KR102125672B1 - Anti-b7-h3 antibody - Google Patents

Abstract

The present invention relates to an antibody having a therapeutic effect against a tumor. That is, the present invention relates to an antibody that exhibits anti-tumor activity by binding to B7-H3. An object of the present invention is to provide a drug having a therapeutic effect on a tumor. An anti-B7-H3 antibody exhibiting anti-tumor activity by binding to B7-H3 is obtained, and a pharmaceutical composition for treating tumors containing the antibody is obtained.

Description

Anti-VII-7-antibody {ANTI-B7-H3 ANTIBODY}

The present invention relates to an antibody that binds B7-H3 useful as a therapeutic and/or prophylactic agent for tumors, and a method for treating and/or preventing tumors using the antibody.

B7-H3 is a protein having a transmembrane structure once (non-patent document 1). Two variants exist in the extracellular region of the N-terminal side of B7-H3, V or C type Ig domains of each two points exist in variant 1, and V or C type of each one point in variant 2 Ig domain exists. 45 amino acids are present in the intracellular region at the C-terminal side.

As a receptor for B7-H3, TLT-2 having a once transmembrane structure has been reported (Non-Patent Document 2). However, there are some papers claiming that TLT-2 is not a receptor for B7-H3 (Non-Patent Document 3). According to the former report, activation of CD8 positive T cells is enhanced by binding of the receptor to B7-H3.

B7-H3 has been clinically reported to be highly expressed in many cancers, particularly non-small cell lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, and pancreatic cancer (non-patent documents 4 to non Patent Document 11). In addition, in prostate cancer, it has been reported that positively correlated with B7-H3 expression intensity and clinical pathological malignancy such as tumor volume, extraprostate infiltration and Gleason score, and progression of cancer (non Patent Document 8). Similarly, B7-H3 expression and non-recurring survival rate are negatively correlated in polymorphic glioma (Non-Patent Document 9), and in pancreatic cancer, it is correlated with B7-H3 expression, lymph node metastasis, and pathological progression ( Non-Patent Document 11). In ovarian cancer, B7-H3 expression correlates with lymph node metastasis and pathological progression.

In addition, by introducing siRNA for the B7-H3 gene into the B7-H3 positive cancer cell line, it has been reported that adhesion to fibronectin decreases and cell migration ability or matrigel infiltration ability decreases (Non-Patent Document 12). ). Moreover, in polymorphic glioma, there is a report that the expression of B7-H3 escapes from cell death caused by NK cells (Non-Patent Document 13).

On the other hand, it has been reported that B7-H3 is expressed not only in cancer cells but also in blood vessels in or around tumors (Non-Patent Document 5, Non-Patent Document 14). It has been reported that if B7-H3 is expressed in ovarian cancer vessels, the survival rate decreases.

The B7 family molecule has been suggested to be related to the immune system. It has been reported that B7-H3 is expressed in monocytes, dendritic cells or activated T cells (Non-Patent Document 15). B7-H3 has been reported to co-stimulate the proliferation of CD4 positive or CD8 positive T cells with activation of cytotoxic T cells. However, it has been reported that B7-H3 does not co-stimulate (Non-Patent Document 1).

There have been reports that the B7-H3 molecule is associated with autoimmune diseases. In rheumatism or other autoimmune diseases, it has been reported that B7-H3 is important for the interaction of fibroblast type synovial cells with activated T cells (non-patent document 16) or when B7-H3 releases cytokines from activated macrophages. It has been reported to be an auxiliary stimulating factor of, and is related to the development of sepsis (Non-Patent Document 17). In addition, it has been reported that by administering an anti-B7-H3 antibody in an induction phase to a mouse asthma model, Th2 cell-induced cytokine production in the lymph node in question is suppressed by administering an anti-mouse B7-H3 antibody, thereby improving asthma. (Non-Patent Document 18).

As for B7-H3, it has been reported that an antibody against mouse B7-H3 promotes CD8 positive T cells that infiltrate tumors and suppresses tumor proliferation (Non-Patent Document 24). In addition, a patent has been described in which an antibody recognizing variant 1 of B7-H3 exhibits an adenocarcinoma in vivo anti-tumor effect (Patent Document 1).

Despite these studies, until now, the epitope of anti-B7-H3 antibodies that exhibit anti-tumor effects in vivo has not been clarified, and a specific amino acid sequence in the extracellular region of B7-H3 is a monoclonal target for cancer treatment. There are no reports that it is useful as an epitope of raw antibodies.

Even if it is an antibody against the same antigen, the properties of the antibody are different because the epitopes and the arrangement of the antibodies are different. And, because these properties are different, when administered to humans in clinical practice, they exhibit different responses in terms of the effectiveness of the drug, the frequency of therapeutic response, and the frequency of side effects and drug resistance.

For antibodies against B7-H3, the creation of antibodies with properties different from those of the prior art has been strongly demanded.

WO2008/066691

The Journal of Immunology 2004, Vol. 172, p.2352-2359 Proceedings of the national Academy of Sciences of the United States of America 2008, Vol. 105, p.10495-10500 European Journal of Immunology 2009, Volume 39, p.1754-1764 Lung Cancer, 2009, Volume 66, p.245-249 Clinical Cancer Research 2008, Volume 14, p.5150-5157 Clinical Cancer Research 2008, Volume 14, p.4800-4808 Cancer Immunology, Immunotherapy 2010, Vol. 59, p.1163-1171 Cancer Research 2007, Vol. 67, p.7893-7900 Histopathology 2008, Volume 53, p.73-80 Modern Pathology 2010, Volume 23, p.1104-1112 British Journal of Cancer 2009, Vol 101, p.1709-1716 Current Cancer Drug Targets 2008, Volume 8, p.404-413 Proceedings of the national Academy of Sciences of the United States of America 2004, Vol. 101, p.12640-12645 Modern Pathology 2010, Volume 23, p.1104-1112 Nature Immunology 2001, Vol. 2, p. 269-274 The Journal of Immunology 2008, Volume 180, p.2989-2998 The Journal of Immunology 2010, Vol. 185, p.3677-3684 The Journal of Immunology 2008, Vol. 181, p.4062-4071

An object of the present invention is to provide an antibody that becomes a pharmaceutical product having a therapeutic effect against a tumor, a functional fragment of the antibody, and a method of treating a tumor using the antibody or a functional fragment of the antibody.

The present inventors conducted a courtesy study to solve the above problems, and as a result, found an antibody specifically binding to B7-H3 and exhibiting anti-tumor activity, thereby completing the present invention. That is, the present invention includes the following inventions.

(1) An antibody or functional fragment of the antibody, which has the following characteristics;

(a) specifically binds to B7-H3

(b) has antibody-dependent cell-mediated phagocytosis (ADCP) activity

(c) Has anti-tumor activity in vivo

(2) The antibody according to (1) above, or a functional fragment of the antibody, wherein B7-H3 is a molecule consisting of the amino acid sequence shown in SEQ ID NO: 6 or 10.

