having the correct sequence was designated as hATR5Hvb / CVIDEC. The nucleotide sequence and corresponding humanized H chain amino acid sequence version b contained in plasmid hATR5Hvb / CVIDEC are shown in SEQ ID NO: 39. The amino acid sequence of version b is also shown in SEQ ID NO: 40.

F3NMFS and F3NMFA, and F3NMBS and F3NMBA were ligated, and were digested with Bali and Xhol, and Ncol and Xhol, respectively. They were introduced into plasmid hATR5Hva / CVIDEC (Ball / Ncol) prepared by digestion with Bali and Ncol, and the nucleotide sequence was determined. The plasmid having the correct sequence was designated as hATR5Hvc / CVIDEC. The nucleotide sequence and the corresponding humanized H chain amino acid sequence version c contained in plasmid hATR5Hvc / CVIDEC are shown in SEQ ID NO: 41. The amino acid sequence of version c is also shown in SEQ ID NO: 42.

(iii) Construction of humanized H chain versions of

The dee versions were generated by replacing the FR3 of version a with FR3 derived from another human antibody using the FR shuffling method. To replace FR3 in version d with a derivative of the human antibody M62723 (DDBJ, Pascual V. et al., J. Clin. Invest., 86: 1320-1328, 1990), four FR3-encoding DNA primers were generated. The FR F3EPS shuffle primer (SEQ ID NO: 43) has a sense DNA sequence and F3EPA (SEQ ID NO: 44) has an antisense DNA sequence, and the 3 'end of the primers has a complementary sequence. 18 bp.

The exogenous primers F3PrS (SEQ ID NO: 45) and F3PrA (SEQ ID NO: 46) have homology to the FR shuffle primers F3EPS and F3EPA, and may also be used for RF shuffling of other FR3. To replace FR3 in the version and a derivative of the human antibody Z80844 (DDBJ, Thomsett AR. Et al., Unpublished), two FR3-encoding DNA primers were generated. 0 F3VHS shuffle primer (SEQ ID NO: 47) has a sense DNA sequence and F3VHA (SEQ ID NO: 48) has an antisense DNA sequence, and the 3 'end of the primers has a complementary sequence of 18 bp F3EPS, F3EPA, F3PrS, F3PrA, F3VHS and F3VHA were synthesized by Pharmacia Biotech.

PCR was performed using KOD DNA polymerase (Toyo Boseki) using the attached buffer provided that it contained 5 pL of each of the F3EPS and F3EPA FR shuffling primers, or 1 pM F3VHS and F3VHA, 0.2 mM dNTPs , MgCl2 1.0 mM, and 2.5 units 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 addition of an additional 100 pmole of exogenous primers F3PrS and F3PrA, PCR was performed for 25 cycles at the same temperature cycle.

DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using a 2% Nu Sieve GTG agarose (FMC BioProducts). Agarose strips containing about 424 bp DNA fragments were excised, to which 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the DNA fragments. . After precipitation of ethanol-purified DNA, one third of the volume was dissolved in 14 µl of water. The obtained PCR reaction mixture was digested with Bali and Ncol, and was introduced into plasmid hATR5Hva / CVIDEC (Bali / Ncol) prepared by digestion with Bali and Ncol and the nucleotide sequence determined.

Plasmids having the correct sequence were designated as hATR5Hvd / CVIDEC and hATR5Hve / CVIDEC. The nucleotide sequence and the corresponding humanized H chain amino acid sequence version d contained in plasmid hATR5Hvd / CVIDEC are shown in SEQ ID NO: 49 and the amino acid sequence of version d is also shown in SEQ ID NO: 50. The nucleotide sequence and corresponding amino acid sequence of the humanized H-chain version and contained in plasmid hATR5Hve / CVIDEC are shown in SEQ ID NO: 51 and the amino acid sequence of version and is also shown in SEQ ID NO: 52.

(iv) Construction of humanized H chain feg versions

The feg versions were generated by replacing FR3 of version a with FR3 derived from another human antibody using the FR shuffling method. To replace FR3 in version f with a derivative of the human antibody L04345 (DDBJ, Hillson JL. Et al., J. Exp. Med., 178: 331-336, 1993) and to replace FR3 in version g with a derivative of human antibody S78322 (DDBJ, Bejcek BE. et al., Cancer Res., 55: 2346-2351, 1995), two primers were synthesized, each encoding an FR3. The F3SSS shuffle primer (SEQ ID NO: 53) of version f has a sense DNA sequence and F3SSA (SEQ ID NO: 54) has an antisense DNA sequence, and the 3 'end of the primers has an 18 bp complementary sequence.

F3CDS (SEQ ID NO: 55) of version g has a sense DNA sequence and F3CDA (SEQ ID NO: 56) has an antisense DNA sequence, and the 3 'end of the primers has a complementary 18 bp sequence . F3SSS, F3SSA, F3CDS, and F3CDA were synthesized and purified by Pharmacia Biotech. PCR was performed using KOD DNA polymerase (Toyo Boseki) using the attached buffer provided that it contained 5 pL of each of the F3SSS and F3SSA FR shuffling primers, or F3CDS and F3CDA 1 μΜ, 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 at a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1

<td>minute, and</td><td>74 ° C during</td><td>1 minute</td><td>. After addition</td><td>too much</td><td> 100</td>

<td>pmole of</td><td>initiators</td><td>exogenous</td><td>F3PrS and F3PrA,</td><td>the PCR</td><td>was</td>

<td>made</td><td>for 25</td><td>cycles</td><td>with the same</td><td>cycle</td><td>in</td>

temperatures.

DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using a 2% NuSieve GTG agarose (FMC BioProducts). Agarose strips containing about 424 bp DNA fragments were excised, to which 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the DNA fragments. DNA. After precipitation of ethanol-purified DNA, one third of the volume was dissolved in 14 µl of water. The obtained PCR reaction mixture was digested with Bali and Ncol and was introduced into plasmid hATR5Hva / CVIDEC (Bali / Ncol) prepared by digestion with Bali and Ncol and the nucleotide sequence was determined.

Plasmids having the correct sequence were designated as hATR5Hvf / CVIDEC and hATR5Hvg / CVIDEC. The nucleotide sequence and corresponding humanized H chain amino acid sequence version f contained in plasmid hATR5Hvf / CVIDEC, and version f amino acid sequence are shown in SEQ ID NO: 57 and 58. The nucleotide sequence and corresponding humanized H chain amino acid sequence version g contained in plasmid hATR5Hvg / CVIDEC, and the amino acid sequence of version g are shown in SEQ ID NO: 59 and 60.

(v) Construction of humanized H chain version h

The h version was generated by replacing version 3 FR3 with FR3 derived from another human antibody using the RF shuffling method. To replace FR3 in version h with a derivative of human antibody Z26827 (DDBJ, van Der Stoep et al., J. Exp. Med., 177: 99107, 1993), two primers each encoding FR3 were synthesized. The FR F3ADS shuffle primer (SEQ ID NO: 61) of version h has a sense DNA sequence and F3ADA (SEQ ID NO: 62) has an antisense DNA sequence, and the 3 'end of the primers has an 18 bp complementary sequence.

F3ADS and F3ADA were synthesized and purified by Pharmacia Biotech. PCR was performed using KOD DNA Polymerase (Toyo Boseki) using the attached buffer provided that it contained 5 pL of each 1 pM FR F3ADS and F3ADA shuffling primers, 0.2 mM dNTPs, 1.0 mM MgCb, and 2.5 units KOD DNA polymerase in 100 µl of the reaction mixture for 5 cycles a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 74 ° C for 1 minute. After adding another 100 pmole of exogenous primers F3PrS and F3PrA,

PCR was performed for 25 cycles with the same temperature cycle. DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using a 2% NuSieve GTG agarose (FMC BioProducts).

Agarose strips containing about 424 bp DNA fragments were excised, to which 3 volumes (ml / g) of TE were added, and then extracted with phenol, phenol / chloroform, and chloroform to purify the DNA fragments. DNA. After precipitation of ethanol-purified DNA, one third of the volume was dissolved in 14 µl of water. The obtained PCR reaction mixture was digested with Bali and Ncol, and was introduced into plasmid hATR5Hva / CVIDEC (Bali / Ncol) prepared by digestion with Bali and Ncol, and the nucleotide sequence was determined. Plasmids having the correct sequence were designated as hATR5Hvh / CVIDEC. The nucleotide sequence and the corresponding humanized H chain amino acid sequence version h contained in plasmid hATR5Hvh / CVIDEC, and the amino acid sequence of version h are shown in SEQ ID NO: 63. The amino acid sequence of version h is shown in SEQ. ID NO: 64.

(vi) Construction of humanized H chain versions iej

The i and j versions were generated by replacing FR3 of version a with FR3 derived from another human antibody using the FR shuffling method. 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. Natl. Acad. USA, 89: 10026-10030, 1992), two primers were synthesized each encoding a

FR3. The FR shuffle primer F3MMS (SEQ ID NO: 65) of version i has a sense DNA sequence and F3MMA (SEQ ID NO: 66) has an antisense DNA sequence, and the 3 'end of the primers has a sequence complementary 18 bp.

F3BMS (SEQ ID NO: 67) of version j has a sense DNA sequence and F3BMA (SEQ ID NO: 68) has an antisense DNA sequence, and the 3 'end of the primers has a complementary 18 bp sequence . F3MMS, F3MMA, F3BMS, and F3BMA were synthesized and purified by Pharmacia Biotech. PCR was performed using Ampli Taq Gold (Perkin-Elmer) using the attached buffer provided it contained 5 µl of each of the F3MMS and F3MMA FR shuffling primers, or 1 pM F3BMS and F3BMA, 0.2 mM dNTPs , 1.0 mM MgCl2 and 2.5 units of Ampli Taq Gold in 100 µL of the reaction mixture for 5 cycles at a temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1

<td>minute, and</td><td>74 ° C during</td><td>1 minute.</td><td>After</td><td>the addition</td><td>too much</td><td> 100</td>

<td>pmole of</td><td>initiators</td><td>exogenous</td><td>F3PrS</td><td>and F3PrA,</td><td>the PCR</td><td>was</td>

<td>made</td><td>for 25</td><td>cycles</td><td>with</td><td>the same</td><td>cycle</td><td>in</td>

temperatures.

DNA fragments amplified by the PCR method were separated by agarose gel electrophoresis using a 2% Nu Sieve GTG agarose (FMC BioProducts). Agarose strips containing about 424 bp DNA fragments were excised, to which 3 volumes (ml / g) of TE were added, and were then extracted with phenol, phenol / chloroform, and chloroform to purify the DNA fragments. DNA. After precipitation of ethanol-purified DNA, one third of the volume was dissolved in 14 µl of water. The obtained PCR reaction mixture was digested with Bali and Ncol and was introduced into plasmid hATR5Hva / CVIDEC (Bali / Ncol) prepared by digestion with Bali and Ncol, and the nucleotide sequence was determined.

Plasmids having the correct sequence were designated as hATR5Hvi / CVIDEC and hATR5Hvj / CVIDEC. The nucleotide sequence and corresponding version I humanized H chain amino acid sequence contained in plasmid hATR5Hvi / CVIDEC, and the version i amino acid sequence are shown in SEQ ID NO: 69 and 70. The nucleotide sequence and corresponding humanized H chain amino acid sequence version j contained in plasmid hATR5Hvj / CVIDEC, and the amino acid sequence of version j are shown in SEQ ID NO: 71 and 72.

(vii) Construction of humanized H chain versions bl and dl

The bl and dl versions were generated by replacing the FR2 of the bed versions with the FR2 derived from another human antibody using the FR shuffling method. To replace FR.2 with a derivative of human antibody P01742 (SWISS-PROT, Cunningham BA. Et al., Biochemistry, 9: 3161-3170, 1970), two DNA primers encoding FR2 were synthesized. The FR shuffle vector, F2MPS (SEQ ID NO: 73) has a sense DNA sequence and F2MPA (SEQ ID NO: 74) has an antisense DNA sequence. They also have a complementary sequence to each other, and have EcoT221 and Bali recognition sequences at both ends thereof.

F2MPS and F2MPA were synthesized and purified by Pharmacia Biotech. F2MPS and F2MPA were ligated and digested with BcoT221 and Bali. HATR5Hvb / CVIDEC (EcoT22I / Bali) and hATR5Hvd / CVIDEC (EcoT221 / Bali) plasmids were prepared by BcoT221 and Bali digestion, and the nucleotide sequence was determined. Plasmids having the correct sequence were designated as hATR5Hvbl / CVIDEC and hATR5Hvdl / CVIDEC. The nucleotide sequence and the corresponding humanized H chain amino acid sequence version 'bl1 contained in plasmid hATR5Hvbl / CVIDEC, the amino acid sequence of the version

76. The sequence bl is shown in SEQ ID NO: nucleotide and the corresponding humanized H chain amino acid sequence version dl contained in plasmid hATR5Hvdl / CVIDEC, and the amino acid sequence of version dl is shown in SEQ ID NO: 77 and 78.

(viii) Construction of humanized H chain versions b3 and d3

Versions b3 and d3 were generated by replacing FR2 from bed versions with FR2 derived from another human antibody using the FR shuffling method. To replace FR2 with a derivative of human antibody Z80844 (DDDJ, Thomsett AR. Et al., Unpublished), two DNA primers encoding FR2 were synthesized. The FR F2VHS shuffling vector (SEQ ID NO: 79) has a sense DNA sequence and F2VHA (SEQ ID NO: 80) has an antisense DNA sequence. They also have a complementary sequence to each other, and have BcoT221 and Bali recognition sequences at both ends of these. The synthesis and purification of F2VHS and F2VHA referred to Pharmacia Biotech.

F2VHS and F2VHA were ligated and digested with Ecol221 and Bali. HATR5Hvb / CVIDEC (Ecol221 / Bali) and hATR5Hvd / CVIDEC (Ecol221 / BalI) plasmids prepared by digestion with Ecol221 and Bali were introduced and the nucleotide sequence determined. Plasmids having the correct sequence were designated as hATR5Hvb3 / CVIDEC and hATR5Hvd3 / CVIDEC. The nucleotide sequence and corresponding version b3 humanized H chain amino acid sequence contained in plasmid hATR5Hvb3 / CVIDEC, and the version b3 amino acid sequence are shown in SEQ ID NO: 81 and 82. The nucleotide sequence and corresponding amino acid sequence of the humanized H chain version d3 contained in plasmid hATR5Hvd3 / CVIDEC, and the amino acid sequence of version d3 are shown in SEQ ID NO: 83 and 84.

(2) Construction of a humanized antibody L chain V region (i) Version a

The L-chain V region of the humanized ATR-5 antibody was generated by CDR grafting using the PCR method. For generation of an L chain of a humanized antibody (version a) having structural regions derived from the human antibody Z37332 (DDBJ, Welschof M. et al., J. Immunol. Methods, 179: 203-214, 1995), were Seven PCR primers were used.

The h5Lv1S (SEQ ID NO: 85) and h5Lv4S (SEQ ID NO: 86) CDR graft primers have a sense DNA sequence, the h5Lv2A (SEQ ID NO: 87), h5Lv3A (SEQ ID CDR) graft primers. NO: 88), and h5Lv5A (SEQ ID NO: 89) have an antisense DNA sequence, and each primer has complementary 20 bp sequences at both ends thereof. The exogenous primers h5LvS (SEQ ID NO: 90) and h5LvA (SEQ ID NO: 91) have homology to the h5LvlS and h5Lv5A CDR graft primers. The synthesis and purification of the CDR primers h5Lv1S, h5Lv4S, h5Lv2A, h5Lv3A, h5Lv5A, h5LvS, and h5LvA referred to Pharmacia Biotech.

PCR solutions contain, in 100 µl, 120 mM Tris-HCl (pH 8.0), 10 mM KCl (NH4)2ONLY4 6 mM, 0.1% Triton X-100, 0.001% BSA, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), MgCl2 1 mM, 2.5 units KOD DNA polymerase (Toyo Boseki), 50 pmole of the h5Lv1S, h5Lv2A, h5Lv3A, h5Lv4S, and h5Lv5A CDR graft primers.

PCR was performed using Thermal Cycler 480 DNA (Perkin-Elmer) for 5 cycles with the temperature cycle of 94 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute to assemble 5 primers. CDR graft. After the addition of an additional 100 pmole of h5LvS and h5LvA exogenous primers to the reaction mixture, PCR was performed for 30 cycles at 94 ° C for 30 seconds, 52 ° C for 1 minute, and 72 ° C for 1 minute to amplify the assembled DNA fragments.

The PCR reaction mixture was separated by agarose gel electrophoresis using a 3% NuSieve GTG agarose (FMC BioProducts), and the agarose strips containing about 400 bp long DNA fragments were excised. Agarose strips were extracted with phenol and chloroform, DNA fragments recovered by ethanol precipitation. The recovered DNA fragments were digested with restriction enzymes Spl1 (Takara Shuzo) and BglII (Takara Shuzo) at 37 ° C for 4 hours. The digestion mixture was extracted with phenol and chloroform, and after ethanol precipitation of the DNA fragments, they are dissolved in 10 µl TE. The SplI-BglII DNA fragment prepared as above encoding the humanized L-chain V region and the CVIDEC vector prepared by Spl1 and BglII digestion were ligated using DNA binding kit ver.2 (Takara Shuzo) to make them. react at 16 ° C for 1 hour according to the instructions attached to the kit.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated on LBA-agar medium and incubated overnight at 37 ° C to obtain an E. coli transformant. The transformant was grown overnight in 3 mL of LBA medium, and plasmid DNA was prepared from the cell fractions using the QIAprep Spin Plasmid Kit (QIAGEN).

corresponding

Shuzo), and the sequence was determined by confirming the nucleotide sequence in both directions. The plasmid containing the gene encoding the humanized antibody L-chain V region that has a BgIII recognition sequence and the 5 'end Kozak sequence, and a 3' end Spl1 recognition sequence has been designated A nucleotide sequence) humanized amino acid sequence) version a is shown in SEQ ID NO: 92. The amino acid sequence of version a is also shown in SEQ ID NO: 93.

(including chain (ii) versions b and c

The b and c versions were generated by replacing (FR shuffling) the FR a of version a. For version b, FR3 derived from human antibody S68699 (DDBJ, Hougs L. et al., Exp. Clin. Immunogenet., 10: 141151, 1993), and for version c, FR3 derived from human antibody was used. P01607 (SWISS-PROT, Epp O et al., Biochemistry, 14: 4943-4952, 1975), respectively.

Primers F3SS (SEQ ID NO: 94) and F3SA (SEQ ID NO: 95) encoding FR3 of version b, or primers F3RS (SEQ ID NO: 96) and F3RA (SEQ ID NO: 97) encoding FR3 of version c have a complementary sequence to each other, and have the restriction enzyme recognition sequences Kpnl and Pstl at both ends thereof. The synthesis and purification of F3SS, F3SA, F3RS, and F3RA referred to Pharmacia Biotech. 100 pmole of each of F3SS and F3SA, or F3RS and F3RA were ligated by treatment at 96 ° C for 2 minutes and at 50 ° C for 2 minutes and the double stranded DNA fragments were generated.

These double stranded DNA fragments were digested with restriction enzyme Kpnl (Takara Shuzo) at 37 ° C for 1 hour, and then with restriction enzyme Pstl (Takara Shuzo) at 37 ° C for 1 hour. The digestion mixture was extracted with phenol and chloroform, then precipitated with ethanol and dissolved in TE.

hATR5Lva / CVIDEC plasmid was digested with restriction enzyme Kpnl (Takara Shuzo) at 37 ° C for 1 hour, and then with restriction enzyme Pstl (Takara Shuzo) at 37 ° C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose strips having DNA fragments of about 3000 bp in length were excised. Agarose strips were extracted with phenol and chloroform, and after the DNA fragments were precipitated with ethanol they were dissolved in TE.

The Kpnl-PstI DNA fragment prepared as above encoding FR3 of hATR5Lva / CVIDEC versions b or ceo vector from which FR3 was removed by digestion with Kpnl and Pstl were ligated using DNA binding kit ver.2 (Takara Shuzo) by having them react at 16 ° C for 1 hour according to the instructions attached to the kit.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated on LBA-agar medium and incubated overnight at 37 ° C to obtain an E. coli transformant. 0 The transformant was grown overnight in 3 mL of LBA medium, and from the cell fractions plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN).

Plasmids containing the gene encoding version b or version c in which the L-chain FR3 of humanized antibody version a was replaced were designated as hATR5Lvb / CVIDEC or hATR5Lvc / CVIDEC, respectively. The nucleotide sequence and corresponding humanized L-chain amino acid sequence version b contained in plasmid hATR5Lvb / CVIDEC and the amino acid sequence of version b are shown in SEQ ID NO: 98 and 99. The nucleotide sequence and corresponding humanized L-chain amino acid sequence version c 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

Versions bl and b2 were generated by replacing FR2 with version b. For the bl version it was

100 FR2 derived from the human antibody S65921 (DDBJ,

Tonge DW et al., Year Immunol., Ί: 56-62, 1993), and for version b2 FR2 derived from human antibody X93625 (DDBJ, Cox JP et al., Eur. J. Immunol., 24) was used. :

827-836, 1994), respectively.

Primers F2SS (SEQ ID NO: 102) and F2SA (SEQ ID NO: 103) encoding FR2 of version bl, or primers F2XS (SEQ ID NO: 104) and F2XA (SEQ ID NO: 105) encoding FR2 of version b2 have a complementary sequence to each other, and have the restriction enzyme recognition sequences AflII and Spel at both ends thereof. F2SS, F2SA, F2XS, and F2XA were synthesized by Pharmacia Biotech. 100 pmole of each of F2SS and F2SA, or F2XS and F2XA were ligated by treatment at 96 ° C for 2 minutes and at 50 ° C for 2 minutes, and the double stranded DNA fragments were generated.

These double stranded DNA fragments were digested with restriction enzymes AflII (Takara Shuzo) and Spel (Takara Shuzo) at 37 ° C for 1 hour. The digestion mixture was extracted with phenol and chloroform, and after the DNA fragments were precipitated with ethanol, they were dissolved in TE.

The hATR5Lvb / CVIDEC plasmid was digested with restriction enzymes AflII (Takara Shuzo) and Spel (Takara Shuzo) at 37 ° C for 1 hour. The digestion mixture was separated by agarose gel electrophoresis using 1.5% NuSieve GTG agarose (FMC BioProducts), and the agarose strips having DNA fragments of about 3000 bp in length were excised. The agarose strip was extracted with phenol and chloroform, and after the DNA fragments were precipitated with ethanol, they were dissolved in TE.

101

The AflII-Spel DNA fragment prepared as above encoding the bl2 or b2 version FR2 and the hATR5Lvb / CVIDEC vector in which the FR2 was removed by digestion with AflII and Spel were ligated using the DNA binding kit ver.2 (Takara Shuzo) by having them react at 16 ° C for 1 hour according to the instructions attached to the kit.

The ligation mixture was added to 100 µl of competent E. coli JM109 (Nippongene) cells and incubated for 30 minutes on ice and 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then E. coli plated on LBA-agar medium and incubated overnight at 37 ° C to obtain an E. coli transformant. The transformant was grown overnight at 37 ° C in 4 mL of LBA medium, and from the cell fractions plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN).

Plasmids containing the gene encoding the bl or b2 version in which the L-chain FR2 of the humanized version b antibody was replaced were designated as

102 hATR5Lvbl / CVIDEC and hATR5Lv2 / CVIDEC, respectively. The nucleotide sequence and corresponding amino acid sequence of the humanized L-chain version bl contained in plasmid hATR5Lvbl / CVIDEC and the amino acid sequence of version bl are shown in SEQ ID NO: 106 and 107. The nucleotide sequence and corresponding amino acid sequence of humanized L-chain version b2 contained in plasmid hATR5Lvb2 / CVIDEC and the amino acid sequence of version b2 are shown in SEQ ID NO: 108 and 109.

(3) Construction of humanized antibody expression vector (i) Combination of humanized H chain with chimeric L chain

The hATR5Hva / CVIDEC plasmid containing an H chain V region was digested with Nhel and Sall, and a humanized H chain V region cDNA fragment was recovered and introduced into chATR5 / N5KG4P (Sall / Nhel) prepared by chATR5 digestion. / N5KG4P, a chATR-5 antibody expression plasmid vector, with Nhel and SalI. The plasmid thus generated was designated as hHva-chLv / N5KG4P.

The hATR5Hvb / 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 chATR5 / N5KG4P (Sall / Nhel) prepared by chATR5 digestion. / N5KG4P, a chATR-5 antibody expression plasmid vector, with Nhel and SalI. The plasmid thus generated was designated as hHvb-chLv / N5KG4P.

The hATR5Hvc / CVIDEC, hATR5Hvd / CVIDEC, and hATR5Hve / CVIDEC plasmids containing an H chain V region were

103 digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into chATR5 / N5KG4P (Sall / Nhel) prepared by chATR5 / N5KG4P digestion, a chATR-5 antibody expression plasmid vector , with Nhel and Sall. The plasmids thus generated were designated as hHvcchLv / N5KG4P, hHvd-chLv / N5KG4P, and hHve-chLv / N5KG4P.

HATR5Hvf / CVIDEC and hATR5Hvh / CVIDEC plasmids containing an H chain V region were digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into chATR5 / N5KG4P (Sall / Nhel) prepared by of chATR5 / N5KG4P digestion, a chATR-5 antibody expression plasmid vector, with Nhel and Sall. The plasmids thus generated were designated as hHvf-chLv / N5KG4P and hHvh-chLv / N5KG4P.

The HATR5Hvi / CVIDEC and hATR5Hvj / CVIDEC plasmids containing the H chain V region were digested with Nhel and Sall, and the humanized H chain V region cDNA fragments were recovered and introduced into chATR5 / N5KG4P (Sall / Nhel) prepared by of chATR5 / N5KG4P digestion, a chATR-5 antibody expression plasmid vector, with Nhel and Sall. The plasmids thus generated were designated as hHvi-chLv / N5KG4P and hHvj-chLv / N5KG4P.

The hATR5Hbl / CVIDEC and hATR5Hvdl / CVIDEC plasmids containing an H chain V region were digested with Nhel and Sall, and the humanized H chain V region cDNA fragments were recovered and introduced into chATR5 / N5KG4P (Sall / Nhel) prepared by chATR5 / N5KG4P digestion, a chATR-5 antibody expression plasmid vector with

104

Nhel and Sall. The plasmids thus generated were designated as hHvbl-chLv / N5KG4P and hHvdl-chLv / N5KG4P.

(ii) Combination of the humanized L chain with a chimeric H chain

Using an N5KG4P antibody expression vector, it 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 restriction enzymes BglII (Takara Shuzo) and Spll (Takara Shuzo) at 37 ° C for 2-3 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 about 400 bp long DNA fragments were excised. The agarose strips were extracted with phenol and chloroform, and after the DNA fragments were precipitated with ethanol, they were dissolved in TE.

The SplI-BglII DNA fragment containing the gene encoding the humanized L-chain V region of each of these versions and the Spl1 and BglII digested hATR5Hv / N5KG4P were ligated using the ver.2 DNA binding kit (Takara Shuzo) making React them at 16 ° C for 1 hour according to the instructions attached to the kit.

The ligation mixture was added to 100 µl of competent E. coil JM109 (Nippongene) cells and incubated for 30 minutes on ice and for 1 minute at 42 ° C. Then 300 Âµl of Hi-Competence Broth (Nippongene) was added, incubated at 37Â ° C for 1 hour, and then

105

E. coli was plated on LBA agar medium and incubated overnight at 37 ° C to obtain an E. coli transformant.

The transformant was cultured overnight at 37 ° C in 250 mL or 500 mL of LBA medium and from plasmid fractions plasmid DNA was prepared using the Plasmid Maxi Kit (QIAGEN). Plasmids into which a gene encoding the chimeric H chain and the humanized L chain were introduced are chHv-hLvb / N5KG4P, chHv-hLvc / N5KG4P, chHv-hLvbl / N5KG4P, and chbv / hv N5KG4P.

(iii) Combination of humanized H chain with humanized L chain

The hATR5Hva / CVIDEC plasmid containing an H chain V region was digested with Nhel and Sall, and a humanized H chain V region cDNA fragment was recovered and introduced into hLva / N5KG4P (Sall / Nhel) prepared by plasmid digestion. chHv-hLva / N5KG4P containing the L chain cDNA sequence of the humanized antibody ATR-5 version a with Nhel and SalI. The plasmid thus generated was designated as hHva-hLva / N5KG4P.

HATR5Hvb / CVIDEC and hATR5Hvc / CVIDEC plasmids containing an H chain V region were digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into hLva / N5KG4P (Sall / Nhel) prepared by chHv-hLva / N5KG4P digestion plasmid containing the L-strand cDNA sequence of the humanized antibody ATR-5 version a with Nhel and SalI. The plasmids thus generated were designated as hHvb-hLva / N5KG4P and hHvc-hLva / N5KG4P.

106

HATR5Hvb / CVIDEC, hATR5Hvd / CVIDEC, and hATR5Hve / CVIDEC plasmids containing an H chain V region were digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall (Nhel) prepared by plasmid digestion chHv-hLvb / N5KG4P containing the L chain cDNA sequence of the humanized antibody ATR-5 version b with Nhel and SalI. The plasmids thus generated were designated as hHvb-hLvb / N5KG4P, hHvd-hLvb / N5KG4P, and hHvehLvb / N5KG4P.

HATR5Hvf / CVIDEC, hATR5Hvg / CVIDEC, and hATR5Hvh / CVIDEC plasmids containing the H chain V region were digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall (Nhel) prepared by digesting plasmid chHv-hLvb / N5KG4P containing the L-strand cDNA sequence of the humanized antibody ATR-5 version b with Nhel and Sall. The plasmids thus generated 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 Sall, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by chHv-hLvb / N5KG4P digestion plasmid containing the humanized antibody ATR-5 version b L chain cDNA sequence with Nhel and SalI. The plasmids thus generated were designated as hHvi-hLvb / N5KG4P and hHvj-hLvb / N5KG4P.

107

The hATR5Hvbl / CVIDEC and hATR5Hvdl / CVIDEC plasmids containing an H chain V region were digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by chHv-hLvb / N5KG4P digestion plasmid containing the humanized antibody ATR-5 version b L chain cDNA sequence with Nhel and SalI. The plasmids thus generated were designated as hHvbl-hLvb / N5KG4P and hHvdl-hLvb / N5KG4P.

The hATR5Hvb3 / CVIDEC and hATR5Hvd3 / CVIDEC plasmids containing an H chain V region were digested with Nhel and Sall, and humanized H chain V region cDNA fragments were recovered and introduced into hLvb / N5KG4P (Sall / Nhel) prepared by chHv-hLvb / N5KG4P digestion plasmid containing the humanized antibody ATR-5 version b L chain cDNA sequence with Nhel and SalI. The plasmids thus generated were designated as hHvb3-hLvb / N5KG4P and hHvd3-hLvb / N5KG4P.

The hATR5Hvb / CVIDEC plasmid containing an H chain V region was digested with Nhel and Sall, and a humanized H chain V region cDNA fragment was recovered and inserted into hLvbl / N5KG4P (Sall / Nhel) and hLvb2 / N5KG4P (Sall (Nhel) prepared by digesting the chHv-hLvbl / N5KG4P and chHv-hLvb2 / N5KG4P plasmids containing the humanized antibody ATR-5 versions bl and b2 cDNA sequence with Nhel and Sall. The plasmids thus generated were designated as hHvb-hLvbl / N5KG4P and hHvbhLvbl / N5KG4P.

The hATR5Hvi / CVIDEC plasmid containing an H chain V region was digested with Nhel and SalI, and a cDNA fragment from

108 Humanized H-chain V-region was recovered and introduced into hLvbl / N5KG4P (Sall / Nhel) and hLvb2 / N5KG4P (Sall / Nhel) prepared by digesting chHv-hLvbl / N5KG4P plasmids and chHv-hLvb2 / N5KG4P sequence containing of the L chain of the humanized antibody ATR-5 versions bl and b2 with Nhel and Sall. The plasmids thus generated were designated as hHvi-hLvbl / N5KG4P and hHvihLvb2 / N5KG4P.

(4) COS-7 cell transfection

To evaluate antigen binding activity and neutralizing activity of the humanized antibody, the above antibody was transiently expressed in COS-7 cells.

The constructed expression plasmid vector was transduced to COS-7 cells by electroporation using the Gene Pulser instrument (Bio Rad). 50 pg or 20 pg of plasmid was added to 0.78 mL of CBS-7 cells suspended in PBS at a cell concentration of 1χ107 cells / mL, which were subjected to 1,500 V and 25 pF capacitance pulses.

After 10 minutes of the recovery period at room temperature, the electroporated cells were suspended in a DMEM medium (GIBCO) containing 5% IgG ultra-low fetal bovine serum (GIBCO), and cultured using a 10 cm culture box or a 15 cm culture box in a CO incubator2 at 5%. After culturing for 24 hours, the culture supernatant was aspirated, and then serum-free HBCHO medium (Irvine Scientific) was added. After culturing for a further 72 hours or 96 hours, the culture supernatant was harvested and centrifuged to remove cell debris.

109 (5) Antibody Purification

From the COS-7 cell culture supernatant, the antibody was purified using the MAPSII AffiGel Protein A kit (Bio Rad) or rProtein A Sepharose Fast Flow (Pharmacia Biotech). Purification using AffiGel Protein MAPSII kit was performed according to the instructions attached to the kit. Purification using rProtein A Sepharose Fast Flow was performed as follows:

ml of rProtein 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 TBS. The adsorbed antibody fraction was eluted through 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 ultrafiltration two or three times to the purified antibody fraction using Centriprep 30 or 100 (amicon), the solvent was replaced with TBS, and was finally concentrated to about 1.5 mL.

Example 4. Antibody Quantification and Activity Evaluation (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 prepared goat anti-human IgGY antibody (BIO SOURCE) to a concentration of 1 pg / mL in the immobilization buffer (0.1 M NaHCO3, 0.02% NaN3, pH 9.6)

110 (hereinafter referred to as CB). After blocking with 200 µL of dilution buffer (50 mM Tris-HCl, MgCl2 1 mM, 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN3, 1% Bovine Serum Albumin, pH 8.1) (hereinafter referred to as DB), the supernatant COS-7 cell culture in which antibody was expressed or purified antibody was serially diluted with DB, and then added to each well.

After incubation at room temperature for 1 hour followed by washing with Dulbecco's PBS containing 0.05% Tween 20 (hereinafter referred to as RB), 100 µl of alkaline phosphatase-conjugated goat anti-human IgGY antibody was added ( Biosource) which was diluted 1000 times with DB. After incubation at room temperature for 1 hour followed by washing with RB, Sigmal04 (p-nitrophenyl phosphate, SIGMA) dissolved in substrate buffer (50 mM NaHCO3, MgCl) was added.2 10 mM, pH 9.8) to 1 mg / ml, and then the absorbance at 405/655 nm was measured using the Microplate Reader (Bio Rad). As a standard for concentration measurement, IgG4K (Binding Site) was used.

(2) Measurement of antigen binding activity

Antigen binding cell ELISA plates were prepared as follows. The cells used were J82 human bladder carcinoma cells (ATCC HTB-1). In 60 wells of a 96-well cell culture plate, 1x102 * * 5 J82 cells. These were grown (RPMI1640 medium containing 10% fetal bovine serum (GIBCO)) for one day in a CO incubator.2 to allow cells to adhere. After discarding the culture liquid, each well was washed twice with 300 µl PBS. 100 µl PBS containing

111 4% paraformaldehyde (hereinafter referred to as PFA / PBS), and placed on ice for 10 minutes to immobilize the cells.

PFA / PBS was discarded, and each well was washed twice with 300 µl PBS, and then blocked with 250 µl DB. Culture supernatant or purified antibody was serially diluted with DB, 100 µl of which was added to each well. After incubation at room temperature for 2 hours followed by RB washing, 100 Âµl of alkaline phosphatase-conjugated goat anti-human IgGY antibody (BioSource) diluted 1000-fold with DB was added. After incubation for 1 hour followed by RB washing, the substrate solution was added, and then the absorbance at 405/655 nm was measured using the Microplate Reader (Bio-Rad).

(3) Measurement of neutralizing activity

Neutralizing activity of mouse antibody, chimeric antibody, and humanized antibody was measured with inhibitory activity against Factor Xa production activity by human placental-derived thromboplastin, Thromborel S (Boehringer AG), as an index. Thus, 60 pL of buffer (TBS containing 5 mM CaCl2 and 0.1% BSA) was added to 10 pL of 1.25 mg / mL Thromborel S and 10 pL of appropriately diluted antibody, which was then incubated in a 96 wells at room temperature for 1 hour. 10 µl each of 3.245 pg / ml human Factor X (Celsus Laboratories) and 82.5 ng / ml human Factor Vila (Enzyme Research) were each added, and then incubated at room temperature for 1 hour.