(3) The antibody according to (1) or (2) above or a functional fragment of the antibody, which binds to the IgC1 and/or IgC2 domains of B7-H3.

(4) IgC1 is a domain consisting of the amino acid sequence of amino acid numbers 140 to 244 in SEQ ID NO: 6, and IgC2 is a domain consisting of the amino acid sequence of amino acid numbers 358 to 456 in sequence number 6, described in (3) above. Antibodies or functional fragments of the antibodies.

(5) The antibody according to any one of (1) to (4) above, or a functional fragment of the antibody, which has a competitive inhibitory activity with the M30 antibody for binding to B7-H3.

(6) The antibody according to any one of (1) to (5) above, or a functional fragment of the antibody, having antibody-dependent cytotoxicity (ADCC) activity and/or complement-dependent cytotoxicity (CDC) activity.

(7) The antibody according to any one of (1) to (6) above, wherein the tumor is cancer, or a functional fragment of the antibody.

(8) The antibody according to (7) above, wherein the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer, or kidney cancer, or a functional fragment of the antibody.

(9) As a complementarity determining region in the heavy chain, CDRH1 consisting of the amino acid sequence of SEQ ID NO: 92, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 93, CDRH3 consisting of the amino acid sequence of SEQ ID NO: 94, and in the light chain Any one of (1) to (8) above having CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 95, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 96 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 97 as a complementarity determining region of The antibody according to claim or a functional fragment of the antibody.

(10) The above having the heavy chain variable region consisting of the amino acid sequence of amino acid numbers 20 to 141 in SEQ ID NO: 51 and the light chain variable region consisting of the amino acid sequence of amino acid numbers 23 to 130 in sequence number 53 ( The antibody according to any one of 1) to (9), or a functional fragment of the antibody.

(11) The antibody according to any one of (1) to (10) above, wherein the constant region is a human-derived normal region, or a functional fragment of the antibody.

(12) The antibody according to (11) above, or a functional fragment of the antibody, having a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 63 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 59.

(13) The antibody according to any one of (1) to (12) above, which is humanized, or a functional fragment of the antibody.

(14) (a) Amino acid sequence of amino acid numbers 20 to 141 in SEQ ID NO: 85, (b) Amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 87, (c) Amino acid number of 20 in SEQ ID NO: 89 To the amino acid sequence of 141 to (d) amino acid sequence of amino acid number 20 to 141 of SEQ ID NO: 91, (e) an amino acid sequence of at least 95% homology to the sequence of (a) to (d), And (f) a heavy chain variable region consisting of an amino acid sequence selected from the group consisting of deletions, substitutions or added amino acid sequences in the sequence of (a) to (d), and (g) sequence numbers. The amino acid sequence of amino acids 21 to 128 in 71, (h) the amino acid sequences of amino acids 21 to 128 in SEQ ID NO: 73, (i) the amino acid sequences of amino acids 21 to 128 in SEQ ID NO: 75, ( j) The amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 77, (k) The amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 79, (l) The amino acid sequences of 21 to 128 in SEQ ID NO: 81 Amino acid sequence, (m) amino acid sequence of amino acid numbers 21 to 128 in SEQ ID NO: 83, (n) amino acid sequence of at least 95% or more homology to the sequence of (g) to (m), and (o) The antibody according to (13) above, which has a light chain variable region consisting of an amino acid sequence selected from the group consisting of deletion, substitution or added amino acid sequence in the sequence of (g) to (m) or the same or Functional fragment of antibody.

(15) The variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 85 and the variable region of the light chain consisting of the amino acid sequences of amino acids 21 to 128 in SEQ ID NO: 71, SEQ ID NO: 85 The variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in, and the variable region of the light chain consisting of the amino acid sequences of amino acid numbers 21 to 128 in SEQ ID NO: 73, amino acid numbers 20 to 20 of SEQ ID NO: To the variable region of the heavy chain consisting of the amino acid sequence described in 141 and the variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 75, to the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 85 The variable region of the heavy chain consisting of the variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 77 of the variable region of the heavy chain, and the variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 85 And the variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 79, the variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 85, and the sequence of 81 The variable region of the light chain consisting of the amino acid sequence of amino acid numbers 21 to 128, the variable region of the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 141 of SEQ ID NO: 85, and the amino acid numbers 21 to 128 of SEQ ID NO: 83. The variable region of the light chain consisting of the amino acid sequence described, the variable region of the heavy chain consisting of the amino acid sequence described in amino acid number 20 to 141 in SEQ ID NO: 91 And the variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 71, the variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 91, and in SEQ ID NO: 73. The variable region of the light chain consisting of the amino acid sequence of amino acid numbers 21 to 128, the variable region of the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 141 of SEQ ID NO: 91, and the amino acid numbers 21 to 128 of SEQ ID NO: 75. The light chain variable region consisting of the amino acid sequence described, and the heavy chain variable region consisting of the amino acid sequence described in amino acid number 20 to 141 in SEQ ID NO: 91 and the amino acid sequence described in amino acid numbers 21 to 128 in SEQ ID NO: 77 The antibody or functional fragment of antibody described in (14) above, which has a heavy chain variable region and a light chain variable region selected from the group consisting of light chain variable regions.

(16) The heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 471 in SEQ ID NO: 85, the light chain consisting of the amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 71, amino acid numbers 20 to 20 of SEQ ID NO: The heavy chain consisting of the amino acid sequence described in 471 and the light chain consisting of the amino acid sequence shown in amino acid number 21 to 233 in SEQ ID NO: 73, the heavy chain consisting of the amino acid sequence shown in amino acid number 20 to 471 in SEQ ID NO: 85 and the sequence number Light chain consisting of the amino acid sequence of amino acids 21 to 233 in 75, heavy chain consisting of the amino acid sequence of amino acids 20 to 471 in SEQ ID NO: 85, and the amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 77 Light chain consisting of, heavy chain consisting of amino acid sequence of amino acid numbers 20 to 471 in SEQ ID NO: 85, light chain consisting of amino acid sequence of amino acid numbers 21 to 233 of SEQ ID NO: 79, amino acid number of SEQ ID NO: 85 The heavy chain consisting of the amino acid sequence of 20 to 471, the light chain consisting of the amino acid sequence of amino acid numbers 21 to 233 in SEQ ID NO: 81, the heavy chain consisting of the amino acid sequence of amino acids 20 to 471 in SEQ ID NO: 85, and The light chain consisting of the amino acid sequence of amino acid numbers 21 to 233 in SEQ ID NO: 83, the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 471 in SEQ ID NO: 91, and the amino acid numbers 21 to 233 of SEQ ID NO: 71 Light chain consisting of amino acid sequence, amino acids set forth in amino acids 20 to 471 in SEQ ID NO: 91 The heavy chain consisting of the sequence and the light chain consisting of the amino acid sequence of amino acid number 21 to 233 in SEQ ID NO: 73, the heavy chain consisting of the amino acid sequence of amino acid number 20 to 471 in SEQ ID NO: 91, and the amino acid in sequence number 75 The light chain consisting of the amino acid sequence of numbers 21-233, the heavy chain consisting of the amino acid sequence of amino acids 20-471 in SEQ ID NO: 91, and the light chain consisting of the amino acid sequences of amino acids 21-233 of SEQ ID NO: 77 The antibody according to (14) or (15) above having a heavy chain and light chain selected from the group consisting of slurries, or functional fragments of the antibody.