112

10 µl of 0.5 M EDTA was added to stop the reaction, to which 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 reaction at room temperature for 1 hour, the absorbance at 405/655 nm was again determined. Neutralizing activity can be determined by calculating residual activity (%) from each change in absorbance with the hourly change in absorbance in the absence of antibody addition as 100% activity.

The chromogenic substrate solution was prepared by dissolving the Testzyme S-2222 chromogenic substrate (Chromogenix) according to the accompanying instructions, diluting 2 times with purified water and mixing with a polybrene solution (0.6 mg / mL bromide). hexadimethylene, SIGMA) at 1: 1.

(4) Activity evaluation (i) Combination of humanized H-chain version A with a chimeric L-chain

An antibody (α-ch) is generated which is the humanized H chain version a combined with a chimeric L chain which was tested for antigen binding activity by cell ELISA. The amount of antigen bound was found to decrease at the high concentration (Figure 1). Neutralizing activity against antigen through inhibition of FXa production was poor compared to that of the positive control chimeric antibody (ch-ch) (Figure 2). Therefore, it was decided to perform the higher version of the humanized H chain through FR shuffling. O

113 Chimeric antibody used herein was that expressed on COS-7 cells, purified and evaluated.

(ii) Combination of humanized version A chain with a chimeric H chain

An antibody (ch-a) which is the humanized version A-chain combined with a chimeric H-chain that was tested for antigen binding activity by cell ELISA was generated. It has been found to have binding activity equal to or greater than that of the chimeric antibody (Figure 1). On the other hand, antigen neutralizing activity was poor compared to that of the positive control chimeric antibody (Figure 2). Therefore, it was decided to perform the upper version of the humanized L chain through FR shuffling. The chimeric antibody used herein was that expressed in purified COS-7 cells and evaluated.

(iii) Combination of humanized H-chain version A with humanized L-chain version A

An antibody (aa) which is humanized H-chain version A was combined with humanized L-chain version which was tested for antigen binding activity by cellular ELISA. The antigen-bound amount was found to decrease on the high concentration side (Figure 3). Neutralizing activity against antigen by inhibiting FXa production was poor compared to that of the positive control chimeric antibody (Figure 4). Therefore, it was decided to perform the higher version of the humanized H chain and the L chain through FR shuffling. The chimeric antibody used herein was that expressed in purified COS-7 cells and evaluated.

114 (iv) Combination of humanized H chain versions b, c, ed with a chimeric L chain

Antibodies (b-ch, c-ch, and d-ch, respectively) that are humanized H chain modified to higher version were generated by RF shuffling combined with a chimeric L chain, and were tested for binding activity. to antigen by cell ELISA, d-ch exhibited binding activity equal to that of the chimeric antibody, and b-ch and c-ch exhibited slightly lower binding activity (Figures 5 and 6). On the other hand, antigen neutralizing activity compared to that of the positive control chimeric antibody was almost equal in b-ch, and slightly weak in d-ch. In the c-ch version, it was significantly weaker than that of the chimeric antibody (Figure 7). Therefore, humanized H chains and b versions were considered to be those of the humanized H chain that exhibited high activity.

(v) Combination of humanized H chain version b with humanized L chain version a

An antibody (ba) which is humanized H-chain version b modified to higher version was generated by FR shuffling combined with humanized L-chain version a, and was tested for antigen binding activity by cell ELISA. . The antigen bound amount was found to decrease at the high concentration (Figure 5). On the other hand, antigen neutralizing activity was significantly weak compared to that of the positive control chimeric antibody (Figure 8). Therefore, ba and aa were those which exhibited high activity. 0 antibody

115 The chimeric used herein was that expressed in COS-7 cells, purified, and evaluated.

(vi) Combination of humanized L chain versions b and c with a chimeric H chain

Antibodies (ch-b and ch-c, respectively) which are the b and c versions of the humanized L chain combined with a chimeric H chain were wanted, and both were found to have antigen binding activity and neutralizing activity against the same antigen as the chimeric antibody (Figures 9 and 10). Therefore, versions b and c were chosen as candidates for a humanized antibody L chain. Mouse antibody-derived version b which has one amino acid less in the number of amino acid residues is considered higher than version c in terms of antigenicity. The chimeric antibody used herein was that expressed in purified CHO DG44 cells and evaluated. In the evaluation below the antibody was used as a positive control.

(vii) Combination of humanized H chain version b with humanized L chain version b and c

Antibodies (bb and bc, respectively) which are humanized H-chain version b combined with humanized L-chain versions b and c were generated, and were tested for antigen binding activity and antigen neutralizing activity. Both had slightly lower activity than the chimeric antibody in both binding and neutralizing activity (Figures 11 and 12).

(viii) Combination of humanized H chain versions b with humanized L chain bed version

116

Antibodies (bb and db, respectively) which are humanized H chain modified to higher version were generated by FR shuffling combined with humanized L chain version b, and were tested for antigen binding activity by cell ELISA. db exhibited binding activity equal to that of the chimeric antibody, and bb exhibited slightly lower binding activity at high concentration (Figure 13). On the other hand, antigen neutralizing activity compared to that of the positive control chimeric antibody was slightly low in bb, and significantly weak in db (Figure 14). Therefore, bb has been shown to be a high neutralizing activity version, while db is a high binding activity version.

(ix) Combination of humanized version H chain with a chimeric L chain and humanized version B chain

Antibodies (e-ch and eb, respectively) were generated which are the humanized L-chain version and combined with a chimeric L-chain and humanized version b, e-ch exhibited an antigen binding activity equal to that of the chimeric antibody, but in and b the amount of antibody expressed was very low and most of the binding activity was lost (Figure 15). The neutralizing activity against the e-ch antigen was significantly low compared to that of the chimeric antibody (Figure 16). Therefore, it was concluded that chain H version and combined with chain L version b did not work well.

(x) Combination of humanized H chain versions f, g and h with humanized L chain version b

117

Antibodies (fb, gb, and hb, respectively) were generated which are the humanized H chain versions f, g, and h combined with the humanized L chain version b. In antibody fb and hb, the amount of antibody expressed was very small. For feh versions, antibodies combined with the chimeric L chain were generated but not expressed. gb reached saturation at a low concentration, and exhibited weaker binding activity than that of the chimeric antibody (Figure 17). The neutralizing activity against gb antigen was significantly weak compared to that of the chimeric antibody (Figure 18).

(xi) Combination of humanized H chain versions bl and dl with humanized L chain version b

Antibodies (bl-b and dl-b, respectively) were generated which are humanized H-chain versions bl and dl combined with humanized L-chain version b. Almost no antibody was expressed in any of them. For these, antibodies combined with a chimeric L chain were generated but not expressed.

(xii) Combination of humanized chain H versions b3 and d3 with humanized chain L version b

Antibodies (b3-b and d3-b, respectively) were generated which are humanized H-chain versions b3 and d3 combined with humanized L-chain version b. The antigen binding activity of d3-b was slightly lower than that of the chimeric antibody, and that of b3-b was much lower (Figure 19). The neutralizing activity against b3-b antigen was higher than bb, but was lower than that of the chimeric antibody, and d3-b and bb remained the same in activity (Figure 20).

118 (xiii) Combination of humanized H chain versions i and j with a chimeric L chain and humanized version B chain

Antibodies (i-ch and j-ch, respectively) that are humanized H-chain versions i and j combined with a chimeric L-chain, and antibodies (ib and jb, respectively) generated with humanized L-chain version b, were generated and tested. for antigen binding activity and antigen neutralizing activity. The binding activity of either antibody was almost equal to that of the chimeric antibody (Figures 21 and 22). i-ch exhibited greater neutralizing activity than the chimeric antibody, and that of jch was significantly lower than that of the chimeric antibody (Figure 23). ib exhibited a neutralizing activity equal to that of the chimeric antibody, and jb exhibited significantly weaker neutralizing activity than that of the chimeric antibody (Figure 24).

(xiv) The humanized L chain versions bl and b2

When antibodies (ch-bl and ch-b2, respectively) that are humanized L-chain versions bl and b2 combined with a chimeric H-chain were generated, both exhibited an antigen binding activity equal to that of the chimeric antibody (Figure 25). For antigen neutralizing activity, ch-bl exhibited a binding activity equal to that of the chimeric antibody, while ch-b2 exhibited slightly higher activity than the chimeric antibody at high concentration (Figure 26). Versions b1 and b2 may be candidates for a humanized antibody L chain, but b2 is superior in that it has stronger activity.

119 (xv) Combination of humanized H chain version b with humanized L chain version b2

An antibody (b-b2) which is humanized H-chain version b combined with humanized L-chain version b2 was generated and tested for antigen binding activity and antigen neutralizing activity. Binding activity was slightly lower than that of the chimeric antibody (Figure 27). Neutralizing activity, although slightly higher than bb, was lower than ib (Figure 28).

(xvi) Combination of humanized H chain version i with humanized L chain version bl or b2

Antibodies (i-bl and i-b2, respectively) were generated which are humanized H-chain version i combined with humanized L-chain version bl or b2, and were tested for antigen binding activity and neutralizing activity against antigen. The binding activity of i-b2 was almost equal to that of the chimeric antibody, and that of i-b1 was slightly lower than that of the chimeric antibody (Figure 29). The neutralizing activity of ibl and i-b2 was higher than that of the chimeric antibody and ib, which was in a decreasing order of i-b2> i-bl (Figure 30).

Example 5. Preparation of humanized antibody produced by CHO cells and evaluation of their activity (1) Establishment of a stably producing CHO cell line

To establish cell lines that stably produce a humanized antibody (bb, ib, and i-b2),

120 an antibody expression gene vector in CHO cells (DG44) acclimatized to a serum free medium.

Plasmid hHvb-hLvb / N5KG4P, hHvi-hLvb / N5KG4P and hHvi-hLvb2 / N5KG4P DNAs were digested with the restriction enzyme SspI (Takara Shuzo) and linearized, extracted with phenol and chloroform, and purified by precipitation and ethanol in precipitation. . The linearized expression gene vector was introduced into DG44 cells using the electroporation instrument (Gene Pulser; Bio Rad). DG44 cells were suspended in PBS at a cell concentration of 1x107 cells / ml, and about 0.8 ml of this suspension was added 10 or 50 pg of DNA, which was subjected to 1,500 V pulses and 25 pF capacitance.

After 10 minutes of the recovery period at room temperature, the treated cells were suspended in a CHOS-SFMII (GIBCO) medium containing hypoxanthine / thymidine (GIBCO) (hereinafter referred to as HT), which was inoculated into two 96-well plates ( Falcon) at 100 µl / well, and grown in a CO2 incubator. Eight to nine hours after the start of culture, 100 µl / well of CHO-S-SFMII medium containing HT and 1 mg / ml of GENETICIN (GIBCO) were added to change to 500 pg / mL GENETICIN selection medium, and The cells into which the antibody gene had been introduced were selected. The medium was changed fresh once every 3-4 days with 1/2 volume. At a time about 2 weeks after switching to the selection medium, an aliquot of the culture supernatant was recovered from the well in which 4-5 days later favorable cell growth was observed. The concentration of antibody expressed in the culture supernatant was measured by the ELISA described above to measure the concentration of

121 antibody, and cells having a high yield of antibody production were selected.

(2) Large-scale purification of humanized antibody

After the selected DG44 cell lines as above which produce the humanized antibody (bb, ib, and ib2) are cultured for a few days in 500 mL / bottle of CHO-S-SFMII medium using a 2 L rolling bottle (CORNING), The culture medium was harvested and fresh CHO-S-SFMII medium was added and cultivated again. The culture medium was centrifuged to remove cell debris, and filtered with a 0.22 pm or 0.45 pm filter. Repeating this yielded a total of about 2 L of each culture supernatant. From the obtained culture supernatant, the antibody was purified by ConSep LC100 system (Millipore) attached to a Protein A (Poros) affinity column.

(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 goat anti-human IgGY antibody (BioSource) prepared at 1 pg / mL concentration with CB. After blocking with 200 µl DB, the culture supernatant of CHO cells in which the antibody had been expressed or purified antibody was serially diluted with DB, and added to each well.

After incubation at room temperature for 1 hour and washing with RB, 100 µl of the alkaline phosphatase-conjugated goat anti-human IgGY antibody (BioSource) diluted 1000 times with DB was added. After incubation at

122 At room temperature for 1 hour and RB wash, 100 µl of substrate solution was added, and then the absorbance at 405/655 nm was measured using the Microplate Reader (Bio Rad). As a standard for concentration measurement, human IgG4K (The Binding Site) was used.

(4) Measurement of antigen binding activity

Antigen binding cell ELISA plates were prepared as follows. The cells used were J82 human bladder carcinoma cells (ATCC HTB-1), which were inoculated into a 96-well cell culture plate at a cell count of 1χ105 cells These were grown (RPMI1640 medium containing 10% fetal bovine serum (GIBCO)) for one day in a CO2 incubator to allow cells to adhere. After discarding the culture liquid, each well was washed twice with PBS. 100 µl 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 PBS and then blocked with 250 µl DB. Based on the above measurement result, the purified antibody was serially diluted with DB starting at 10 pg / ml in a factor of 2.100 pL from which they were added to each well. After incubation at room temperature for 2 hours and washing with RB, 100 µl of alkaline phosphatase-conjugated goat anti-human IgGY antibody (BioSource) diluted 1000 times with DB was added. After incubation at room temperature for 1 hour and washing with RB, 100 µl of substrate solution was added, and then

123 Absorbance at 405/655 nm was measured using the Microplate Reader (Bio-Rad).

(5) Measurement of neutralizing activity against TF (Factor Inhibiting Activity against FXa production)

Factor Xa production inhibiting activity of the humanized antibody was measured with inhibition activity against Factor Xa production activity by human placenta-derived thromboplastin, Thromborel S (Boehringer AG) as Index. Thus, 60 pL of the buffer (TBS containing 5 mM CaCl2 and 0.1% BSA) was added to 10 pL of 5 mg / ml Thromborel S and 10 pL of the antibody, which was then incubated in a 96-well plate at room temperature. room for 1 hour. The antibody was serially diluted with the buffer starting at 200 pg / ml by a factor of 5.

10 µl each of 3.245 pg / ml human Factor X (Celsus Laboratories) and 82.5 ng / ml human Factor Vila (Enzyme Research) were added each, 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 solution was added and the absorbance at 405/655 nm was determined by Microplate Reader (Bio Rad). After reaction at room temperature for 30 minutes, the absorbance at 405/655 nm was measured again. Residual activity (%) was determined from each change in absorbance with the change in absorbance for 30 minutes in the absence of antibody addition as 100% activity.

The chromogenic substrate solution was prepared by dissolving the Testzyme S-2222 (Chromogenix) chromogenic substrate according to the attached instructions, and

124 mixing with a 1: 1 polybrene solution (0.6 mg / mL hexadimethylene bromide, SIGMA).

(6) Measurement of neutralizing activity against TF (inhibition activity against FX binding)

Inhibitory activity against FX binding of the humanized antibody was measured using human placenta-derived thromboplastin, Thromborel S (Boehringer AG), in which a TF and Factor Vila complex had been previously formed and inhibitory activity against binding. FX was measured with the production activity of TF-FVlla complex Factor Xa as an index. Thus, 60 pL of buffer (TBS containing 5 mM CaCl2 and 0.1% BSA) was added to 10 pL of 5 mg / mL Thromborel S and 10 pL of 82.5 ng / mL human Factor Vila (Enzyme Research ), which was preincubated in a 96-well plate 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 pg / ml human Factor X (Celsus Laboratories) added and further incubated at room temperature for 45 minutes. . The antibody was serially diluted with the buffer starting at 200 pg / ml by a factor of 2. 10 µl of 0.5 M EDTA was added to stop the reaction. 50 µl of the chromogenic substrate solution was added and the absorbance at 405/655 nm was determined by Microplate Reader (Bio Rad). After reacting at room temperature for 30 minutes, the absorbance at 405/655 nm was measured again. Residual activity (%) was determined from each change in absorbance with the change in absorbance for 30 minutes in the absence of antibody addition as 100% activity.

125

The chromogenic substrate solution was prepared by dissolving the Testzyme S-2222 chromogenic substrate (Chromogenix) according to the attached instructions, and mixing with a polybrene solution (0.6 mg / mL hexadimethylene bromide, SIGMA) at 1 ° C. :1.

(7) Measurement of neutralizing activity against inhibition activity against (plasma coagulation)

The neutralizing activity against TF (plasma coagulation inhibitory activity) of the humanized antibody was measured using, as an index, the prothrombin time determined using the human placenta-derived thromboplastin, Thromborel S (Boehringer AG). Thus, 100 µl of human plasma (Cosmo Bio) was placed in a sample beaker, to which 50 µl of diluted antibody was added at various concentrations, and heated at 37 ° C for 3 minutes. 50 µl of 1.25 mg / ml Thromborel S was added and preheated to 37 ° C to initiate plasma coagulation. Coagulation time was measured using Amelung KC-10A attached to Amelung CR-A (both from MC Medical).

Antibody was serially diluted with TBS containing 0.1% BSA (hereinafter referred to as BSA-TBS) starting at 80 pg / mL by a factor of 2. With no antibody addition coagulation time as 100% of TF plasma clotting activity, residual TF activity was calculated from each clotting time on antibody addition based on a standard curve obtained by plotting Thromborel S concentration and clotting time.

126

The standard curve was created from the various Thromborel S concentrations and the clotting time was measured. 50 µl BSA-TBS was added to 50 µl of appropriately diluted Thromborel S, which was heated at 37 ° C for 3 minutes, 100 µl of preheated human plasma at 37 ° C was added to initiate coagulation, and coagulation time. It was determined. Thromborel S was serially diluted with Hank's buffer (GIBCO) containing 25 mM CaCl 2 starting at 6.25 mg / mL in a factor of 2. Thromborel S concentration was represented on the abscissa, and clotting time was ordered on log-log paper, which gave a standard curve.

(8) Activity assessment

All humanized antibodies bb, ib, and i-b2 had an activity equal to or greater than that of the chimeric antibody (Figure 31). For inhibition activity against FXa production, as well as FX binding inhibition activity, and inhibition activity against plasma coagulation, humanized antibodies, bb, ib, and i-b2 had an equal or greater activity. that of the chimeric antibody, and activity was of a decreasing order i-b2> ib> bb (Figures 32, 33, and 34).