(17) The heavy chain consisting of the amino acid sequence of SEQ ID NO: 85, the light chain consisting of the amino acid sequence of SEQ ID NO: 85, the heavy chain consisting of the amino acid sequence of SEQ ID NO: 85, and the light chain consisting of the amino acid sequence of SEQ ID NO: 73 , A heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 85, a light chain consisting of the amino acid sequence shown in SEQ ID NO: 75, a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 85, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 77, the arrangement The heavy chain consisting of the amino acid sequence of No. 85, the light chain consisting of the amino acid sequence of SEQ ID NO: 79, the heavy chain consisting of the amino acid sequence of SEQ ID NO: 85, and the light chain consisting of the amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 85 The heavy chain consisting of the amino acid sequence described in SEQ ID NO: 83, the light chain consisting of the amino acid sequence shown in SEQ ID NO: 91, the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 91, the light chain consisting of the amino acid sequence shown in SEQ ID NO: 71, SEQ ID NO: 91 Heavy chain consisting of amino acid sequence, light chain consisting of amino acid sequence shown in SEQ ID NO: 73, heavy chain consisting of amino acid sequence shown in SEQ ID NO: 91, light chain consisting of amino acid sequence shown in SEQ ID NO: 75, and amino acid shown in SEQ ID NO: 91 The antibody according to any one of (14) to (16) above, or a functional fragment of the antibody, having a heavy chain and a light chain selected from the group consisting of a heavy chain consisting of an array and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 77. .

(18) The functional fragment of the antibody according to any one of (1) to (17) above, wherein the functional fragment is selected from the group consisting of Fab, F(ab)2, Fab' and Fv.

(19) The polynucleotide encoding the antibody according to any one of (1) to (18) above, or a functional fragment of the antibody.

(20) The polynucleotide according to (19) above, having a nucleotide sequence of 58 to 423 in SEQ ID NO: 50, and a nucleotide sequence of 67 to 390 in SEQ ID NO: 52.

(21) The polynucleotide according to (19) or (20) above, which is the nucleotide sequence of SEQ ID NO: 62 and the nucleotide sequence of SEQ ID NO: 58.

(22) (a) the nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 84, (b) the nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 86, (c) the nucleotide number 58 of SEQ ID NO: 88 A polynucleotide consisting of the nucleotide sequence described in 423 to (d) the nucleotide sequence described in nucleotide numbers 58 to 423 in SEQ ID NO: 90, and (e) a nucleotide sequence complementary to the nucleotide sequence described in (a) to (d). A nucleotide sequence selected from the group consisting of the nucleotide sequence of the polynucleotide hybridized under stringent conditions, and (f) the nucleotide sequences described in nucleotide numbers 61 to 384 in SEQ ID NO: 70, and (g) in SEQ ID NO: 72. The nucleotide sequence of nucleotide numbers 61 to 384 in (h) the nucleotide sequence of nucleotide numbers 61 to 384 in sequence number 74, (i) the nucleotide sequence of nucleotide numbers 61 to 384 in sequence number 76, (j) The nucleotide sequence of nucleotide numbers 61 to 384 in SEQ ID NO: 78, (k) the nucleotide sequence of nucleotide numbers 61 to 384 in SEQ ID NO: 80, (l) the nucleotide sequence of nucleotide numbers 61 to 384 in SEQ ID NO: 82 , And (m) a nucleotide sequence selected from the group consisting of a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence described in (f) to (l) and a nucleotide sequence of a polynucleotide hybridized under stringent conditions. The polynucleotide according to (19) or (20) above.

(23) The nucleotide sequence shown in nucleotide numbers 58 to 423 in SEQ ID NO: 84 and the nucleotide sequence shown in nucleotide numbers 61 to 384 in SEQ ID NO: 70, the nucleotide sequence shown in nucleotide numbers 58 to 423 in SEQ ID NO: 84 and the sequence number The nucleotide sequence of nucleotide numbers 61 to 384 at 72, the nucleotide sequence of nucleotide numbers 58 to 423 at SEQ ID NO: 84 and the nucleotide sequence of nucleotide numbers 61 to 384 at SEQ ID NO: 74, the nucleotide number of SEQ ID NO: 84 The nucleotide sequence of 58 to 423 and the nucleotide sequence of nucleotide numbers 61 to 384 of SEQ ID NO: 76, the nucleotide sequence of nucleotide numbers 58 to 423 of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 78 of nucleotides of 61 to 384 Nucleotide sequence, nucleotide sequence of nucleotide number 58-423 in sequence number 84, and nucleotide sequence of nucleotide number 61-384 in sequence number 80, nucleotide sequence and sequence number of nucleotide number 58-423 in sequence number 84. The nucleotide sequence of nucleotide numbers 61 to 384 in 82, the nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 90 and the nucleotide sequence of nucleotide numbers 61 to 384 in SEQ ID NO: 70, the nucleotide number in SEQ ID NO: 90 The nucleotide sequence of 58 to 423 and the nucleotide sequence of nucleotide numbers 61 to 384 of SEQ ID NO: 72, the nucleotide sequence of nucleotide numbers 58 to 423 of SEQ ID NO: 90 And in the group consisting of the nucleotide sequence set forth in nucleotide numbers 61 to 384 in SEQ ID NO: 74, and the nucleotide sequence set forth in nucleotide numbers 58 to 423 in SEQ ID NO: 90 and the nucleotide sequence set forth in nucleotide numbers 61 to 384 in SEQ ID NO: 76. The polynucleotide according to (22) above, having a nucleotide sequence of choice.

(24) The nucleotide sequence as set forth in nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and the nucleotide sequence as shown in nucleotide numbers 61 to 699 in SEQ ID NO: 70, the nucleotide sequence as shown in nucleotide numbers 58 to 1413 in SEQ ID NO: 84 Nucleotide sequence shown in nucleotide number 61-699 in 72, nucleotide sequence shown in nucleotide number 58-1413 in sequence number 84, and nucleotide sequence shown in nucleotide number 61-699 in sequence number 74, nucleotide number in sequence number 84 The nucleotide sequence of 58 to 1413 and the nucleotide sequence of nucleotide numbers 61 to 699 at SEQ ID NO: 76, the nucleotide sequence of nucleotide numbers 58 to 1413 at SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 78 to nucleotide numbers 61 to 699 Nucleotide sequence, nucleotide sequence of nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and nucleotide sequence of nucleotide numbers 61 to 699 in SEQ ID NO: 80, nucleotide sequence and sequence number of nucleotide numbers 58 to 1413 in SEQ ID NO: 84 The nucleotide sequence shown in nucleotide number 61-699 in 82, the nucleotide sequence shown in nucleotide number 58-1413 in sequence number 90, the nucleotide sequence shown in nucleotide number 61-699 in sequence number 70, the nucleotide number in sequence number 90 The nucleotide sequence described in 58-1413 and the nucleotide sequence described in nucleotide numbers 61-699 in SEQ ID NO: 72, the nucleotide sequence described in nucleotide numbers 58-1413 in SEQ ID NO: 90 Consisting of the nucleotide sequence shown in nucleotide numbers 61 to 699 in nucleotide sequence and SEQ ID NO: 74, and the nucleotide sequence shown in nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and the nucleotide sequence shown in nucleotide numbers 61 to 699 in SEQ ID NO: 76 The polynucleotide according to (22) or (23) above, having a nucleotide sequence selected from the group.

(25) The nucleotide sequence of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 70, the nucleotide sequence of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 72, the nucleotide sequence of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 74 , The nucleotide sequence shown in SEQ ID NO: 84 and the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 84 and the nucleotide sequence shown in SEQ ID NO: 78, the nucleotide sequence shown in SEQ ID NO: 84 and the nucleotide sequence shown in SEQ ID NO: 80 Nucleotide sequence shown in number 84 and nucleotide sequence shown in sequence number 82, nucleotide sequence shown in sequence number 90 and nucleotide sequence shown in sequence number 70, nucleotide sequence shown in sequence number 90, nucleotide sequence shown in sequence number 72, sequence number 90 The nucleotide sequence according to any one of (22) to (24) above, which is a nucleotide sequence selected from the group consisting of the nucleotide sequence described in SEQ ID NO: 74 and the nucleotide sequence described in SEQ ID NO: 90 and the nucleotide sequence described in SEQ ID NO: 76. The polynucleotide described in.

(26) An expression vector containing the polynucleotide according to any one of (19) to (25) above.

(27) A host cell transformed with the expression vector according to (26) above.

(28) The host cell according to (27) above, wherein the host cell is a eukaryotic cell.

(29) A step characterized by comprising the step of culturing the host cell according to (27) or (28) above, and a step of collecting a target antibody or functional fragment of the antibody from the culture obtained in the step. Methods for the production of antibodies or fragments.

(30) An antibody or functional fragment of the antibody, which is obtained by the production method of (29) above.

(31) The functional fragment of the antibody according to (30) above, wherein the functional fragment is selected from the group consisting of Fab, F(ab)2, Fab' and Fv.

(32) The antibody according to any one of (1) to (18), (30), or (31), wherein the sugar chain modification is regulated to enhance antibody-dependent cytotoxic activity, or a functional fragment of the antibody.

(33) A pharmaceutical composition comprising at least one of the antibody according to (1) to (18), (30) to (32) or a functional fragment of the antibody.

(34) The pharmaceutical composition according to (33) above, for the treatment of tumors.

(35) A pharmaceutical composition for treating a tumor, comprising the antibody according to (1) to (18), (30) to (32), or at least one functional fragment of the antibody, and at least one cancer therapeutic agent. .

(36) The pharmaceutical composition according to (34) or (35) above, wherein the tumor is cancer.

(37) The pharmaceutical composition according to the above (36), wherein the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, stomach cancer, esophageal cancer, or kidney cancer.

(38) A method of treating a tumor, characterized in that at least one of the antibody (1) to (18), (30) to (32) or a functional fragment of the antibody is administered to an individual.

(39) The antibody according to (1) to (18), (30) to (32), or at least one of the functional fragments of the antibody, and at least one therapeutic agent for cancer are administered to an individual simultaneously, separately, or continuously. Treatment method of a tumor, characterized in that.

(40) The treatment method according to (38) or (39) above, wherein the tumor is cancer.

(41) The treatment method according to the above (40), wherein the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, stomach cancer, esophageal cancer, or kidney cancer.

According to the present invention, it is possible to obtain a therapeutic agent for cancer or the like containing an antibody having anti-tumor activity against cancer cells by binding to B7-H3.