Example 6. Kinetic Analysis of TF and Anti-TF Antibody Interaction Using BIACORE

Kinetic analysis of the antigen-antibody reaction was performed using BIACORE. Recombinant G protein was immobilized on a sensor chip to which the antibody was bound. Purified recombinant TF (a soluble TF in which the FLAG peptide was labeled at 1-219) was used as antigen, and soluble TF prepared at various concentrations was used for analysis. From

127 For the obtained sensogram, the kinetic parameters (dissociation rate constants kdiss, and binding rate constants kass) were calculated. For kinetic analysis reference was 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) Protein G immobilization on sensor chip

Protein G (ZYMED) is immobilized on the CM5 sensor chip (BIACORE).

The running buffer was HBS-EP buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% (v / v) polysorbate) (BIACORE), and flow rate was 5 pL / min. Carboxymethylxtran carboxyl groups on the CM5 sensor chip were activated by injection of 0.05 M N-hydroxysuccinimide (NHS) / N-ethyl N '(3-dimethylaminopropyl) carbodiimide hydrochloride (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 pg / mL with 10 mM sodium acetate buffer (pH 4.0). .

An additional 100 µl of 1.0 M ethanolamine hydrochloride (pH 8.5) was injected to block excess active groups. For this purpose 10 µl 0.1 M glycine-hydrochloric acid buffer (pH 2.5) and 10 µl 10 mM hydrochloric acid buffer were injected to wash covalently unbound substances. Doing this for each flow cell and injecting 10

128 pL of 72 nM version ib2 humanized anti-TF antibody, about 1000 RU was confirmed to bind.

(2) Interaction of immobilized anti-TF antibody and human TF

Human TF to which the FLAG peptide had been attached to the C-terminus of amino acid sequence 1-219 was expressed in CHO cells and was purified. This was used as soluble TF.

As running buffer, HBS-EP buffer was used. 10 pL of 72 nM antibody solution was injected at a flow rate of 20 pL / min to immobilize the antibody. The antibody was diluted with HBS-EP buffer. 40 µl of the soluble human TF solution was injected there at various concentrations at a flow rate of 30 µl / min. In the analysis, 80 seconds were set for injection as the binding phase and then switched to HBS-EP buffer to establish the 120 second dissociation phase. After the dissociation phase was completed, 10 µl of 20 mM hydrochloric acid was injected to reconstitute the sensor chip. Binding, dissociation, and reconstitution were established as one cycle, and for each antibody the sensogram was obtained. Soluble human TF solution was prepared at 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 blank the sensogram obtained by injecting the HBS-EP buffer used for dilution was used.

The above procedure was performed for each flow cell No. 1 to 3.

(3) Kinetic analysis of interaction

129 The data file of interest was read, and the comparison of reaction patterns by inscription was done using the HBS-EP buffer sensogram as a threshold. In addition, using a BIAevaluation 2.1 (Pharmacia) analytical application software exclusively for BIACORE which calculates the kinetic parameters (kass link rate constants and kdiss dissociation rate constants) by curve fitting, kinetics analysis was performed. of interaction. To determine the kass link rate constants, the type 4 analysis model (BIAevaluation 2.1 Software Hand book, A1-A5) was used. Based on the values calculated from each flow cell, the kinetic parameters for each antibody were obtained. The result (average of values calculated from each flow cell + standard deviation) is shown in Table 6.

Table 6

<td colspan="5">Kinetic parameters of chimeric and humanized anti-TF antibody (n = 3)</td>

<td></td><td>chimeric</td><td>bb</td><td>ib</td><td>i-b2</td>

<td>kdiss [x10 4 1 / s]</td><td> 5,06 + 0,12</td><td> 9,52+0,22</td><td> 6,49+0,17</td><td> 6,35 +0,15</td>

Example 7. Measurement of reactivity of humanized anti-TF antibody to human TF

Using the dot-blot hybridization method (Protein Experimental Method for Molecular Biological Research, Revised, Yodosha, edited by Takenawa Tadaomi, p. 101), reactivity to undenatured TF, denatured TF under non-reducing conditions, and denatured TF was investigated. under reducing conditions. TF was used in which the FLAG had been directed to the extracellular region, expressed in CHO cells and purified (shTF). ShTF was diluted with

130 each of the three buffers (buffer A: 10 mM Tris-HCl, pH 8.0; buffer B: 10 mM Tris-HCl (pH 8.0), 8 M urea; buffer C: 10 mM Tris-HCl, pH 8 .0, 8 M urea, 5 mM DTT). Undenatured TF was treated with buffer A, while non-denatured TF was treated with buffer B, and denatured TF was treated with buffer C. Each sample was treated for 24 hours at room temperature. After treatment, the sample was transferred to a nitrocellulose membrane (Bio Rad). 0.5 pL, 1 pL, and 2 pL of the sample (3 pg / mL) were transferred to the membrane, and the membrane was air dried. It was blocked with DB (50 mM Tris-HCl, pH 8.1, 0.15 M NaCl, 1 mM MgCl 2, 0.05% (v / v) Tween 20, NaN3 0.02% (w / v), 1% (w / v) BSA). The membrane was reacted in the DB containing the humanized anti-TF antibody or the DB (control). After washing with PBS containing 0.05% (v / v) Tween 20, it reacted with DB containing peroxidase labeled anti-human IgG (DAKO) antibody. After washing with PBS containing 0.05% (v / v) Tween 20, it was treated by ECL Western Blotting reagent (Amersham), and was exposed to an X-ray film for 30 seconds.

<td>As shown in Figure 35,</td><td>the antibody</td><td>anti-TF</td>

<td>chimeric and anti-TF antibodies</td><td>humanized</td><td>(versions</td>

<td colspan="2">bb, ib, and ib2) reacted with everyone from</td><td>TF no</td>

<td colspan="2">denatured, TF not reducibly denatured,</td><td>and TF</td>

<td>denatured reductively.</td><td></td><td></td>

<td>Example 8. Effect Confirmation</td><td colspan="2">antithrombotic drugs in</td>

acute IHD mouse models - for information only

The antithrombotic effects of the anti-TF antibody were confirmed in a thromboplastin-induced IHD model using mice. Thus, a human thromboplastin solution was continuously injected into the vein of SD rats.

131 40 mg / kg for 3 hours to create a model of IHD. The anti-TF antibody (the chimeric humanized anti-TF antibody i-b2) was administered intravenously at a dose of 0.2 mg / kg five minutes before the start of thromboplastin solution injection. Fifteen minutes after the end of continuous injection of thromboplastin solution, blood was drawn with the addition of abdominal aorta citrate, from which platelets were counted, activated thromboplastin partial time (aPTT), fibrinogen concentration (Fib) was measured. , 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 lower platelet count, prolonged aPTT, lower fibrinogen concentration, higher sFMC and TAT concentrations, and an evident hypercoagulated state. In contrast, both chimeric and humanized anti-TF antibodies inhibited these changes equally strongly.

The result revealed that the humanized anti-TF antibody is useful as an antithrombotic agent.

132

Table 7

<td>Measurement Item</td><td>Normal group without thromboplastin administration</td><td>Solvent Administration Control Group</td><td>Antibody Administration Group chimeric</td><td>Humanized Antibody Administration Group</td>

<td>Count of platelets (x104/ mm3)</td><td> 115,5+11,8</td><td> 82,9+14,3</td><td> 100,7+12,9</td><td> 96,1+13,3</td>

<td>Concentration in fibrinogen (normal group = 100%)</td><td> 100,0+4,2</td><td> 64,8+20,0</td><td> 101,0 + 6, 6The’</td><td> 98, 9+5,7The’</td>

<td>SFMC concentration (pg / mL)</td><td> 74,2+5,5</td><td> 3517+3645</td><td> 129, 9 + 46, 8The’</td><td> 66,5+23, 0The’</td>

<td>TAT concentration (ng / mL)</td><td> 3,4+0,6</td><td> 29,6+31,0</td><td> 3, 8 + 0,7B)</td><td> 4,2+0,9</td>

<td colspan="5">(Mean 1 standard deviation)</td>

Significance of relative difference with solvent administration control group: a): p <0.01, b): p <0.05

Reference Example 1. Preparation of anti-TF monoclonal antibody

1. Human TF Purification

Purification of TF from human placenta was performed according to the method of Ito (Ito, T. et al., J. Biol. Chem., 114: 691-696, 1993). Thus, human placenta was homogenized in Tris-buffered saline (TBS, pH 7.5) containing 1.0mM benzamidine hydrochloride, 1mM phenylmethylsulfonyl fluoride, 1mM diisopropylfluorophosphate, and 0.02% sodium azide, and then the precipitate was degreased with cold acetone. The obtained degreased powder was suspended in the above buffer containing 2% Triton X-100 to solubilize TF.

133 The supernatant was subjected to affinity oromatography using a Concanavalin A-Sepharose 4B column (Pharmacia) and a Sepharose 4B-bound anti-TF antibody column (Pharmacia), and purified TF was obtained. This was concentrated with an ultrafiltration membrane (PM-10, Amicon) and stored as the purified sample at 4 ° C.

TF content in the purified sample was quantified by Sandwich ELISA which combined a commercially available anti-TF monoclonal antibody (American Diagnostica) and a polyclonal antibody (American Diagnostica) with recombinant TF as standard.

Purity in the purified sample was confirmed by subjecting the sample to SDS-PAGE using a 4-20% density gradient polyacrylamide gel, and staining the product with silver.

2. Immunization and the preparation of hybridoma

After mixing purified human TF (about 70 pg / ml) with an equal volume of complete Freund's adjuvant (Difco), it was subcutaneously immunized in the abdomen of 5 week old male Balb / c mice (Nippon Charles River) at 10 ° C. TF / mouse pg. At day 12, 18, and 25, TF mixed with incomplete Freund's adjuvant was boosted subcutaneously to 5 pg TF / mouse, and as final immunization the PBS diluted TF solution was given intraperitoneally at 5 pg / mouse on day 32.

Three days after the final immunization, spleen cells were prepared from four mice, and were fused to the mouse myeloma cell line P3U1 at one-fifth of its cell count by the method of

134 polyethylene glycol. The fused cells were suspended in RPMI-1640 medium (hereinafter referred to as RPMI medium) (Lifetech Oriental) containing 10% fetal bovine serum, which was inoculated into 400 wells per mouse (about 400 cells / well) of a plate. 96 wells. On day 1, 2, 3, and 5 after the fusion, half the volume of medium was changed to RPMI (hereinafter referred to as HAT medium) containing HAT (Dainippon Seiyaku) and Condimed Hl (Boehringer Mannheim GmbH) media for selection. Hybridoma HAT.

Hybridomas selected by the screening method described below were cloned by conducting limiting dilution twice.

For limiting dilution, 0.8 cells per well were inoculated into two 96-well plates. For wells in which individual colonies were confirmed by microscopic examination, clones were selected by measuring TF binding activity and TF neutralizing activity. The obtained clones were acclimatized from HAT medium to RPMI medium. Upon confirmation of the absence of reduction in antibody production capacity due to acclimatization, limiting dilution for complete cloning was again performed. By the above procedure, hybridomas producing six antibodies (ATR-2, 3, 4, 5, 7, and 8) which strongly inhibit the binding of the TF / Factor Vila complex to Factor X were established.

3 Ascites formation and antibody purification

Ascites formation of established hybridomas was performed according to the standard method. So 106 hybridomas that were subcultured in vitro were

135 grafted intraperitoneally to male BALB / c mice that had previously received intravenous mineral oil administration twice. Ascites were harvested from mice that showed a swollen abdomen 1-2 weeks after graft.

Antibody purification from ascites was performed using the ConSepLC100 system (Millipore) equipped with the Protein A column (Nippon Gaishi).

4 Cell ELISA

J82 human bladder carcinoma cells (Fair DS et al., J. Blol. Chem., 262: 11692-11698, 1987) known to express TF at a high level were obtained from ATCC, and subcultured and maintained in the RPMI medium under the condition of 37 ° C, C02 at 5% and humidity at 100%.

Cell ELISA plates were prepared by inoculating J82 cells in a 96-well plate at 10 ° C.5 cells / well, culture for one day under the above condition, removing the medium and then washing twice with phosphate buffered saline (PBS), adding a 4% paraformaldehyde solution (PFA), and letting stand on ice for 10 minutes for immobilization. After PFA was removed, the plate was washed with PBS, Tris buffer (Blocking buffer) containing 1% BSA and 0.02% sodium azide was added thereto, and the plate was stored at 4 ° C until the utilization.

Cell ELISA was performed as follows. Thus, the Blocking buffer was removed from the plate prepared as above, to which was added an antiTF antibody solution or a hybridoma culture supernatant that reacted to the

136 room temperature for 1.5 hours. After washing with PBS containing 0.05% Tween 20, alkaline phosphatase-conjugated goat anti-mouse IgG (H + L) (Zymed) was reacted for 1 hour. After washing, 1 mg / ml disodium pyrophenyl 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 to J82 cells.

5 TF neutralization activity assay system with Factor Xa activity as index

To 50 µl of Tris-buffered saline (TBS: pH 7.6) containing 5 mM CaCl2 and 0.1% bovine serum albumin, 10 µl of a human placenta-derived thromboplastin solution (5 mg / mL) was added. ) (Thromborel S) (Boehring) and 10 µl of a Factor Vila solution (82.5 ng / mL) (American Diagnostics), which reacted at room temperature for 1 hour to allow formation of the TF / Factor Vila complex. After 10 µl of a predetermined concentration of a diluted anti-TF antibody solution or hybridoma culture supernatant were added and 10 µl of a Factor X solution (3.245 pg / mL) (Celsus Laboratories) and reacted for 45 minutes. , 10 µl of 0.5 M EDTA was added to stop the reaction. 50 µl of a 2 mM S-2222 solution (Daiichi Kagaku Yakuhin) was added, and the changes in absorbance at 405/655 nm over 30 minutes were measured and established as the Factor X production activity. TF. In this method antibody activity that inhibits the binding of the TF / Factor Vila complex to Factor X can be determined.

6 Plasma coagulation inhibition activity assay system

137

50 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 37 ° C for 3 minutes. Then 50 µl of human placenta-derived thromboplastin solution (1.25 mg / ml) was added, and the plasma clotting time was measured using the plasma coagulation measuring instrument (CR-A: Amelung).

7 Determination of Antibody Isotype

For hybridoma culture supernatant and purified antibody, the mouse monoclonal antibody isotyping kit (manufactured by Amersham) was used to confirm the antibody isotype. The result is shown below.

Table 8

Anti-TF monoclonal antibody immunoglobulin isotype

Reference Example 2. Method of preparing soluble human TF

Soluble human TF (shTF) was prepared as follows.

The gene encoding the human TF penetrating region in which amino acids at position 220 and following had been replaced by the FLAG M2 peptide was inserted into a mammalian cell expression vector (containing the gene

138 resistant to neomycin and the DHFR gene) and introduced into CHO cells. For the human TF cDNA sequence, reference was made to an article by James H. Morrissey et al. (Cell (1987) 50: 129-135). The gene sequence and amino acid sequence of this soluble human TF are shown in SEQ ID NO: 151. After G418 drug selection, the expressed cells were selected, which were then subjected to methotrexate expression amplification, and shTF expressing cells were established.

Cells were cultured in serum-free CHO-S-SFMII (GIBCO) medium to obtain a culture supernatant containing shTF. It was diluted 2-fold with an equal volume of 40 mM Tris-HCl buffer (pH 8.5), which was added to the QSepharose Fast Flow column (100 ml, Pharmacia Biotech) equilibrated with 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 obtained shTF fraction, ammonium sulfate was added to a final concentration of 2.5 M, and centrifuged (10,000 rpm, 20 minutes) to precipitate the contaminating proteins. The supernatant was added to Butyl TOYOPEARL (30 mL, TOSOH), and then was washed with a 50 mM Tris-HCl buffer (pH 6.8) containing 2.5 M ammonium sulfate. In 50 mM Tris-HCl buffer (pH 6.8), the ammonium sulfate concentration was linearly reduced from 2.5 M to 0 M to allow elution of shTF. Peak fractions containing shTF were concentrated by Centri-Prep 10 (Amicon). The concentrate was added to the TSKgel G3000SWG column (21.5 x 600 mm, TOSOH) equilibrated with a 20 mM Tris-HCl buffer (pH 7.0) containing 150 mM NaCl, and the shTF peak fraction was collected. It was filtered for sterilization with a 0.22 pm membrane filter and the product was

139 established as soluble human TF (shTF). The sample concentration was calculated assuming that the molar extinction coefficient of the sample ε = 40.130 and the molecular weight = 43.210.

Free string listing text

The content in the <223> string listing is as follows:

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 1:</td><td>Initiator</td><td>MHC-G1</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 2 :</td><td>Initiator</td><td>MHC-G2a</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 3:</td><td>Initiator</td><td>Mkc</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 4 :</td><td>Initiator</td><td>M4 from M13</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 5:</td><td>Initiator</td><td>Ml3 RV</td>

SEQ ID NO: 6: H-chain V-region amino acid sequence of anti-TF ATR-2 mouse monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 7: H-chain V-region amino acid sequence of the anti-TF ATR-3 mouse monoclonal antibody and the nucleotide sequence encoding it

SEQ ID NO: 8: H-chain V region amino acid sequence of the anti-TF ATR-4 mouse monoclonal antibody and the nucleotide sequence encoding it

SEQ ID NO: 9: H-chain V-region amino acid sequence of mouse anti-TF ATR-5 monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 10: H-chain V region amino acid sequence of anti-TF ATR.-7 mouse monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 11: H-chain V region amino acid sequence of mouse anti-TF ATR.-8 monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 12: L-chain V-region amino acid sequence of mouse anti-TF ATR-2 monoclonal antibody and nucleotide sequence encoding it

140

SEQ ID NO: 13: L-chain V-region amino acid sequence of mouse anti-TF ATR-3 monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 14: L-chain V-region amino acid sequence of mouse anti-TF ATR-4 monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 15: L-chain V-region amino acid sequence of anti-TF ATR-5 mouse monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 16: L-chain V-region amino acid sequence of anti-TF ATR-7 mouse monoclonal antibody and nucleotide sequence encoding it

SEQ ID NO: 17: L-chain V region amino acid sequence of mouse anti-TF ATR-8 monoclonal antibody and

<td>the sequence</td><td>nucleotide</td><td>coding</td><td>The</td><td>same</td>

<td>SEQ ID NO:</td><td>18: Initiator</td><td>ch5HS</td><td></td><td></td>

<td>SEQ ID NO:</td><td>19: Initiator</td><td>ch5HA</td><td></td><td></td>

<td>SEQ ID NO:</td><td>20: Initiator</td><td>ch5LS</td><td></td><td></td>

<td>SEQ ID NO:</td><td>21: Initiator</td><td>ch5LA</td><td></td><td></td>

<td>SEQ ID NO:</td><td>22: Initiator</td><td>graft</td><td>in</td><td>CDR hR5HvlS</td>

<td>SEQ ID NO:</td><td>23: Initiator</td><td>graft</td><td>in</td><td>CDR hR5Hv28</td>

<td>SEQ ID NO:</td><td>24: Initiator</td><td>graft</td><td>in</td><td>CDR hR5Hv4S</td>

<td>SEQ ID NO:</td><td>25: Initiator</td><td>graft</td><td>in</td><td>CDR hR5Hv3A</td>

<td>SEQ ID NO:</td><td>26: Initiator</td><td>graft</td><td>in</td><td>CDR hR5Hv5A</td>

<td>SEQ ID NO:</td><td>27: Initiator</td><td>hR5HvPrS</td><td></td><td></td>

<td>SEQ ID NO:</td><td>28: Initiator</td><td>hR5HvPrA</td><td></td><td></td>

<td>SEQ ID NO:</td><td colspan="4">29: Region Amino Acid Sequence</td>

humanized H chain version a and nucleotide sequence encoding the same

SEQ ID NO: 30: Humanized H-chain V-region amino acid sequence version a

SEQ ID NO: 31: FR F3RFFS Shuffle Initiator SEQ ID NO: 32: FR F3RFBS Shuffle Initiator

141

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 33:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3RFFA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 34 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3RFBA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 35:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3NMFS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 36:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3NMBS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 37 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3NMFA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 38 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3NMBA</td>

<td>SEQ</td><td>ID</td><td>AT THE</td><td> : 39</td><td colspan="2">: Sequence</td><td colspan="2">of amino acids</td><td>gives</td><td>region</td>

humanized H chain version b and nucleotide sequence encoding the same

SEQ ID NO: 40: Humanized H chain version V amino acid sequence version b

SEQ ID NO: 41: Humanized H-chain V-region amino acid sequence version c and nucleotide sequence encoding same

SEQ ID NO: 42: Humanized H-chain V-region amino acid sequence version c

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 43:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3EPS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 44 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3EPA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 45 :</td><td>Initiator</td><td colspan="2">F3PrS</td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 4 6 :</td><td>Initiator</td><td colspan="2">F3PrA</td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 47 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3VHS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 48 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3VHA</td>

<td>SEQ ID</td><td>NO: 49: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>jail</td><td>H humanized version</td><td>d</td><td colspan="2"> and the sequence</td><td colspan="3">nucleotide</td>

<td colspan="2">encoding the same</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ ID</td><td>NO: 50: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>jail</td><td>H humanized version</td><td>d</td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ ID</td><td>NO: 51: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>jail</td><td>H humanized version</td><td>and</td><td colspan="2"> and the sequence</td><td colspan="3">nucleotide</td>

<td colspan="2">encoding the same</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ ID</td><td>NO: 52: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>jail</td><td>H humanized version</td><td>and</td><td></td><td></td><td></td><td></td><td></td>

SEQ ID NO: 53: FR F3SSS Shuffle Initiator

142

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>54: Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3SSA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>55: Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3CDS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>56: Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F3CDA</td>

<td>SEQ</td><td>ID</td><td>AT THE</td><td colspan="2">: 57: Sequence</td><td colspan="2">of amino acids</td><td>gives</td><td>region V of</td>

humanized H chain f version and nucleotide sequence encoding the same

SEQ ID NO: 58: Humanized H-chain V-region amino acid sequence version f

SEQ ID NO: 59: Humanized H-chain V-region amino acid sequence version g and nucleotide sequence encoding the same

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>60: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td>g</td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>61: Initiator of</td><td colspan="2">shuffle of</td><td>FR</td><td>F3ADS</td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>62: Initiator of</td><td colspan="2">shuffle of</td><td>FR</td><td>F3ADA</td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>63: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td>H</td><td colspan="2"> and the sequence</td><td colspan="3">nucleotide</td>

encoding the same

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>64: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td>H</td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>65: Initiator of</td><td colspan="2">shuffle of</td><td>FR</td><td>F3MMS</td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>66: Initiator of</td><td colspan="2">shuffle of</td><td>FR</td><td>F3MMA</td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>67: Initiator of</td><td colspan="2">shuffle of</td><td>FR</td><td>F3BMS</td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>68: Initiator of</td><td colspan="2">shuffle of</td><td>FR</td><td>F3BMA</td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>69: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td>i</td><td colspan="2"> and the sequence</td><td colspan="3">nucleotide</td>

<td colspan="3">coding</td><td>the same</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>70: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td>Ϊ́ ΐ H</td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>71: Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td>j</td><td colspan="2"> and the sequence</td><td colspan="3">nucleotide</td>

encoding the same

143

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>72: Sequence</td><td>of amino acids</td><td>gives</td><td>region V</td><td>gives</td>

<td colspan="2">jail SEQ ID</td><td colspan="2">H humanized version NO: 73: Initiator of</td><td>IIj shuffle of</td><td>FR</td><td>F2MPS</td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>74: Initiator of</td><td>shuffle of</td><td>FR</td><td>F2MPA</td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>75: Sequence</td><td>of amino acids</td><td>gives</td><td>region V</td><td>gives</td>

<td colspan="2">jail</td><td colspan="2">H humanized version</td><td colspan="2">bl and the sequence</td><td colspan="2">nucleotide</td>

encoding the same

<td>SEQ ID</td><td>AT THE:</td><td> 76:</td><td>Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>jail</td><td colspan="3">H humanized version</td><td>bl</td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ ID</td><td>AT THE:</td><td> 77 :</td><td>Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>jail</td><td colspan="3">H humanized version</td><td>dl</td><td colspan="2"> and the sequence</td><td colspan="3">nucleotide</td>

<td colspan="2">coding</td><td colspan="2">the same</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>SEQ ID</td><td>AT THE:</td><td> 78 :</td><td>Sequence</td><td>in</td><td>amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

H chain humanized dl version

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 79:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F2VHS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 80 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F2VHA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 81</td><td colspan="2">: Sequence</td><td colspan="2">of amino acids</td><td>gives</td><td>region V of</td>

humanized H chain version b3 and the nucleotide sequence encoding the same

SEQ ID NO: 82: Humanized H chain V-region amino acid sequence version b3

SEQ ID NO: 83: Humanized H chain V region amino acid sequence version d3 and nucleotide sequence encoding it

SEQ ID NO: 84: Humanized H chain V region amino acid sequence version d3

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 85:</td><td>Vector</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>h5LvlS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 86:</td><td>Vector</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>h5Lv4S</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 87 :</td><td>Vector</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>h5Lv2A</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 88 :</td><td>Vector</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>h5Lv3A</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 89:</td><td>Vector</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>h5Lv5A</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 90 :</td><td colspan="2">Initiator</td><td>h5LvS</td><td></td><td></td><td></td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 91:</td><td colspan="2">Initiator</td><td>h5LvA</td><td></td><td></td><td></td>

144

SEQ ID NO: 92: Humanized L-string sequence encoding the same

SEQ ID NO: 93: Humanized L-String Sequence Version

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>94: Initiator</td><td>in</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>95: Initiator</td><td>in</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>96: Initiator</td><td>in</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td>97: Initiator</td><td>in</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td colspan="2">98: Sequence</td>

humanized L chain encoding version the same

SEQ ID NO: 99: Humanized L chain sequence version SEQ ID NO: 100: Humanized L chain sequence version encoding the same.

SEQ ID NO: 101: Humanized L-chain sequence V-region amino acid version of nucleotide sequence

<td>of amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>shuffle of</td><td>FR</td><td>F3SS</td><td></td><td></td>

<td>shuffle of</td><td>FR</td><td>F3SA</td><td></td><td></td>

<td>shuffle of</td><td>FR</td><td>F3RS</td><td></td><td></td>

<td>shuffle of</td><td>FR</td><td>F3RA</td><td></td><td></td>

<td>of amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td colspan="2">good sequence</td><td colspan="3">nucleotide</td>

<td>of amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

<td>of amino acids</td><td>gives</td><td>region</td><td>V</td><td>gives</td>

of the V region nucleotide amino acid sequence of the

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 102 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F2SS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 103:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F2SA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 104 :</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F2XS</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 105:</td><td>Initiator</td><td>in</td><td>shuffle</td><td>in</td><td>FR</td><td>F2XA</td>

<td>SEQ</td><td>ID</td><td>AT THE:</td><td> 106</td><td colspan="2">: Sequence</td><td colspan="3">amino acids</td><td>region</td>

humanized L-chain version bl and the nucleotide sequence encoding the same

SEQ ID NO: 107: Humanized L-chain V-region amino acid sequence version bl

SEQ ID NO: 108: Humanized L-chain V-region amino acid sequence version b2 and nucleotide sequence encoding it

SEQ ID NO: 109: Humanized L-chain V-region amino acid sequence version b2

145

String SEQ ID NO string SEQ ID NO

SEQ ID NO of string SEQ ID NO of string SEQ ID NO of string

String SEQ ID NO string SEQ ID NO

String SEQ ID NO

110: Whole Version Humanized H Amino Acid Sequence

111: Humanized H amino acid sequence versions aaj

112: Humanized H amino acid sequence versions bl and dl

113: Humanized H amino acid sequence versions b3 and d3

114: Humanized H amino acid sequence version a

115: Humanized H amino acid sequence versions b, bl, and

116: Humanized H amino acid sequence version c

117: Humanized H amino acid sequence versions d, dl, e

118: Humanized H amino acid sequence version e

119: Humanized H amino acid sequence version f

120: Humanized H amino acid sequence version g

121: Humanized H amino acid sequence version h

122: Humanized H amino acid sequence version i

123: Humanized H amino acid sequence version j

124: Humanized H amino acid sequence all versions

125: Humanized L amino acid sequence all versions

FR1 da

FR2 da

RF2 from

FR3 da

B3 fr3

FR3 da

D3 FR3

FR3 da

FR4 from

FR1 region V region V region V region V region V region V region V region V region V region V region V region V region V region V region V

146

126: Humanized V-region FR2 amino acid sequence versions a, b and c

127: Humanized V-region FR2 amino acid sequence version bl

128: Humanized V-region FR2 amino acid sequence version b2

129: Humanized V-region FR3 amino acid sequence version a

130: Humanized V-region FR3 amino acid sequence version b, bl and b2

131: Humanized V-region FR3 amino acid sequence version c

132: Humanized V-region FR4 amino acid sequence all versions

133: Humanized H-region CDR1 amino acid sequence all versions

134: Humanized H-region CDR2 amino acid sequence all versions

135: Humanized H-region CDR3 amino acid sequence all versions

136: Humanized L-region CDR1 amino acid sequence all versions

137: Humanized L-region CDR2 amino acid sequence all versions

138: Humanized L-region CDR3 amino acid sequence all versions

139: Anti-TF mouse monoclonal antibody V-region amino acid sequence ATR-2 140: Anti-TF mouse monoclonal antibody V-region amino acid sequence ATR-3 141: Anti-TF mouse monoclonal antibody V-region amino acid sequence anti-TF mouse ATR-4

SEQ ID NO:

from string L SEQ ID NO:

of chain L

SEQ ID NO:

from chain L SEQ ID NO: from chain L

SEQ ID NO:

from string L SEQ ID NO:

of chain L

SEQ ID NO:

from string L SEQ ID NO:

V from string SEQ ID NO:

Chain V

SEQ ID NO:

V from string SEQ ID NO:

Chain V

SEQ ID NO:

V from string SEQ ID NO:

Chain V

SEQ ID NO:

string H of SEQ ID NO:

chain H of

SEQ ID NO:

chain H of

147

SEQ ID NO: 142: Anti-TF mouse monoclonal antibody H-chain V region amino acid sequence ATR-5 SEQ ID NO: 143: Anti-TF ATR mouse monoclonal antibody H-chain V region amino acid sequence SE7 ID NO: 144: Anti-TF mouse monoclonal antibody H-chain V region amino acid sequence ATR-8 SEQ ID NO: 145: Anti-mouse mouse monoclonal antibody L-chain V region amino acid sequence TF ATR-2 SEQ ID NO: 146: Anti-TF mouse monoclonal antibody L-chain V region amino acid sequence ATR-3 SEQ ID NO: 147: Anti-TF mouse monoclonal antibody L-chain V region amino acid sequence SEQ ID NO: 148: L-chain V-chain amino acid sequence of anti-TF mouse monoclonal antibody ATR-5 SEQ ID NO: 149: L-chain V-region amino acid sequence of anti-TF mouse monoclonal antibody ATR-7 SEQ ID NO: 150: AT-8 mouse monoclonal antibody L-chain V region amino acid sequence ATR-8 SEQ ID NO: 151: Soluble human TF amino acid sequence and nucleotide sequence encoding the same SEQ ID NO: 152: Anti-TF mouse amino acid sequence Soluble human TF

SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA <120> Humanized antibodies against human tissue factor (TF) and process for producing humanized antibodies <130> Order Number: 99912098.3 <150> JP 10-91850 <151> 1998-04 -03 <160> 152

148 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220>

<223> MHC-G1 Primer <400> 1 ggatcccggg ccagtggata gacagatg <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220>

<223> MHC-G2a Primer <400> 2 ggatcccggg agtggataga ccgatgg <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220>

<223> MKC Primer <400> 3 ggatcccggg tggatggtgg gaagatg <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220>

<223> M13 M4 Primer <400> 4 gttttcccag tcacgac <210> 5 <211> 17 <212> DNA <213> Artificial Sequence

149 <220>

<223> M13 RV Primer <400> 5 caggaaacag ctatgac 17 <210> 6 <211> 411 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (57] <220>

<221> mat-peptide <222> (55) ... (441) <223> Nucleotide sequence encoding the anti-TF mouse monoclonal antibody ATR-2 H-chain V region <4 0 0> 6

<td>atg</td><td>hunt</td><td>tgg</td><td>age</td><td>tgg</td><td>up until</td><td>ttt</td><td>ctc</td><td>ttc</td><td>ctc</td><td>ctg</td><td>tea</td><td>gga</td><td>act</td><td>a CA</td><td>ggt</td><td> 48</td>

<td>gtc</td><td>cac</td><td>tct</td><td>gag</td><td>up until</td><td>cag</td><td>ctg</td><td>cag</td><td>cag</td><td>tct</td><td>gga</td><td>cct</td><td>gag</td><td>ctg</td><td>gtg</td><td>the AG</td><td> 96</td>

<td>Pro</td><td>Gly</td><td>Allah</td><td>To be</td><td>Val</td><td>Lys</td><td>Val</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Tyr</td><td>To be</td><td>Phe</td><td></td>

<td>gag</td><td>tgg</td><td>Att</td><td>gga</td><td>tat</td><td>Att</td><td>gat</td><td>cct</td><td>tac</td><td>aat</td><td>ggt</td><td>ggt</td><td>act</td><td>up until</td><td>tac</td><td>aac</td><td> 240</td>

<td>Gin</td><td>Lys</td><td>Phe</td><td>Lys</td><td>Gly</td><td>Lys</td><td>Allah</td><td>Thr</td><td>Read</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>To be</td><td>To be</td><td>To be</td><td></td>

<td>Thr</td><td>Allah</td><td>Phe</td><td>Met</td><td>His</td><td>Read</td><td>Asn</td><td>To be</td><td>Read</td><td>Thr</td><td>To be</td><td>Glu</td><td>Asp</td><td>To be</td><td>Allah</td><td>Val</td><td></td>

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Allah</td><td>Arg</td><td>Gly</td><td>Gly</td><td>Glu</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Phe</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td></td>

<210> 7 <211> 411 <212> DNA <213> Mouse

150 <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (441) <223> Nucleotide sequence encoding anti-TF mouse monoclonal antibody ATR-3 H-chain V region <400> 7

<td>atg</td><td>gaa</td><td>tgg</td><td>age</td><td>tgg</td><td>up until</td><td>ttt</td><td>ctc</td><td>ttc</td><td>ctc</td><td>ctg</td><td>tea</td><td>gga</td><td>act</td><td>a CA</td><td>ggt</td><td> 48</td>

<td>QtC</td><td>cac</td><td>LCt</td><td>gag</td><td>up until</td><td>cag</td><td>ctg</td><td>cag</td><td>cag</td><td>tet</td><td>gga</td><td>cct</td><td>gag</td><td>ctg</td><td>gtg</td><td>the AG</td><td> 96</td>

<td>Pro</td><td>Glv</td><td>Allah</td><td>To be</td><td>Val</td><td>Lys</td><td>Val</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Tyr</td><td>To be</td><td>Phe</td><td></td>

<td>gaç</td><td>tgg</td><td>Att</td><td>gga</td><td>tat</td><td>Att</td><td>gat</td><td>cct</td><td>tac</td><td>aat</td><td>ggt</td><td>ggt</td><td>act</td><td>up until</td><td>tac</td><td>aac</td><td> 240</td>

<td>Thr</td><td>Allah</td><td>Phe</td><td>Met</td><td>Kis</td><td>Read</td><td>Asn</td><td>To be</td><td>Read</td><td>Thr</td><td>To be</td><td>Glu</td><td>Asso</td><td>To be</td><td>Allah</td><td>Val</td><td></td>

<210> 8 <211> 408 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (408) <223> Nucleotide sequence encoding the anti-TF mouse monoclonal antibody ATR-4 H chain H region

151 <400> 8

<td>atg</td><td>aaa</td><td>tgc</td><td>age</td><td>tgg</td><td>gtc</td><td>up until</td><td>ttc</td><td>ttc</td><td>ctg</td><td>atg</td><td>gea</td><td>gtg</td><td>gtt</td><td>a CA</td><td>ggg</td><td> 43</td>

<td>Met</td><td>Lys</td><td>Cys</td><td>To be</td><td>Trp</td><td>Val</td><td>Ile</td><td>Phe</td><td>Phe</td><td>Read</td><td>Met</td><td>Allah</td><td>Val</td><td>val</td><td>Thr</td><td>Gly</td><td></td>

<td>Go</td><td>Asn</td><td>To be</td><td>Glu</td><td>Val</td><td>Gin</td><td>Read</td><td>Gin</td><td>Gin</td><td>To be</td><td>Gly</td><td>Allah</td><td>Glu</td><td>Read</td><td>Val</td><td>Arg</td><td></td>

<td>Pro</td><td>Gly</td><td>Allah</td><td>Read</td><td>Val</td><td>Lys</td><td>Read</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>lie</td><td></td>

<td>Pro</td><td>Lys</td><td>Phe</td><td>Gin</td><td>Gly</td><td>Lvs</td><td>Allah</td><td>To be</td><td>I le</td><td>Thr</td><td>Allah</td><td>Asp</td><td>Thr</td><td>To be</td><td>To be</td><td>Asn</td><td></td>