1 is a diagram showing the presence or absence of ADCP activity on NCI-H322 cells of an anti-B7-H3 antibody. The error bar in the figure represents the standard error (n = 3).
2 is a diagram showing the presence or absence of ADCP activity on NCI-H322 cells of a commercial anti-B7-H3 antibody.
3 is a diagram showing the presence or absence of ADCC activity on the empty vector of the M30 antibody and B7-H3 expressing 293 cells. The error bar in the figure represents the standard error (n = 3). In addition, "mock" in the figure means the ADCC activity of M30 for 293 cells expressing the empty vector, and "B7H3" means the ADCC activity of M30 for 293 cells expressing B7-H3.
4 is a diagram showing the presence or absence of CDC activity on NCI-H322 cells of an anti-B7-H3 antibody. The error bar in the figure represents the standard error (n = 3).
5A is a diagram showing the reactivity of M30 antibody to B7-H3 deletion variant (B7-H3 IgV1). The binding of the control antibody is indicated by the dotted line, and the binding of the M30 hybridoma culture supernatant is indicated by the solid line.
5B is a diagram showing the reactivity of the M30 antibody to the B7-H3 deletion variant (B7-H3 IgC1). The binding of the control antibody is indicated by the dotted line, and the binding of the M30 hybridoma culture supernatant is indicated by the solid line.
5C is a diagram showing the reactivity of the M30 antibody to the B7-H3 deletion variant (B7-H3 IgV2). The binding of the control antibody is indicated by the dotted line, and the binding of the M30 hybridoma culture supernatant is indicated by the solid line.
5D is a diagram showing the reactivity of the M30 antibody to the B7-H3 deletion variant (B7-H3 IgC2). The binding of the control antibody is indicated by the dotted line, and the binding of the M30 hybridoma culture supernatant is indicated by the solid line.
5E is a diagram showing the reactivity of M30 antibody to B7-H3 deletion variant (B7-H3 IgC1-V2-C2). The binding of the control antibody is indicated by the dotted line, and the binding of the M30 hybridoma culture supernatant is indicated by the solid line.
5F is a diagram showing the reactivity of the M30 antibody to the B7-H3 deletion variant (B7-H3 IgV2-C2). The binding of the control antibody is indicated by the dotted line, and the binding of the M30 hybridoma culture supernatant is indicated by the solid line.
6 is a diagram showing the anti-tumor activity of anti-B7-H3 antibody against mice implanted with NCI-H322 cells. The error bar in the figure represents the standard error (n = 10).
7 is a diagram showing the anti-tumor effect of M30 antibody when phagocytic cells are removed from the living body. The error bar in the figure represents the standard error (n = 8). In addition, "mm＾3" means "㎣".
8 is a diagram showing ADCP activity of M30 antibody and cM30 antibody on NCI-H322 cells. The error bar in the figure represents the standard error (n = 4).
9 is a diagram showing the anti-tumor effect of M30 antibody and cM30 antibody on mice transplanted with MDA-MB-231 cells. The error bars in the figures represent standard errors (n = 9).
FIG. 10A is a diagram showing the contention inhibitory activity of cM30 antibody and M30-H1-L4 antibody to the extracellular region polypeptide antigen binding of B7-H3 variant 1 antigen of M30. The error bar in the figure represents the standard error (n = 3).
10B is a diagram showing the contention inhibitory activity of cM30 antibody and M30-H1-L4 antibody to the extracellular region polypeptide antigen binding of B7-H3 variant 2 antigen of M30. The error bar in the figure represents the standard error (n = 3).
11 is a diagram showing ADCP activity against NCI-H322 cells of M30 antibody, cM30 antibody, and M30-H1-L4 antibody. The error bar in the figure represents the standard error (n = 4).
12 is a diagram showing ADCC activity against NCI-H322 cells of cM30 antibody and M30-H1-L4 antibody. The error bar in the figure represents the standard error (n = 3).
13A shows the nucleotide sequence of B7-H3 variant 1 (SEQ ID NO: 5).
13B shows the amino acid sequence of B7-H3 variant 1 (SEQ ID NO: 6).
14A shows the nucleotide sequence of B7-H3 variant 2 (SEQ ID NO: 9).
14B shows the amino acid sequence of B7-H3 variant 2 (SEQ ID NO: 10).
15A shows the nucleotide sequence of B7-H3 IgV1 (SEQ ID NO: 20).
15B shows the amino acid sequence of B7-H3 IgV1 (SEQ ID NO: 21).
16A shows the nucleotide sequence of B7-H3 IgC1 (SEQ ID NO: 22).
16B shows the amino acid sequence of B7-H3 IgC1 (SEQ ID NO: 23).
17A shows the nucleotide sequence of B7-H3 IgV2 (SEQ ID NO: 24).
17B shows the amino acid sequence of B7-H3 IgV2 (SEQ ID NO: 25).
18A shows the nucleotide sequence of B7-H3 IgC2 (SEQ ID NO: 26).
18B shows the amino acid sequence of B7-H3 IgC2 (SEQ ID NO: 27).
19A shows the nucleotide sequence of B7-H3 IgC1-V2-C2 (SEQ ID NO: 28).
19B shows the amino acid sequence of B7-H3 IgC1-V2-C2 (SEQ ID NO: 29).
20A shows the nucleotide sequence of B7-H3 IgV2-C2 (SEQ ID NO: 30).
20B shows the amino acid sequence of B7-H3 IgV2-C2 (SEQ ID NO: 31).
21A shows the nucleotide sequence of the M30 antibody heavy chain (SEQ ID NO: 50).
21B shows the amino acid sequence of the M30 antibody heavy chain (SEQ ID NO: 51).
22A shows the nucleotide sequence of the M30 antibody light chain (SEQ ID NO: 52).
22B shows the amino acid sequence of the M30 antibody light chain (SEQ ID NO: 53).
23 shows the nucleotide sequence of human κ chain secretion signal, human κ chain normal region and human polyA addition signal (SEQ ID NO: 56).
24 shows the human IgG1 signal sequence and the nucleotide sequence of the constant region (SEQ ID NO: 57).
25A shows the nucleotide sequence of the M30 antibody chimeric type light chain (SEQ ID NO: 58).
25B shows the amino acid sequence of the M30 antibody chimeric type light chain (SEQ ID NO: 59).
26A shows the nucleotide sequence of the M30 antibody chimeric type heavy chain (SEQ ID NO: 62).
26B shows the amino acid sequence of the M30 antibody chimeric type heavy chain (SEQ ID NO: 63).
27A shows the nucleotide sequence of the M30-L1 type light chain (SEQ ID NO: 70).
27B shows the amino acid sequence (arrangement number 71) of the M30-L1 type light chain.
28A shows the nucleotide sequence of the M30-L2 type light chain (SEQ ID NO: 72).
28B shows the amino acid sequence of the M30-L2 type light chain (SEQ ID NO: 73).
29A shows the nucleotide sequence of the M30-L3 type light chain (SEQ ID NO: 74).
29B shows the amino acid sequence of the M30-L3 type light chain (SEQ ID NO: 75).
30A shows the nucleotide sequence of the M30-L4 type light chain (SEQ ID NO: 76).
30B shows the amino acid sequence of the M30-L4 type light chain (SEQ ID NO: 77).
31A shows the nucleotide sequence of the M30-L5 type light chain (SEQ ID NO: 78).
31B shows the amino acid sequence of the M30-L5 type light chain (SEQ ID NO: 79).
32A shows the nucleotide sequence of M30-L6 type light chain (SEQ ID NO: 80).
32B shows the amino acid sequence of the M30-L6 type light chain (SEQ ID NO: 81).
33A shows the nucleotide sequence of the M30-L7 type light chain (SEQ ID NO: 82).
33B shows the amino acid sequence of the M30-L7 type light chain (SEQ ID NO: 83).
Figure 34A shows the nucleotide sequence of the M30-H1 type heavy chain (SEQ ID NO: 84).
Figure 34b shows the amino acid sequence of the M30-H1 type heavy chain (SEQ ID NO: 85).
35A shows the nucleotide sequence of the M30-H2 type heavy chain (SEQ ID NO: 86).
35B shows the amino acid sequence of the M30-H2 type heavy chain (SEQ ID NO: 87).
36A shows the nucleotide sequence of the M30-H3 type heavy chain (SEQ ID NO: 88).
36B shows the amino acid sequence of the M30-H3 type heavy chain (SEQ ID NO: 89).
37A shows the nucleotide sequence of M30-H4 type heavy chain (SEQ ID NO: 90).
37B shows the amino acid sequence of the M30-H4 type heavy chain (SEQ ID NO: 91).
38 is a diagram showing the anti-tumor effect of humanized M30 (M30-H1-L4) antibody against mice transplanted with MDA-MB-231 cells. The error bar in the figure represents the standard error (n = 6).

In this specification, "cancer" and "tumor" are used in the same sense.

In the present specification, the word "gene" includes not only DNA, but also its mRNA, cDNA, and its cRNA.

In this specification, the word "polynucleotide" is used in the same sense as nucleic acid, and includes DNA, RNA, probes, oligonucleotides, and primers.

In the present specification, "polypeptide" and "protein" are used without distinction.

In the present specification, "cells" also include cells in animal individuals and cultured cells.

In the present specification, "B7-H3" is used in the same sense as the B7-H3 protein, and also means B7-H3 variant 1 and/or B7-H3 variant 2.