<td>Thr</td><td>Allah</td><td>Tyr</td><td>Read</td><td>Gin</td><td>Read</td><td>To be</td><td>To be</td><td>Read</td><td>Thr</td><td>To be</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Allah</td><td>Val</td><td></td>

<td>yr</td><td>Tyr</td><td>Cys</td><td>Asp</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td></td>

<210> 9 <211> 408 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (408) <223> Nucleotide sequence encoding the anti-TF mouse monoclonal antibody ATR-5 H-chain V region <400> 9

<td>atg</td><td>aaa</td><td>tgc</td><td>age</td><td>tgg</td><td>gtc</td><td>up until</td><td>ctc</td><td>ttc</td><td>ctg</td><td>atg</td><td>gea</td><td>gtg</td><td>gtt</td><td>a CA</td><td>ggg</td>

<td>Met</td><td>Lys</td><td>Cys</td><td>To be</td><td>Trp</td><td>Val</td><td>Ile</td><td>Phe</td><td>Phe</td><td>Read</td><td>Met</td><td>Allah</td><td>Val</td><td>Val</td><td>Thr</td><td>Gly</td>

<td>Pro</td><td>Gly</td><td>Allah</td><td>Read</td><td>Val</td><td>Lys</td><td>Read</td><td>To be</td><td>Cys</td><td>Lys</td><td>Gly</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile</td>

144

192

152

<td>Glu</td><td>Trp</td><td>Ile</td><td>Gly</td><td>Gly</td><td>Asn</td><td>Asp</td><td>ro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>His</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td></td>

<td>ro</td><td>Lys</td><td>Phe</td><td>Gin</td><td>Gly</td><td>Lys</td><td>Allah</td><td>To be</td><td>Ile</td><td>Thr</td><td>Allah</td><td>Asp</td><td>Thr</td><td>To be</td><td>To be</td><td>Asn</td><td></td>

<td>Tyr</td><td>Phe</td><td>Cys</td><td>Allah</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td></td>

<210> 10 <211> 411 <212> DNA <213> Mouse <220>

<221> six-peptide <222> (1) ... (57) <220>

<221> mat-peptic <222> (58) ... (411) <223> Nucleotide sequence encoding anti-TF mouse monoclonal antibody ATR-7 H-chain V region

<td>atg</td><td>gaa</td><td>tgg</td><td>age</td><td>tgg</td><td>up until</td><td>ttt</td><td>ctc</td><td>ttc</td><td>ctc</td><td>ctg</td><td>tca</td><td>gga</td><td>act</td><td>a CA</td><td>ggt</td><td> 43</td>

<td>gtc</td><td>cac</td><td>tet</td><td>gac</td><td>up until</td><td>cag</td><td>ctg</td><td>cag</td><td>cag</td><td>tet</td><td>gga</td><td>cct</td><td>gag</td><td>ctg</td><td>gtg</td><td>the AG</td><td> 96</td>

<td>Pro</td><td>Gly</td><td>To be</td><td>To be</td><td>Go</td><td>Lys</td><td>Go</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Tyr</td><td>To be</td><td>Phe</td><td></td>

<td>Gin</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Asp</td><td>Lys</td><td>Allah</td><td>Thr</td><td>Read</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>To be</td><td>To be</td><td>To be</td><td></td>

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Allah</td><td>Arg</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>Tyr</td><td>Asp</td><td>Cys</td><td>Tyr</td><td>Trp</td><td>Gly</td><td></td>

<td>Gin</td><td>Gly</td><td>Thr</td><td>Read</td><td>Go</td><td>Thr</td><td>Go</td><td>To be</td><td>Allah</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 11

153 <211> 411 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (411) <223> Nucleotide sequence encoding the anti-TF mouse monoclonal antibody ATR-8 H chain H region

<td>gtc</td><td>cac</td><td>tct</td><td>gac</td><td>up until</td><td>cag</td><td>ctg</td><td>cag</td><td>cag</td><td>tct</td><td>gga</td><td>cct</td><td>gag</td><td>ctg</td><td>gtg</td><td>the AG</td><td> 96</td>

<td>Pro</td><td>Gly</td><td>Allah</td><td>To be</td><td>val</td><td>Lys</td><td>Val</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Tyr</td><td>To be</td><td>Phe</td><td></td>

<td>a CA</td><td>gcc</td><td>ttc</td><td>atg</td><td>cat</td><td>ctc</td><td>aac</td><td>steel</td><td>ctg</td><td>a CA</td><td>tct</td><td>gag</td><td>gac</td><td>tct</td><td>gea</td><td>gtc</td><td> 336</td>

<td>Gin</td><td>Gly</td><td>Thr</td><td>Read</td><td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>Allah</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <213></td><td>little mouse</td>

<td> <221></td><td>sig-peptide</td>

<td> <221></td><td>peptide mat</td>

154 <223> Nucleotide sequence encoding the L-chain V region of the mouse anti-TF ATR-2 monoclonal antibody <400> 12

<td>Met</td><td>Read</td><td>Thr</td><td>Gin</td><td>Read</td><td>Read</td><td>Gly</td><td>Read</td><td>Read</td><td>Read</td><td>Read</td><td>Trp</td><td>Phe</td><td>Allah</td><td>Gly</td><td>Gly</td><td></td>

<td>Lys</td><td>cys</td><td>Asp</td><td>Ile</td><td>Gin</td><td>Met</td><td>Thr</td><td>Gin</td><td>To be</td><td>Pro</td><td>Allah</td><td>To be</td><td>Gin</td><td>To be</td><td>Allah</td><td>To be</td><td></td>

<td>ctg</td><td>gga</td><td>gaa</td><td>agt</td><td>gtc</td><td>acc</td><td>up until</td><td>a CA</td><td>tgc</td><td>ctg</td><td>gea</td><td>agt</td><td>cag</td><td>acc</td><td>Att</td><td>ggt</td><td> 144</td>

<td>Read</td><td>Gly</td><td>Glu</td><td>To be</td><td>Val</td><td>Thr</td><td>Xle</td><td>Thr</td><td>Cys</td><td>Read</td><td>Allah</td><td>To be</td><td>Gin</td><td>Thr</td><td>Ile</td><td>C-ly</td><td></td>

<td>Thr</td><td>Trp</td><td>Read</td><td>Allah</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Lys</td><td>Pro</td><td>Gly</td><td>Lys</td><td>To be</td><td>Pro</td><td>Gin</td><td>Val</td><td></td>

<td>Read</td><td>Ile</td><td>Tyr</td><td>Allah</td><td>Allah</td><td>Thr</td><td>To be</td><td>Read</td><td>Allah</td><td>Asp</td><td>Gly</td><td>Val</td><td>Pro</td><td>To be</td><td>Arg</td><td>Phe</td><td></td>

<td>agt</td><td>ggt</td><td>agt</td><td>gga</td><td>tct</td><td>ggc</td><td>a CA</td><td>aaa</td><td>ttt</td><td>tct</td><td>ttc</td><td>the AG</td><td>up until</td><td>age</td><td>age</td><td>cta</td><td> 288</td>

<td>Gin</td><td>Allah</td><td>Glu</td><td>Asp</td><td>Phe</td><td>Val</td><td>To be</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Read</td><td>Tyr</td><td>To be</td><td>Thr</td><td></td>

95_100_105 <210> 13 <211> 375 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (54) <220>

<221> mat-peptide <222> (55) ... (375) <223> Nucleotide sequence encoding the anti-TF mouse monoclonal antibody ATR-3 L-chain V region <400> 13

<td>Lys</td><td>Cys</td><td>Asp</td><td>ile</td><td>Gin</td><td>Met</td><td>Thr</td><td>Gin</td><td>To be</td><td>Pro</td><td>Allah</td><td>To be</td><td>Gin</td><td>To be</td><td>Allah</td><td>To be</td><td></td>

<td>ctg</td><td>gga</td><td>gaa</td><td>agt</td><td>gtc</td><td>acc</td><td>up until</td><td>a CA</td><td>tgc</td><td>ccg</td><td>gea</td><td>agt</td><td>cag</td><td>acc</td><td>Att</td><td>ggt</td><td> 144</td>

<td>Read</td><td>Gly</td><td>Glu</td><td>To be</td><td>Val</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Cys</td><td>Read</td><td>Allah</td><td>To be</td><td>Gin</td><td>Thr</td><td>Ile</td><td>Gly</td><td></td>

155

<210> 14 <211> 387 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (66) <220>

<221> mat-peptide <222> (67) ... (387) <223> Nucleotide sequence encoding anti-TF mouse monoclonal antibody ATR-4 L-chain V region <400> 14

<td>atg</td><td>gac</td><td>atg</td><td>agg</td><td>gcc</td><td>cct</td><td>gct</td><td>cag</td><td>ttt</td><td>ttt</td><td>ggg</td><td>up until</td><td>ttg</td><td>ttg</td><td>ctc</td><td>tgg</td><td> 48</td>

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>Allah</td><td>Pro</td><td>Allah</td><td>Gin</td><td>Phe</td><td>Phe</td><td>Gly</td><td>Ile</td><td>Read</td><td>Read</td><td>Read</td><td>Trp</td><td></td>

<td>ttt</td><td>cca</td><td>ggt</td><td>up until</td><td>now</td><td>tgt</td><td>gac</td><td>up until</td><td>the AG</td><td>atg</td><td>acc</td><td>cag</td><td>tct</td><td>cca</td><td>tcc</td><td>tcc</td><td> 96</td>

<td>atg</td><td>tat</td><td>gcc</td><td>teg</td><td>ctg</td><td>gga</td><td>gag</td><td>now</td><td>gtc</td><td>act</td><td>up until</td><td>act</td><td>tgc</td><td>the AG</td><td>gcg</td><td>agt</td><td> 144</td>

<td>Met</td><td>Tyr</td><td>Allah</td><td>To be</td><td>Read</td><td>Gly</td><td>Glu</td><td>Arg</td><td>Val</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td></td>

<td>tct</td><td>cct</td><td>the AG</td><td>acc</td><td>ctg</td><td>up until</td><td>tat</td><td>tac</td><td>gca</td><td>a CA</td><td>age</td><td>ttg</td><td>gca</td><td>gat</td><td>ggg</td><td>gtc</td><td> 240</td>

<td>To be</td><td>Pro</td><td>Lys</td><td>Thr</td><td>Read</td><td>Ile</td><td>Tyr</td><td>Tyr</td><td>Allah</td><td>Thr</td><td>To be</td><td>Read</td><td>Allah</td><td>Asp</td><td>Gly</td><td>Val</td><td></td>

<td>Ile</td><td>To be</td><td>To be</td><td>Read</td><td>Glu</td><td>To be</td><td>Asp</td><td>Asp</td><td>To be</td><td>Allah</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Read</td><td>Gin</td><td></td>

aaa 387

Lys <210> 15

156 <211> 381 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (60) <220>

<221> mat-peptide <222> (61) ... (381) <223> Nucleotide sequence encoding the anti-TF mouse monoclonal antibody ATR-5 L-chain V region

<td>atg</td><td>agg</td><td>gee</td><td>cct</td><td>gct</td><td>cag</td><td>ttt</td><td>ttt</td><td>ggg</td><td>up until</td><td>ttg</td><td>ttg</td><td>ctc</td><td>tgg</td><td>ttt</td><td>cca</td><td> 48</td>

<td>Met</td><td>Arg</td><td>Allah</td><td>Pro</td><td>Allah</td><td>Gin</td><td>Phe</td><td>Phe</td><td>Gly</td><td>Ile</td><td>Read</td><td>Read</td><td>Read</td><td>Trp</td><td>Phe</td><td>Pro</td><td></td>

<td>ggt</td><td>up until</td><td>now</td><td>tgt</td><td>gac</td><td>up until</td><td>the AG</td><td>atg</td><td>acc</td><td>cag</td><td>tet</td><td>cca</td><td>tcc</td><td>tet</td><td>atg</td><td>tat</td><td> 96</td>

<td>gea</td><td>teg</td><td>ctg</td><td>gga</td><td>gag</td><td>now</td><td>gtc</td><td>act</td><td>up until</td><td>act</td><td>tgc</td><td>the AG</td><td>gcg</td><td>agt</td><td>cag</td><td>gac</td><td> 144</td>

<td>Allah</td><td>To be</td><td>Read</td><td>Gly</td><td>Glu</td><td>Arg</td><td>Go</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gin</td><td>Asp</td><td></td>

<td>the AG</td><td>acc</td><td>ctg</td><td>up until</td><td>tat</td><td>tat</td><td>gea</td><td>a CA</td><td>age</td><td>ttg</td><td>gea</td><td>gat</td><td>ggg</td><td>gtc</td><td>cca</td><td>tea</td><td> 240</td>

<td>Lys</td><td>Thr</td><td>Read</td><td>Ile</td><td>Tyr</td><td>Tyr</td><td>Allah</td><td>Thr</td><td>To be</td><td>Read</td><td>Allah</td><td>Asp</td><td>Gly</td><td>Go</td><td>Pro</td><td>To be</td><td></td>

<td>now</td><td>ttc</td><td>agt</td><td>ggc</td><td>agt</td><td>gga</td><td>tet</td><td>ggg</td><td>ca a</td><td>gat</td><td>tat</td><td>tet</td><td>cta</td><td>acc</td><td>up until</td><td>aac</td><td> 288</td>

<td>Asn</td><td>Read</td><td>Glu</td><td>To be</td><td>Asp</td><td>Asp</td><td>Thr</td><td>Allah</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Read</td><td>Gin</td><td>His</td><td>Gly</td><td></td>

100 <210> 16 <211> 393 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (394) <223> Nucleotide sequence encoding the L-chain V region of the anti-TF mouse monoclonal antibody ATR-7

157 <400> 16

<td>Met</td><td>To be</td><td>Pro</td><td>Allah</td><td>Gin</td><td>Pn <=</td><td>Read</td><td>Phe</td><td>Read</td><td>Read</td><td>Go</td><td>Read</td><td>Trp</td><td>Ile</td><td>Arg</td><td>Glu</td><td></td>

<td>up until</td><td>aac</td><td>ggt</td><td>gat</td><td>gtt</td><td>gtg</td><td>ctg</td><td>acc</td><td>cag</td><td>act</td><td>cca</td><td>etc</td><td>act</td><td>ttg</td><td>teg</td><td>gtt</td><td> 96</td>

<td>Thr</td><td>Ile</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Allah</td><td>To be</td><td>Go</td><td>To be</td><td>Cys</td><td>Lys</td><td>To be</td><td>To be</td><td>Gin</td><td>To be</td><td>Read</td><td></td>

<td>ggc</td><td>cag</td><td>tet</td><td>cca</td><td>the AG</td><td>ege</td><td>erg</td><td>up until</td><td>tat</td><td>cct</td><td>gtg</td><td>tet</td><td>aaa</td><td>ctg</td><td>gac</td><td>tet</td><td> 240</td>

<td>ctg</td><td>aaa</td><td>up until</td><td>age</td><td>now</td><td>gtg</td><td>gag</td><td>gct</td><td>gag</td><td>gat</td><td>ttg</td><td>gga</td><td>gtt</td><td>tat</td><td>tat</td><td>-gt</td><td> 336</td>

<td>Read</td><td>Lys</td><td>Ile</td><td>To be</td><td>Arg</td><td>Go</td><td>Glu</td><td>Allah</td><td>Glu</td><td>Asp</td><td>Read</td><td>Gly</td><td>Go</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td></td>

gaa ata aaa 393

Glu Ile Lys 110 <210> 17 <211> 393 <212> DNA <213> Mouse <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (393) <223> Nucleotide sequence coding for the L-chain V region of the anti-TF mouse monoclonal antibody ATR-8 <400> 17

<td>Met</td><td>To be</td><td>Pro</td><td>Allah</td><td>Gin</td><td>Phe</td><td>Read</td><td>Phe</td><td>Read</td><td>Read</td><td>Val</td><td>Read</td><td>Trp</td><td>Ile</td><td>Arg</td><td>Asp</td><td></td>

<td>up until</td><td>aac</td><td>ggt</td><td>gat</td><td>gct</td><td>gta</td><td>ctg</td><td>acc</td><td>cag</td><td>act</td><td>cca</td><td>ctc</td><td>act</td><td>ttg</td><td>teg</td><td>gtt</td><td> 96</td>

158

<td>dCD</td><td>up until</td><td>gga</td><td>hunt</td><td>cca</td><td>gee</td><td>tec</td><td>gtc</td><td>tet</td><td>tgc</td><td>the AG</td><td>tea</td><td>agt</td><td>cag</td><td>age</td><td>etc</td><td> 144</td>

<td>Thr</td><td>Ile</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Allah</td><td>To be</td><td>Val</td><td>To be</td><td>Cys</td><td>Lys</td><td>To be</td><td>To be</td><td>Gin</td><td>To be</td><td>Read</td><td></td>

<td>gcc</td><td>cag</td><td>tet</td><td>cca</td><td>the AG</td><td>ege</td><td>cta</td><td>up until</td><td>tac</td><td>ctg</td><td>gtg</td><td>tet</td><td>aaa</td><td>ctg</td><td>gac</td><td>tet</td><td> 240</td>

<td>Read</td><td>Lys</td><td>Ile</td><td>To be</td><td>Arg</td><td>Val</td><td>Glu</td><td>Allah</td><td>Glu</td><td>Asp</td><td>Read</td><td>Gly</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td></td>

<td>tgg</td><td>hunt</td><td>gat</td><td>a CA</td><td>cat</td><td>ttt</td><td>blind</td><td>gac</td><td>acg</td><td>ttc</td><td>gga</td><td>ggg</td><td>ggg</td><td>acc</td><td>the AG</td><td>ctg</td><td> 334</td>

<210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220>

<223> ch5HS Primer <400> 18 gtctgtcgac ccaccatgaa atgcagctgg gtcat 35 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220>

<223> Primer ch5HA <400> 19 tgttgctagc tgaggagacg gtgactga 28 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220>

<223> ch5LS Initiator <400> 20 gtctagatct ccaccatgag ggcccctgct cagtt

159

<td> <213></td><td>Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>ch5LA</td>

<400> 21 tgttcgtacg ttttatttcc agcttggc

<td> <213></td><td>Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>hR5HvlS CDR graft</td>

ttctgtcgac ccaccatgaa atgcagctgg gtcatcttct tcctgatggc agtggttaca 60 ggggtcaact cacaggtgca gctgttggag tccggagctg tgcc 104