"Cell injury" in the present specification refers to a pathological change in a cell in any form, and does not remain in direct trauma, but does not remain in direct trauma, DNA cleavage, base dimer formation, chromosome cleavage, cell division device It refers to the damage to the structure and function of all cells, such as damage to the enzyme and reduction of various enzyme activities.

The term "cytotoxic activity" in the present specification means to cause the cytotoxicity.

"Antibody-dependent cell-mediated phagocytic activity" in the present specification means "antibody dependent cell phagocytosis (ADCP) activity", and means a functional activity in which a single or macrophage cell phagocytes target cells such as tumor cells through an antibody. Also called "antibody-dependent phagocytic activity".

"Antibody-dependent cellular cytotoxicity" in the present specification means "antibody dependent cellular cytotoxicity (ADCC) activity", and refers to an action activity in which NK cells injure target cells such as tumor cells through antibodies.

The term "complement dependent cytotoxicity (CDC) activity" in the present specification means "complement dependent cytotoxicity (CDC) activity", and refers to an activity activity in which complement damages target cells such as tumor cells through an antibody.

The term "functional fragment of an antibody" in the present specification is a partial fragment of an antibody having an antigen-binding activity, and refers to a fragment that retains all or part of the function of the antibody, and Fab, F(ab ')2, scFv, and the like. In addition, Fab', a monovalent fragment of the variable region of the antibody treated with F(ab')2 under reducing conditions, is also included in the functional fragment of the antibody. However, it is not limited to these molecules as long as it has the ability to bind antigen. In addition, these functional fragments include not only the full-length molecule of the antibody protein treated with an appropriate enzyme, but also a protein produced in a suitable host cell using a genetically modified antibody gene.

"Fab'" in the present invention is a monovalent fragment of the variable region of an antibody treated with F(ab')2 under reducing conditions as described above. However, Fab' produced using a genetically modified antibody gene is also included in Fab' in the present invention.

"Epitope" in the present specification means a partial peptide or partial conformation of B7-H3 to which a specific anti-B7-H3 antibody binds. The epitope, which is a partial peptide of B7-H3, can be determined by methods well known to those skilled in the art, such as an immunoassay method, for example, the following method. First, various partial structures of antigens are prepared. In the preparation of the partial structure, known oligopeptide synthesis techniques can be used. For example, a series of polypeptides sequentially shortened to a suitable length from the C-terminal or N-terminal of B7-H7 is prepared using genetic recombination techniques well known to those skilled in the art, and then the reactivity of the antibody to them is examined, approximately After determining the recognition site, epitopes can be determined by synthesizing shorter peptides and examining their reactivity with the peptides. In addition, an epitope, which is a partial stereoscopic structure of an antigen to which a specific antibody binds, can be determined by specifying an amino acid residue of an antigen adjacent to the antibody by X-ray structural analysis.

The term "antibody that binds to the same epitope" in the present specification means a different antibody that binds to a common epitope. When the second antibody binds to the partial peptide or partial conformation to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope. Further, by confirming that the second antibody competes with the first antibody for binding to the antigen (that is, the second antibody interferes with the binding of the first antibody and the antigen), the specific epitope sequence or structure has not been determined. Even if it can be determined that the first antibody and the second antibody bind to the same epitope. In addition, when the first antibody and the second antibody bind to the same epitope and the first antibody has special effects such as anti-tumor activity, it can be expected that the second antibody also has the same activity. Therefore, when the second anti-B7-H3 antibody binds to the partial peptide to which the anti-B7-H3 antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope as B7-H3. In addition, by confirming that the second anti-B7-H3 antibody competes for the binding of the anti-B7-H3 antibody to B7-H3, the first antibody and the second antibody bind to the same epitope as B7-H3. Can be judged.

"CDR" in this specification means a complementarity determining region (CDR: Complemetarity deterring region). It is known that the heavy and light chains of the antibody molecule each have 3 points of CDR. CDR is also referred to as a hypervariable domain, and in the variable regions of the heavy and light chains of the antibody, is a site having a particularly high variability of the primary structure, and on the primary structure of the heavy and light chain polypeptide chains, Each is divided into three points. In the present specification, for the CDR of the antibody, the CDRs of the heavy chain are denoted as CDRH1, CDRH2, CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain CDRL1 from the amino terminal side of the light chain amino acid sequence, It is referred to as CDRL2, CDRL3. These sites are close to each other on the three-dimensional structure, determining the specificity for the binding antigen.

In the present invention, "hybridize under stringent conditions" means a hybridizing solution at 68°C or a DNA-fixed filter in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech). After hybridization was carried out at 68° C. in the presence of 0.7-1.0 M NaCl using 0.1 to 2-fold SSC solution (1-fold SSC is composed of 150 mM NaCl, 15 mM sodium citrate), Refers to hybridizing under conditions that can be identified by washing at 68°C or equivalent conditions.

1.B7-H3

B7-H3 is a member of the B7 family expressed as an auxiliary stimulatory molecule in antigen-presenting cells, and is thought to promote or inhibit immune action by acting on a receptor on T cells.

B7-H3 is a protein having a transmembrane structure once, but there are two variants in the extracellular region on the N-terminal side of B7-H3. B7-H3 variant 1 (4Ig-B7-H3) has V or C type Ig domains at each 2 points, and B7-H3 variant 2 (2Ig-B7-H3) has V or C type at each 1 point. Ig domain exists.

The B7-H3 used in the present invention can be directly purified from B7-H3 expressing cells of humans, non-human mammals (rats, mice, etc.) or used as a preparation of cell membrane fractions of the cells. Can be obtained by synthesis in vitro or by production in a host cell by genetic manipulation. In genetic manipulation, specifically, B7-H3 cDNA is inserted into an expressible vector, and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic or eukaryotic host cells. The protein can be obtained by expressing B7-H3 by transforming.

The nucleotide sequence of the open reading frame (ORF) of the human B7-H3 variant 1 gene is described in SEQ ID NO: 5 of the sequence table, and the amino acid sequence is described in sequence number 6 of the sequence table. In addition, the arrangement of the arrangement numbers 5 and 6 is described in FIG.

The nucleotide sequence of the ORF of the human B7-H3 variant 2 gene is set forth in SEQ ID NO: 9 of the Sequence Listing, and its amino acid sequence is shown in SEQ ID NO: 10 of the Sequence Listing. In addition, the arrangement of the arrangement numbers 9 and 10 is described in FIG.

In addition, in each amino acid sequence of B7-H3, one or several amino acids are composed of substituted, deleted and/or added amino acid sequences, and proteins having biological activity equivalent to the protein are also included in B7-H3.

The mature human B7-H3 variant 1 from which the signal sequence has been removed corresponds to an amino acid sequence consisting of the 27th to 534th amino acid residues of the amino acid sequence shown in SEQ ID NO:6. In addition, the mature human B7-H3 variant 2 from which the signal sequence was removed corresponds to an amino acid sequence consisting of the 27th to 316th amino acid residues of the amino acid sequence shown in SEQ ID NO:6.

In B7-H3 variant 1, each domain is present in the order of IgV1-IgC1-IgV2-IgC2 from the N-terminus, in sequence number 6 of the sequence table, IgV1 corresponds to amino acid numbers 27 to 139, and IgC1 is an amino acid It corresponds to number 140-244, IgV2 corresponds to amino acid number 245-357, and IgC2 corresponds to amino acid number 358-456. In addition, in B7-H3 variant 2, each domain is present in the order of IgV1-IgC2 from the N-terminus, in sequence number 10 of the sequence table, IgV1 corresponds to amino acid numbers 27 to 140, and IgC2 is amino acid number 141 To 243.

The cDNA of B7-H3 is a polymerase chain reaction (hereinafter referred to as "PCR") using primers that specifically amplify the cDNA of B7-H3, using, for example, a cDNA library expressing the cDNA of B7-H3 as a template. (Saiki, RK, et al., Science, (1988) 239, 487-49). In addition, a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 5 or 9 in the sequence table, hybridized under stringent conditions, and further encoding a protein having a biological activity equivalent to B7-H3. It is included in the cDNA of Figure B7-H3. In addition, a splicing variant transcribed from the human or mouse B7-H3 locus or a polynucleotide hybridized under conditions stringent thereto, and also encodes a protein having a biological activity equivalent to B7-H3 The polynucleotide described above is also included in the cDNA of B7-H3.

Further, in the amino acid sequence shown in SEQ ID NO: 6 or 10 in the sequence table, or in the amino acid sequence in which the signal sequence is removed from these sequences, one or several amino acids are substituted, deleted, or added to the amino acid sequence, B7-H3 B7-H3 also includes proteins having biological activity equivalent to. In addition, in the amino acid sequence coded as a splicing variant that is transcribed from the human or mouse B7-H3 locus or its amino acid sequence, one or several amino acids are composed of substituted, deleted, or added amino acid sequences, and also B7 Proteins having a biological activity equivalent to -H3 are also included in B7-H3.

2. Preparation of anti-B7-H3 antibodies

Antibodies against B7-H3 of the present invention can be obtained by immunizing animals with any polypeptide selected from the amino acid sequence of B7-H3 or B7-H3 using conventional methods, and harvesting and purifying antibodies produced in vivo. have. The species of B7-H3 to be an antigen is not limited to humans, and B7-H3 derived from animals other than humans such as mice and rats can also be immunized to animals. In this case, an antibody applicable to a human disease can be selected by testing the cross-linkage between the obtained heterologous B7-H3 and the human B7-H3.

In addition, known methods (e.g., Kohler and Milstein, Nature (1975) 256, p.495-497, Kennet, R. ed., Monoclonal Antibodies, p.365-367, Plenum Press, NY (1980)) According to this, hybridomas can be established by fusing antibody-producing cells and myeloma cells producing antibodies against B7-H3 to obtain monoclonal antibodies.

In addition, B7-H3 as an antigen can be obtained by producing a B7-H3 gene in a host cell by genetic manipulation.

Specifically, a vector capable of expressing the B7-H3 gene may be prepared, introduced into a host cell to express the gene, and the expressed B7-H3 may be purified. Hereinafter, the method of obtaining the antibody specifically for B7-H3 will be described.

(1) Preparation of antigen

Antigens for producing anti-B7-H3 antibodies include B7-H3 or a polypeptide composed of at least six consecutive partial amino acid sequences thereof, or derivatives to which an arbitrary amino acid sequence or carrier is added.

B7-H3 can be directly purified from human tumor tissue or tumor cells, and can also be obtained by synthesizing B7-H3 in vitro or producing it in a host cell by genetic manipulation.

In the genetic manipulation, specifically, after inserting the cDNA of B7-H3 into an expressible vector, it is synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic or eukaryotic host cells can be synthesized. An antigen can be obtained by expressing B7-H3 by transformation.

It is also possible to obtain an antigen as a secreted protein by expressing a fusion protein in which the extracellular region of the membrane protein B7-H3 and the normal region of the antibody are expressed in an appropriate host/vector system.

The cDNA of B7-H3 is, for example, a cDNA library expressing the cDNA of B7-H3 as a template, and a polymerase chain reaction (hereinafter referred to as ``PCR'') using primers that specifically amplify the B7-H3 cDNA. ) (See Saiki, RK, et al. Science (1988) 239, p.487-489)).

Examples of the in vitro synthesis of the polypeptide include, but are not limited to, Rapid Translation System (RTS) manufactured by Roche Diagnostics.

Examples of the host of prokaryotic cells include Escherichia coli, Bacillus subtilis, and the like. To transform the gene of interest in these host cells, the host cell is transformed with a plasmid vector containing a replicon from a species compatible with the host, namely a replication origin and a regulatory sequence. Moreover, as a vector, it is preferable to have the arrangement|positioning which can provide the selectivity of a expression trait (phenotype) to a transformed cell.

Eukaryotic host cells include cells such as vertebrates, insects, and yeast, and vertebrate cells include, for example, COS cells (Gluzman, Y. Cell (1981) 23, p.175-, which are cells of monkeys). 182, ATCC CRL-1650), a mouse fibroblast NIH3T3 (ATCC No. CRL-1658) or Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydro folic acid reductase deletion strain (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1980) 77, p.4126-4220) and the like are frequently used, but are not limited to these.

The transformant obtained as described above can be cultured according to a conventional method, and the target polypeptide is produced intracellularly or extracellularly by the culture.

The medium used for the culture can be appropriately selected from various types commonly used depending on the host cell employed. If it is E. coli, for example, LB medium can be used by adding antibiotics such as ampicillin or IPMG, if necessary. .

By the above culture, the recombinant protein produced in or outside the transformant can be separated and purified by various known separation manipulation methods using the physical or chemical properties of the protein.

As the method, specifically, various liquid chromatography such as treatment with a conventional protein precipitating agent, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. Grafting, dialysis, and combinations thereof.

In addition, by linking a histidine tag consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. Alternatively, by linking the Fc region of IgG to the recombinant protein to be expressed, the protein A column can be efficiently purified.

By combining the above methods, the desired polypeptide can be easily produced in large quantities with high yield and high purity.

(2) Preparation of anti-B7-H3 monoclonal antibody

Examples of antibodies that specifically bind to B7-H3 include monoclonal antibodies that specifically bind to B7-H3, but the method of obtaining them is as described below.

In the production of monoclonal antibodies, the following working steps are generally required.

In other words,

(a) purification of biopolymers used as antigens,

(b) after immunization by injecting an antigen to an animal, blood is collected, the antibody titer is assayed to determine the timing of spleen extraction, and then antibody producing cells are prepared;

(c) preparation of myeloma cells (hereinafter referred to as "myeloma"),

(d) cell fusion of antibody producing cells with myeloma,

(e) selection of a hybridoma group producing the target antibody,

(f) split into single cell clones (cloning),

(g) In some cases, cultivation of hybridomas for mass production of monoclonal antibodies, or breeding of animals transplanted with hybridomas,

(h) Physiological activity of the monoclonal antibody thus prepared, and examination of its binding specificity, or assay of properties as a labeling reagent.