<td> <210> <211> <212> <213></td><td>23 108 DNA Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>hR5Rv28 CDR graft</td>

acaggtgcag ctgttggagt ctggagctgt gctggcaagg cctgggactt ccgtgaagat 60 ctcctgcaag gcttccggat tcaacattaa agactactat atgcattg 108

<td> <210> <211> <212> <213></td><td>24 108 DNA Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>hR5Hv4S CDR graft</td>

gaatggccac agtatgtatg acccgaaatt ccagggcagg gccaaactga ctgcagccac 60 atccgccagt attgcctact tggagttctc gagcctgaca aatgagga 108 <210> 25

160

<td> <211> <212> <213></td><td>110 DNA Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>hR5Hv3A CDR graft</td>

tcatacatac tatggccatt cgcaggatca ttcccaccaa tccattctag accctgtcca 60 ggcotctgtt ttacccaatg catatagtag tcttcaatgt tgaatccgga 110

<td> <210> <211> <212> <213></td><td>26 110 DNA Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>hR5Hv5A CDR graft</td>

agaagctagc tgaggagacg gtgaccaggg tgccttggcc ccagtagccc acggcatagc 60 ccçagtctct tgcacagtaa tagaccgcag aatcctcatt tgtcaggctc 110 <210> 27 <211> 19 <212> Artificial <213> Sequence

<223> hR5HvPrS Primer <400> 27 ttctgtcgac ccaccatga <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220>

<223> Primer hR5HvPrA <400> 28 agaagctagc tgaggagac <210> 29 <211> 415 <212> DNA <213> Artificial Sequence

161 <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (415) <223> Nucleotide sequence encoding the humanized H chain V-region version <400> 29

<td>Val</td><td>Asn</td><td>To be</td><td>Gin</td><td>val</td><td>Gin</td><td>Read</td><td>Read</td><td>Glu</td><td>To be</td><td>Gly</td><td>Allah</td><td>Val</td><td>Read</td><td>Allah</td><td>Arg</td><td></td><td></td>

<td>cct</td><td>ggg</td><td>act</td><td>tcc</td><td>gtg</td><td>the AG</td><td>up until</td><td>tcc</td><td>tgc</td><td>the AG</td><td>gct</td><td>tcc</td><td>gga</td><td>ttc</td><td>aac</td><td>ar t</td><td> 1</td><td> 44</td>

<td>Pro</td><td>Gly</td><td>Thr</td><td>To be</td><td>Val</td><td>Lys</td><td>Ile</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile</td><td></td><td></td>

<td>Glu</td><td>Trp</td><td>Ile</td><td>Gly</td><td>Gly</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>Hls</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td></td><td></td>

<td>ro</td><td>Lys</td><td>Phe</td><td>Gin</td><td>Gly</td><td>Arg</td><td>Allah</td><td>Lys</td><td>Read</td><td>Thr</td><td>Allah</td><td>Allah</td><td>Thr</td><td colspan="2">Be Wing S</td><td>er</td><td></td><td></td>

<td>ile</td><td>Allah</td><td>Tyr</td><td>Read</td><td>Glu</td><td>Phe</td><td>To be</td><td>To be</td><td>Read</td><td>Thr</td><td>Asn</td><td>Glu</td><td>Asp</td><td>It will be</td><td>la V</td><td>al</td><td></td><td></td>

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Allah</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td colspan="2">Tyr Trc G</td><td>ly G</td><td>lr.</td><td></td><td></td>

<td>Glv</td><td>Thr</td><td>Read</td><td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 30 <211> 119 <212> PRT <213> Artificial Sequence <220>

<223> A-amino acid sequence of humanized H-chain V-region version <400> 30

162

<td colspan="2">Gin Vai 1</td><td>Gin</td><td>Read</td><td>Read 5th</td><td colspan="3">Glu Ser Gly</td><td>Allah</td><td>Go 10</td><td>Read</td><td>Allah</td><td>Arg</td><td>Pro</td><td>Gly 15</td><td>Thr</td>

<td>To be</td><td>Go</td><td>Lys</td><td>Ile</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile</td><td>Lys</td><td>Asp</td><td>Tyr</td>

<td>Gly</td><td>Gly</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>His</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>Lys</td><td>Phe</td>

<td>Gin</td><td>Gly</td><td>Arg</td><td>Allah</td><td>Lys</td><td>Read</td><td>Thr</td><td>Allah</td><td>Allah</td><td>Thr</td><td>To be</td><td>Allah</td><td>To be</td><td>Ile</td><td>Allah</td><td>Tyr</td>

<td>Read</td><td>Glu</td><td>Phe</td><td>To be</td><td>To be</td><td>Read</td><td>Thr</td><td>Asn</td><td>Glu</td><td>Asp</td><td>To be</td><td>Allah</td><td>Go</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td>Al a</td><td>Arg</td><td>Asp</td><td>To be</td><td>GLy</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Read</td>

<td>Go</td><td>Thr</td><td>Go</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

115 <210> 31 <211> 100 <212> DNA <213> Artificial Sequence <220>

<223> FR F3RFFS Shuffle Initiator <400> 31 ttcttggcca tagtacgtat gacccgaaat tccagggccg agtcacaatc actgcagaca 60 catccacgaa cacagcctct cgagtctgag 100 <210> <210> <2> 75 <2> Sequence <210> <2> 75 <2> Sequence

<223> FR F3RFBS Shuffle Initiator <400> 32 ggagctctcg agtctgagat ctgaggacac agccatttat tactgtgcaa gagactcggg 60 ctatgccatg gttct 75 <210> 33 <211> 100 <212> Sequence Art <213> Sequence <213>

<223> F3RFFA FR Shuffle Initiator <400> 33

163 ctcagactcg agagctccat gtaggctg-g ttcgtggatg tgtctgcagt gattgtgact 60 cggccctgga atttcgggtc atacatacta tggccaagaa 100 <210> 34 <211> 75 <212> DNA <2132> Artificial <2132> Sequence

<223> FR F3RFBA Shuffle Initiator <400> 34 agaaccatgg catagcccga gtctcttçca cagtaataaa tggctgcgcc ctcagatctc 60 agactcgaga gctcc 75

<td> <210> <211> <212> <213></td><td>35 100 DNA Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>F3NMFS Shuffle Cards</td>

100 ttcttggcca tagtacgtat gacccgaaat tccagggccg agtcacaatg ctçgtagaca catccaagaa ccagttctcc ctgaggctct cgagtgtgac

<td> <210> <211> <212> <213></td><td>36 75 DNA Sequence</td><td colspan="2">Artificial</td>

<td> <220> <223></td><td>Initiator</td><td>shuffle</td><td>from FR F3NMBS</td>

<td colspan="2">gaçgctctcg agtgtgacag</td><td>ccgcggacac agccgtatat</td><td>tactgtgcaa gagactcggg</td>

ctatgccatg gttct 75

<td> <210> <211> <212> <213></td><td>37 100 DNA Sequence</td><td>Artificial</td>

<td> <220> <223></td><td>Initiator</td><td>F3NMFA Shuffle Cards</td>

<400> 37

164 gtcacactcg agagcctcag ggagaactgg ttcttggatg tgtctaccag cattgtgact cçgccctgga atttcgggcc atacacacta tggccaagaa

<td> <210> <211> <212> <213></td><td>38 75 DNA Sequence</td><td>Artificial</td>

<td> <223></td><td>Initiator</td><td>F3NMBA Shuffling Machine</td>

agaaccatga catagcccga gtctcttçca cagtaatata cggctgtgtc cgcggctgtc acactcgaga gcctc

100 <210> 39 <211> 414 <212> DNA <213> Artificial Sequence <220>

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (414) <223> Nucleotide sequence encoding the humanized H-chain V-region version b <400> 39

<td>Val</td><td>Asn</td><td>To be</td><td>Gin</td><td>Val</td><td>Gin</td><td>Read</td><td>Read</td><td>Glu</td><td>To be</td><td>Gly</td><td>Allah</td><td>Val</td><td>Read</td><td>Allah</td><td>Arg</td><td></td>

<td>Pro</td><td>Gly</td><td>Thr</td><td>To be</td><td>Va 1</td><td>Lys</td><td>Ile</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile</td><td></td>

<td>Glu</td><td>Trp</td><td>Ile</td><td>Gly</td><td>Gly</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>Behold</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td></td>

<td>ccg</td><td>aaa</td><td>ttc</td><td>cag</td><td>ggc</td><td>cga</td><td>gtc</td><td>a CA</td><td>up until</td><td>act</td><td>gea</td><td>gac</td><td>a CA</td><td>tcc</td><td>acg</td><td>aac</td><td> 238</td>

<td>Pro</td><td>Lys</td><td>Phe</td><td>Gin</td><td>Gly</td><td>Arg</td><td>Val</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Allah</td><td>Asp</td><td>Thr</td><td>To be</td><td>Thr</td><td>Asn</td><td></td>

<td>Thr</td><td>Allah</td><td>Tyr</td><td>Met</td><td>Glu</td><td>Read</td><td>To be</td><td>To be</td><td>Read</td><td>Arg</td><td>Ssr</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Allah</td><td>lie</td><td></td>

165

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Allah</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Mer</td><td>Asp</td><td>Tyr</td><td>Trp Gly Gin</td><td></td>

<td>GLy</td><td>Thr</td><td>Read</td><td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td></td><td></td><td></td><td></td><td></td>

<210> 40 <211> 119 <212> PRT <213> Artificial Sequence <220>

<223> Humanized H chain region V version b amino acid sequence <400> 40

<td colspan="2" rowspan="2">Gin Val 1</td><td rowspan="2">Gin</td><td rowspan="2">Read</td><td colspan="6">Read Glu Ser Gly Wing Val</td><td rowspan="2">Read</td><td rowspan="2">Allah</td><td rowspan="2">Arg</td><td rowspan="2">Pro</td><td rowspan="2">Gly 15</td><td rowspan="2">Thr</td>

<td>To be</td><td>Val</td><td>Lys</td><td>Ile</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile</td><td>Lys</td><td>Asp</td><td>Tyr</td>

<td>Gly</td><td>Gly</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>His</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>Lys</td><td>Phe</td>

<td>Gin</td><td>Gly</td><td>Arg</td><td>Val</td><td>Thr</td><td>Ile</td><td>Thr</td><td>Allah</td><td>Asp</td><td>Thr</td><td>To be</td><td>Thr</td><td>Asn</td><td>Thr</td><td>Allah</td><td>Tyr</td>

<td>Met</td><td>Glu</td><td>Read</td><td>To be</td><td>To be</td><td>Read</td><td>Arg</td><td>To be</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Allah</td><td>Ile</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td>Allah</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Read</td>

<td>Val</td><td>Thr</td><td>Go</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 41 <211> 414 <212> DNA <213> Artificial Sequence <220>

<221> siq-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (414) <223> Nucleotide sequence encoding the c-version of humanized H-chain V region <400> 41

166

<td>Go</td><td>Asn</td><td>Sar</td><td>Gin</td><td>Val</td><td>Gin</td><td>Read</td><td>Read</td><td>Glu</td><td>To be</td><td>Gly</td><td>Allah</td><td>Val</td><td>Read</td><td>Allah</td><td>Arg</td><td></td>

<td>Glu</td><td>Trp</td><td>Ile</td><td>Giy</td><td>Gly</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>His</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td></td>

<td>Gin</td><td>Phe</td><td>To be</td><td>Read</td><td>Arg</td><td>Read</td><td>To be</td><td>To be</td><td>val</td><td>Thr</td><td>Allah</td><td>Allah</td><td>Asp</td><td>Thr</td><td>Allah</td><td>Val</td><td></td>

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Allah</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td></td>

<td>Gly</td><td>Thr</td><td>Read</td><td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 42 <211> 119 <212> PRT <213> Artificial Sequence <220>

<223> Humanized H-chain version V region c amino acid sequence <400> 42

<td>Gin 1</td><td>Val</td><td>Gin</td><td>Read</td><td>Read 5th</td><td>Glu</td><td>To be</td><td>Gly</td><td>Allah</td><td>Val 10</td><td>Read</td><td>Allah</td><td>Arg</td><td>Pro</td><td>Gly 15</td><td>Thr</td>

<td>To be</td><td>Val</td><td>Lvs</td><td>lie</td><td>To be</td><td>Cys</td><td>Lys</td><td>Allah</td><td>To be</td><td>Gly</td><td>Phe</td><td>Asn</td><td>Ile</td><td>Lys</td><td>Asp</td><td>Tyr</td>

<td>Gly</td><td>Gly</td><td>Asn</td><td>Asp</td><td>Pro</td><td>Allah</td><td>Asn</td><td>Gly</td><td>His</td><td>To be</td><td>Met</td><td>Tyr</td><td>Asp</td><td>Pro</td><td>Lys</td><td>Phe</td>

<td>Read</td><td>Arg</td><td>Read</td><td>To be</td><td>To be</td><td>Val</td><td>Thr</td><td>Allah</td><td>Allah</td><td>Asp</td><td>Thr</td><td>Allah</td><td>Val</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td>Allah</td><td>Arg</td><td>Asp</td><td>To be</td><td>Gly</td><td>Tyr</td><td>Allah</td><td>Met</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Read</td>

<td>Val</td><td>Thr</td><td>Val</td><td>To be</td><td>To be</td><td>Allah</td><td>To be</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

167 <210> 43 <211> 100 <212> DNA <213> Artificial Sequence <220>

<223> FR F3EPS Shuffle Initiator <400> 43 ttcttggcca tagtatgtat gacccgaaat tccagggcag agtcacgatt actgcggacg 60 aatccacgag cacagcctac atggagctct cgagtctgag 100 <210> <210> <210> <210> <210> <210> <210> <210> <1210> <210> <210> <210>

<223> FR F3EPA Shuffle Initiator <400> 44 agaaccatgg catagcccga gtctctcgca cagaaatata cggccgagtc ctcagatctc 60 agactcgaga gctcc 75 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <213>

<223> F3PrS Primer <400> 45 ttctcqqcca taqtatqtat <210> 46 <211> 18 <212> DNA <213> Artificial Sequence <220>

<223> F3PrA Primer <4 0 0> 4 6 aqaaccatqq cataqccc <210> 47 <211> 100 <212> DNA

168 <213> Artificial Sequence <220>

<223> FR F3vHS Shuffle Initiator <400> 47 ttcttggcca tagtatgtat gacccgaaat tccagggcag agtctcgatt accgcggacg 60 agtcaacgaa atggacctca acagtctgag 100 <210> <210> <210> <210> <210> <210> <210> <210> <210> <210> <210>

<223> FR F3vHA Shuffle Initiator <400> 48 agaaccatgg catagcccga gtctctcgca cagaaataaa cggccgtgtc ctcagatctc 60 agactqttga gctcc 75 <210> 49 <211> 414 <212> DNA <213> Sequence Art <2132> Sequence

<221> sig-peptide <222> (1) ... (57) <220>

<221> mat-peptide <222> (58) ... (414) <223> Nucleotide sequence encoding the humanized H chain V-region version d <400> 49

169

<210> 50 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão d da humanizada

<210> 51 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide

170 <222> (58)...(414) <223> Sequência nucleotídica codificando para a versão e da região V da cadeia H humanizada

<210> 52 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão e da região V da cadeia H humanizada <400> 52

171

Cys

Leu <210> 53 <211> 100 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3SSS <400> 53 ttcttggcca tagtatgtat gacccgaaat tccagggcag agtcacgatt accgcggaca 60 catccacgag cacagcctac atggagctca ggagcctçag 100 <210> 54 <211> 75 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3SSA <400> 54 agaaccatgg catagcccga gtctctcgca cagtaataca cggccgtgtc gtcagatctc 60 aggctcctga gctcc 75 <210> 55 <211> 100 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3CDS <400> 55 ttcttggcca tagtatgtac gacccgaaat tccagggcaa agccaccctg actgcagacg 60 aatcctccag cacagcctac atgcaactct cgagcctacg 100 <210> 56 <211> 75 <212> ADN <213> Sequência Artificial <220>

172 <223> Iniciador de baralhamento de FR F3CDA <400> 56 agaaccatgg catagcccga gcctcttgca caagaataga ccgcagagtc ctcagatcgt 60 aggctcgaga gttgc 75 <210> 57 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (58)...(414) <223> Sequência nucleótidica codificando para a versão f da região V da cadeia H humanizada <400> 57

<210> 58 <211> 119 <212> PRT <213> Sequência Artificial

173 <220>

<223> Sequência de aminoácidos da versão f da reqião V da cadeia H humanizada <400> 58

115 <210> 59 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> siq-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (53)...(414) <223> Sequência nucleótidica codificando para a versão q da reqião V da cadeia H humanizada <400> 59

174

<210> 60 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão g da região V da cadeia H humanizada <4 0 0> 60

100 105 110

Val Thr Val Ser Ser Ala Ser

115 <210> 61 <211> 100 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3ADS <400> 61 ttcttggcca tagtatgtat gacccgaaat tccagcgccg cgtcaccatg tcagccgaca 60 agtcctccaç cgccgcctat ttacagtgga ccagccttaa 100 <210> 62

175 <211> 75 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3ADA <400> 62 agaaccatgg catagcccga gtcrctcgcg cagaaatata tggcggtgcc cgaggcctca 60 aggctggtcc actgt 75 <210> 63 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (58)...(414) <223> Sequência nucleótidica codificando para a versão h da cadeia H humanizada

176

<210> 64 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão h da reqião da cadeia H humanizada <4 0 0> 64

<210> 65 <211> 100 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3MMS <400> 65 ttcttggcca tagtatgtat gacccgaaat tccagggcag agtcacgatt accgcggaca 60 catcgacgag cacagtcttc atggaactga gcagcctgag 100 <210> 66 <211> 75

177 <212> ADN <213> Sequência Artificial <220>

<td> <223></td><td>Iniciador</td><td>de</td><td>baralhamento</td><td>de</td><td>FR</td><td colspan="2">F3MMA</td>

<td colspan="2">agaaccatgg catagcccga</td><td colspan="2">gtctctcgca cagtaataca</td><td colspan="3">cggccgtgtc ttcagatctc</td><td> 60</td>

<td colspan="2">aggctgctca gttcc</td><td></td><td></td><td></td><td></td><td></td><td> 75</td>

<td> <213></td><td>Sequência</td><td colspan="2">Artificial</td><td></td><td></td><td></td><td></td>

<td> <223></td><td>Iniciador</td><td>de</td><td>baralhamento</td><td>de</td><td>FR</td><td>F3BMS</td><td></td>

<td colspan="2">ttcttggcca cagtatgtat catccgcgaa cacagcctac</td><td>gacccgaaat tccagçgcag atggagctga ggagcctcag</td><td>agtcaccttt</td><td>accçcggaca</td><td> 60 100</td>

<td> <213></td><td>Sequência</td><td>Artificial</td><td></td><td></td><td></td>

<td> <223></td><td>Iniciador</td><td>de baralhamento</td><td colspan="2">de FR F3BMA</td><td></td>

<td colspan="2">agaaccatgg catagcccga</td><td>gtctctcgca caataataaa</td><td>cagccgtgtc</td><td>tgcagatccg</td><td> 60</td>

aggctcctca actcc 75

<td> <213></td><td>Sequência Artificial</td>

<td> <221></td><td>siq-peptide</td>

<td> <221></td><td>mat-peptide</td>

178 <223> Sequência nucleotídica codificando para a versão i da região V da cadeia H humanizada

<210> 70 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão i da região V da cadeia H humanizada <400> 70

179 <210> 71 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (58)...(414) <223> Sequência nucleotídica codificando para a versão j da região V da cadeia H humanizada <400> 71

<210> 72 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão j da região V da cadeia H humanizada <400> 72

180

<210> 73 <211> 79 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F2MPS <400> 73 ttctargcat tgggtgcgcc aggctccagg acagggcctg gagtggatgg gagggaatga 60 tcctgcgaat ggccattct 79 <210> 74 <211> 79 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F2MPA <400> 74 agaatggcca ttcgcaggat cattccctcc catccactcc aggccctgtc ctggagcctg 60 gcgcacccaa tgcatagaa 79 <210> 75 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

181 <221> mat-peptide <222> (58)...(414) <223> Sequência nucleótidica codificando para a versão bl da região V da cadeia H humanizada <400> 75

<210> 76 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão bl da região V da cadeia H humanizada <400> 76

182

Ala Arg Asp Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Leu 100 105 L1Ó

Val Thr Val Ser Ser Ala Ser

115 <210> 77 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (S 8) ... (414) <223> Sequência nucleótidica codificando para a versão dl da região V da cadeia H humanizada <400> 77

<210> 78 <211> 119 <212> PRT <213> Sequência Artificial <220>

183 <223> Sequência de aminoácidos da versão dl da região V da cadeia H humanizada <400> 78

<td> <210> <211> <212> <213></td><td>79 79 ADN Sequência</td><td colspan="2">Artificial</td>

<td> <220> <223></td><td>Iniciador</td><td>de baralhamento</td><td>de FR F2VHS</td>

<td colspan="2">ttctatgcat tgggtgcgac</td><td>aggcccctgg acaagggctt</td><td>gagtggattg gagggaatga</td>

tcctgcgaat ggccatctt 79

<td> <210> <211> <212> <213></td><td>80 79 ADN Sequência</td><td colspan="2">Artificial</td>

<td> <220> <223></td><td>Iniciador</td><td>de baralhamento</td><td>de FR F2VHA</td>

<td colspan="2">aagatggcca ttcgcaggat</td><td>cattccctcc aatccactca</td><td>agcccttgtc caggggcctg</td>

tcgcacccaa tgcatagaa 79

<td> <213></td><td>Sequência Artificial</td>

<220>

184 <221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (58)...(414) <223> Sequência nucleotidica codificando para a versão b3 da região V da cadeia H humanizada <400> 81

<210> 82 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão b3 da região V da cadeia H humanizada <400> 82

185

<210> 83 <211> 414 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(57) <220>

<221> mat-peptide <222> (58)...(419) <223> Sequência nucleotídica codificando para a versão d3 da região V da cadeia H humanizada

186

<210> 84 <211> 119 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão d3 da região V da cadeia H humanizada <400> 84

100

Val Thr Val Ser 115

105

110

Ser Ala Ser <210> 85 <211> 98 <212> ADN <213> Sequência Artificial <220>

<223> Vector de baralhamento de FR h5LvlS <400> 85 gtctagatct ccaccatgag ggcccctgct cagttttttg ggatcttgtt gctctggttt ccagggatcc gatgtgacat ccagatgacc caçtctcc <210> 86 <211> 98 <212> ADN <213> Sequência Artificial

187 <220>

<223> Vector de baralhamento de FR h5Lv4S <400> 86 ttggcagatg gggtcccatc aaggttcagt ggctccggat ctggtaccga tttcactctc 60 accatctcga gtctgcaacc tgaagatttt gcaactta 98 <210> 87 <211> 98 <212> ADN <213> Sequência Artificial <220>

<223> Vector de baralhamento de FR h5Lv2A <400> 87 cttaagaagc ttttaatgtc ctgtgaggcc ttgcacgtga cggtgactct gtctcctaca gatgcagaca gçgaggatgg agactgggtc atctggat <210> 88 <211> 98 <212> ADN <213> Sequência Artificial <220>

<223> Vector de baralhamento de FR h5Lv3A <400> 88 gatgggaccc catctgccaa actagttgca taacagatca ggagcttagg ggctttccct 60 ggtttctgct gataccaacc taagaagctt ttaatgtc 98 <210> 89 <211> 94 <212> ADN <213> Sequência Artificial <220>

<223> Vector de baralhamento de FR h5Lv5A <400> 89 tgttcgtaçg tctgatctcc accttggtcc ctccgccgaa cgtgtacggg ctctcaccat gctgcagaca gtagtaagtt gcaaaatctt cagg <210> 90 <211> 20 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador h5LvS <400> 90 gtctagatct ccaccatgag 20 <210> 91 <211> 19 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador h5LvA <400> 91 tgttcgtacg tttgatctc 19 <210> 92 <211> 381 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(60) <220>

<221> mat-peptide <222> (61)...(381) <223> Sequência nucleótidica codificando para a versão a da região V da cadeia L humanizada <400> 92

189

gag age ccg tac acg ttc ggc gga ggg acc aag gtg gag ate aaa 331

Glu Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

95_100_105 <210> 93 <211> 107 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão a da reqião V da cadeia L humanizada <400> 93

100 105 <210> 94 <211> 77 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3SS <400> 94 gtctggtacc gattacactc tcaccatctc gagcctccag cctgaagatt trgcaactta ctattgtctg cagaaca <210> 95 <211> 77 <212> ADN

190 <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3SA <400> 95 tgtcctgcag acaatagtaa gttgcaaaat cttcaggctg gaggctcgag atggtgagag 50 tgtaatcgqt accagac 77 <210> 96 <211> 77 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3RS <4 0 0> 9 6 gtctggtacc gattacactc tcaccatctc gagcctccag cctgaagata ttgcaactta 60 ctattgtctg cagaaca 77 <210> 97 <211> 77 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F3RA <400> 97 tgttctgcag acaatagtaa gttgcaatat cttcaggctg gaggctcgag atggtgagag 6C tgtaatcggt accagac 77 <210> 98 <211> 381 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(60) <220>

191 <221> mat-peptide <222> (61) ... (381) <223> Sequência nucleótidica codificando para a versão b da região V da cadeia L humanizada <400> 98

<210> 99 <211> 107 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão b da região V da cadeia L humanizada <400> 99

192 <210> 100 <211> 381 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1) ... (60) <220>

<221> mat-peptide <222> (61)...(381) <223> Sequência nucleótidica codificando para a versão c da região V da cadeia L humanizada <400> 100

<210> 101 <211> 107 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos da versão c da região V da cadeia L humanizada <400> 101

193

<210> 102 <211> 72 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F2SS <400> 102 gtctcttaag ttggttccag cagaaaccag gçaaatctcc taagaccctg atctactatg 60 caactagtaa ca 72 <210> 103 <211> 72 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F2SA <400> 103 tgttactagt tgcatagtag atcagggtct taçgagattt ccccçgtttc tgctggaacc 60 aacttaagag ac 72 <210> 104 <211> 72 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F2XS <400> 104 gtctcttaag ttggtatcag cagaaaccag agaaagcccc taagtccctg atctattatg 60 caactagtaa ca 72

194 <210> 105 <211> 72 <212> ADN <213> Sequência Artificial <220>

<223> Iniciador de baralhamento de FR F2XA <400> 105 tgttactagt tgcataatag atcagggact taggggcttt ctctggttrc tgctgatacc 60 aacttaagag ac 72 <210> 106 <211> 381 <212> ADN <213> Sequência Artificial <220>

<221> sig-peptide <222> (1)...(60) <220>

<221> mat-peptide <222> (61)...(381) <223> Sequência nucleótidica codificando para a versão bl da região V da cadeia L humanizada <400> 106

<210> 107

195 <211> 107 <212> PRT <213> Sequência Artificial <220> Sequência de aminoácidos da versão bl da reqião V da cadeia L humanizada <400> 107

<210> 108 <211> 381 <212> ADN <213> Sequência Artificial <220>

<221> siq-peptide <222> (1)...(60) <220>

<221> mat-peptide <222> (61)...(381) <223> Sequência nucleótidica codificando para a versão b2 da reqião V da cadeia L humanizada

196 <400> 108

<210>

<211>

<212>

<213>

109

107

PRT

Sequência Artificial <220>

<223> Sequência de aminoácidos da versão b2 da região V da cadeia L humanizada <400> 109

<210> 110 <211> 30 <212> PRT <213> Sequência Artificial <220>

197 <223> Sequência de aminoácidos de FRl de todas as versões da região V da cadeia H humanizada <400> 110

<210> 111 <211> 14 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR2 das versões a a j da região V da cadeia H humanizada <400> 111

Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile Gly 5 10 <210> 112 <211> 14 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR2 das versões bl e dl da região V da cadeia H humanizada <400> 112

Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met Gly 5 10 <210> 113 <211> 14 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR2 das versões b3 e d3 da região V da cadeia H humanizada <400> 113

Trp Val Arq Gin Ala Pro Gly Gin Gly Leu Glu Trp Ile Gly 5 10 <210> 114 <211> 32

198 <212> PRT <213> Sequência Artificial <220>

<td><223> região</td><td>Sequência de V da cadeia H</td><td>aminoácidos humanizada</td><td>de</td><td>FR3</td><td>da</td><td>versãi</td><td>o a da</td>

<td> <213></td><td colspan="2">Sequência Artificial</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="2">Sequência de aminoácidos de</td><td>FR3</td><td colspan="3">das versões</td><td>(b), (bl)</td>

e (b3) da região V da cadeia H humanizada <400> 115

<210> 116 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão c da região V da cadeia H humanizada <400> 116

<210> 117 <211> 32 <212> PRT <213> Sequência Artificial <220>

199 <223> Sequência de aminoácidos de FR3 das versões d, dl e d3 da região V da cadeia H humanizada <400> 117

<210> 118 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão e da região V da cadeia H humanizada <400> 118

<210> 119 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão f da região V da cadeia H humanizada <400> 119

<210> 120 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão g da região V da cadeia H humanizada <400> 120

200

<210> 121 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão h da região V da cadeia H humanizada <400> 121

<210> 122 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão i da região V da cadeia H humanizada <400> 122

<210> 123 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão j da região V da cadeia H humanizada <400> 123

<210> 124 <211> 13

201 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR4 de todas as versões da região V da cadeia H humanizada <400> 124

Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser 5 10 <210> 125 <211> 23 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FRl de todas as versões da região V da cadeia L humanizada <400> 125

Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 5 10 15

Asp Arg Val Thr Ile Thr Cys 20 <210> 126 <211> 15 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR2 das versões a, b e c da região V da cadeia L humanizada <400> 126

Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 5 10 15 <210> 127 <211> 15 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR2 da versão bl da região V da cadeia L humanizada <400> 127

202

Trp Phe Gin Gin Lys Pro Gly Lys Ser Pro Lys Thr Lee Ile Tyr 5 10 15 <210> 128 <211> 15 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR2 da versão b2 da região V da cadeia L humanizada <400> 128

Trp Tyr Gin Gin Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile Tyr 5 10 15 <210> 129 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão a da região V da cadeia L humanizada <400> 129

<210> 130 <211> 32 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 das versões b, bl e b2 da região V da cadeia L humanizada <400> 130

<210> 131 <211> 32

203 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR3 da versão c da reqião V da cadeia L humanizada <400> 131

<210> 132 <211> 10 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de FR4 de todas as versões da reqião V da cadeia L humanizada <400> 132

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 5 10 <210> 133 <211> 5 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de CDRl de todas as versões da reqião V da cadeia H humanizada <400> 133

Asp Tyr Tyr Met His <210> 134 <211> 17 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de CDR2 de todas as versões da reqião V da cadeia H humanizada <400> 134

204

Gly Asn Asp Pro Ala Asn Gly His Ser Met Tyr Asp 5 10

Gly <210> 135 <211>

<212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de CDR.3 de da região V da cadeia H humanizada <400> 135

Asp Ser Gly Tyr Ala Met Asp Tyr 5 <210> 136 <211> 11 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de CDRl de da região V da cadeia L humanizada <400> 136

Lys Ala Ser Gin Asp Ile Lys Ser Phe Leu Ser 5 10 <210> 137 <211> 7 <212> PRT <213> Sequência Artificial <220>

<223> Sequência de aminoácidos de CDR2 de da região V da cadeia L humanizada <400> 137

Tyr Ala Thr Ser Leu Ala Asp 5

Pro Lys Phe Gin 15 todas as versões todas as versões todas as versões <210> 138 <211> 9 <212> PRT <213> Sequência Artificial <220>

205 <223> Sequência de aminoácidos de CDR3 de todas as versões da região V da cadeia L humanizada <400> 138

Leu Gin His Gly Glu Ser Pro Tyr Thr 5 <210> 139 <211> 118 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia H do anticorpo monoclonal de ratinho anti-TF ATR-2 <400> 139

<210> 140 <211> 118 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia H do anticorpo monoclonal de ratinho anti-TF ATR-3 <400> 140

206

115 <210> 141 <211> 117 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia H do anticorpo monoclonal de ratinho anti-TF ATR-4 <400> 141

<210> 142 <211> 117 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia H do anticorpo monoclonal de ratinho anti-TF ATR-5 <400> 142

207

<210> 143 <211> 118 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da reqião V da cadeia H do anticorpo monoclonal de ratinho anti-TF ATR-7 <400> 143

Asp Ile Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ser 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Pro Asp Tyr

115 <210> 144 <211> 118 <212> PRT <213> Ratinho <220>

208 <223> Sequência de aminoácidos da região V da cadeia H do anticorpo monoclonal de ratinho anti-TF ATR-8 <400> 144

<210> 145 <211> 107 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia L do anticorpo monoclonal de ratinho anti-TF ATR-2 <400> 145

<210> 146 <211> 107 <212> PRT <213> Ratinho <220>

209 <223> Sequência de aminoácidos da reqião V da cadeia L do anticorpo monoclonal de ratinho anti-TF ATR-3 <400> 146

<210> 147 <211> 107 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da reqião V da cadeia L do anticorpo monoclonal de ratinho anti-TF ATR-4 <400> 147

<210> 148 <211> 107 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da reqião V da cadeia L do anticorpo monoclonal de ratinho anti-TF ATR-5 <400> 148

210

<210> 149 <211> 112 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia L do anticorpo monoclonal de ratinho anti-TF ATR-7 <400> 149

<210> 150 <211> 112 <212> PRT <213> Ratinho <220>

<223> Sequência de aminoácidos da região V da cadeia L do anticorpo monoclonal de ratinho anti-TF ATR-8 <400> 150

211

<210> 151 <211> 780 <212> ADN <213> Homo sapiens <220>

<223> ADN codificando para TF humano solúvel <400> 151

212

<td>tea</td><td>agt</td><td>tea</td><td>gga</td><td>aag</td><td>ãLclâ.</td><td>aca</td><td>gcc</td><td>aaa</td><td>aca</td><td>aac</td><td>act</td><td>aat</td><td>gag</td><td>ttt</td><td>ttg</td><td> 624</td>

225 <210> 152 <211> 259 <212> PRT <220>

<223> Sequência de aminoácidos de TF humano solúvel <400> 152

213

Lisboa,

39 members in 19 offices

This recordMember, by publication datePriority claim9 more offices are in the list below

Priority claims4

Priority claims
Document	Office	Kind	Date
9185098	Japan	A	1998-04-03
9185098	Japan	A	1998-04-03
9185098	–	–	–
JP19980091850	–	–	–

Members39

Family members, oldest first
Document	Office	Kind
CA2325346A1	Canada	A1	1999-10-14
WO9951743A1	World Intellectual Property Organization (WIPO)	A1	1999-10-14
AU3055699A	Australia	A	1999-10-25
NO20004946D0	Norway	D0	2000-10-02
NO20004946L	Norway	L	2000-12-04
BR9909382A	Brazil	A	2000-12-05
BR9909382A	Brazil	A	2000-12-05
TR2000002885T2	Türkiye	T2	2000-12-21
TR200002885T2	Türkiye	T2	2000-12-21
EP1069185A1	European Patent Office (EPO)	A1	2001-01-17
KR20010042435A	Republic of Korea	A	2001-05-25
CN1303431A	China	A	2001-07-11
SK14812000A3	Slovakia	A3	2001-08-06
PL343322A1	Poland	A1	2001-08-13
HU0101160A2	Hungary	A2	2001-08-28
HUP0101160A2	Hungary	A2	2001-08-28
IL138801A0	Israel	A0	2001-10-31
TR2001001541T2	Türkiye	T2	2002-06-21
TR200101541T2	Türkiye	T2	2002-06-21
AU752730B2	Australia	B2	2002-09-26
US6677436B1	United States of America	B1	2004-01-13
US2004044187A1	United States of America	A1	2004-03-04
JP2004121253A	Japan	A	2004-04-22
EP1069185A4	European Patent Office (EPO)	A4	2005-01-19
JP2005330292A	Japan	A	2005-12-02
JP2007186522A	Japan	A	2007-07-26
JP3998419B2	Japan	B2	2007-10-24
JP4014558B2	Japan	B2	2007-11-28
JP2008161198A	Japan	A	2008-07-17
JP4180106B2	Japan	B2	2008-11-12
US7494647B2	United States of America	B2	2009-02-24
EP1069185B1	European Patent Office (EPO)	B1	2011-06-08
AT512225T	Austria	T	2011-06-15
ATE512225T1	Austria	T1	2011-06-15
DK1069185T3	Denmark	T3	2011-06-27
PT1069185EThis record	Portugal	E	2011-08-11
ES2364266T3	Spain	T3	2011-08-30
EP2363484A2	European Patent Office (EPO)	A2	2011-09-07
EP2363484A3	European Patent Office (EPO)	A3	2012-04-25

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

Abstract

Term

DESCRIPTION

O

O

O

II

FRY CPR1 FS C0R2

D3 FR3

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

SEQ ID NO:

PRT

Priority claims4

Members39

Numbers

Titles2

Classification