Hereinafter, the method for producing a monoclonal antibody will be described in detail according to the above steps, but the method for producing the antibody is not limited to this, and for example, antibody producing cells other than non-cells and myeloma may be used.

(a) Purification of antigen

As an antigen, B7-H3 prepared by the method as described above or a part thereof can be used.

In addition, a membrane fragment prepared from a B7-H3 expressing recombinant cell, or a B7-H3 expressing recombinant cell itself, or a partial peptide of the protein of the present invention chemically synthesized using methods well known to those skilled in the art, can also be used as an antigen.

(b) Preparation of antibody producing cells

The antigen obtained in step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant, or Cali alum, and immunized to the experimental animal as an immunogen. Experimental animals can be used without difficulty in the animals used in the known hybridoma production method. Specifically, for example, mice, rats, goats, sheep, cows, and horses can be used. However, it is preferable to make the mouse or rat an immunized animal from the viewpoint of availability of myeloma cells to be fused with the extracted antibody producing cells.

In addition, there are no particular restrictions on the actually used mouse and rat strains, and for mice, for example, each strain A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, 129, etc., also for rats, for example Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer and the like can be used.

These mice and rats can be obtained from, for example, experimental animal breeding vendors such as Nippon Klea and Nippon Charles River.

Of these, considering the compatibility with the myeloma cells described later, the BALB/c line in mice is particularly preferred as the immunized animal as the Wistar and Low line in rats.

In addition, in consideration of homology in humans and mice, which are antigens, it is also preferable to use mice in which the biomechanism for removing autoantibodies is reduced, that is, autoimmune disease mice.

In addition, the age at the time of immunization of these mice or rats is preferably 5 to 12 weeks of age, more preferably 6 to 8 weeks of age.

To immunize an animal with B7-H3 or this recombinant, for example, Weir, D. M., Handbook of Experimental Immunology Vol. I. Ⅱ. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Illinois (1964) and the like can be used.

Among these immunization methods, specifically, the preferred method in the present invention is as follows.

That is, the membrane protein fraction, which is an antigen, or cells expressing the antigen are first administered intradermally or intraperitoneally in the animal.

However, in order to increase immunity efficiency, the combination of both is preferred, and intradermal administration is performed in the first half, and intraperitoneal administration is performed only in the second half or the last time, and especially the immunity efficiency can be increased.

The antigen administration schedule varies depending on the type of immunized animal, individual differences, etc., but in general, the antigen administration frequency is 3 to 6 times, the administration interval is 2 to 6 weeks, and the administration frequency is 3 to 4 times, the administration interval is 2 to 4 times. Stocks are more preferred.

In addition, although the dose of the antigen varies depending on the type of animal, individual differences, etc., it is generally set to about 0.05 to 5 mg, preferably about 0.1 to 0.5 mg.

The additional immunization is performed after 1 to 6 weeks, preferably 2 to 4 weeks, and more preferably 2 to 3 weeks after administration of the above antigen.

In addition, the antigen dose at the time of immunization differs depending on the type and size of the animal, but in general, for example, in the case of a mouse, 0.05 to 5 mg, preferably 0.1 to 0.5 mg, more preferably 0.1 It should be about 0.2 mg.

After 1 to 10 days, preferably 2 to 5 days, and more preferably 2 to 3 days after the additional immunization, spleen cells or lymphocytes including antibody producing cells are aseptically taken out from the immunized animal. At that time, if the antibody titer is measured and an animal with a sufficiently high antibody titer is used as a source of antibody-producing cells, subsequent operational efficiency can be improved.

Examples of the method for measuring the antibody value used herein include RIA method or ELISA method, but are not limited to these methods.

The antibody titer in the present invention can be measured, for example, by the procedure described below according to the ELISA method.

First, a purified or partially purified antigen is adsorbed onto a solid surface such as a 96-hole plate for ELISA, and a protein having no antigen-associated solid surface, such as bovine serum albumin (hereinafter referred to as "BSA"). After washing with the surface, and contacting the sample (for example, mouse serum) diluted stepwise as the first antibody, the antigen in the sample is bound to the antigen.

In addition, by adding an antibody to an enzyme-labeled mouse antibody as a second antibody, the antibody is bound by binding to a mouse antibody, and after washing, the substrate of the enzyme is added to measure changes in absorbance due to color development based on substrate decomposition. Calculate

Isolation of antibody-producing cells from spleen cells or lymphocytes of immunized animals is known by methods (e.g., Kohler et al., Nature (1975) 256, p.495,; Kohler et al., Eur. J. Immunol .(1977) 6, p.511,; Milstein et al., Nature (1977), 266, p.550,; Walsh, Nature, (1977) 266, p.495). For example, in the case of splenocytes, a general method of isolating antibody producing cells can be adopted by finely cutting the spleen, filtering the cells with a stainless steel mesh, and then suspending them in Eagle Minimum Essential Medium (MEM).

(c) Preparation of myeloma cells (hereinafter referred to as "myeloma")

The myeloma cells used for cell fusion are not particularly limited and can be appropriately selected from known cell lines and used. However, in consideration of convenience when selecting hybridomas from fusion cells, it is preferable to use HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) deletion strains in which the selection procedure is established.

That is, mouse-derived X63-Ag8 (X63), NS1-ANS/1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63.653), SP2/0-Ag14 (SP2/0) ), MPC11-45.6TG1.7 (45.6TG), FO, S149/5XXO, BU.1, etc., rat derived 210.RSY3.Ag.1.2.3 (Y3), human derived U266AR (SKO-007) , GM1500·GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), 8226AR/NIP4-1 (NP41). These HGPRT-deficient strains can be obtained from, for example, American Type Culture Collection (ATCC).

These cell lines are 8- in a medium containing a suitable medium, for example, 8-azaguanine medium [glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine serum (hereinafter referred to as "FCS") to the RPMI-1640 medium. Azaguanine added medium], passage culture in Iscove's Modified Dulbecco's Medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle Medium (hereinafter referred to as "DMEM") However, 3-4 days before cell fusion, the cells were cultured in a normal medium [for example, ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co., Ltd.)] to obtain a cell count of 2 x 10 ⁷ or more on the day of fusion. Secure it.

(d) cell fusion

Fusion of antibody producing cells and myeloma cells is a known method (Weir, DM, Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, EA and Mayer, MM, Experimental Immunochemistry , Charles C Thomas Publisher Springfield, Illinois (1964), etc., can be appropriately carried out under conditions that do not extremely decrease the cell viability.

As such a method, for example, a chemical method of mixing antibody producing cells and myeloma cells in a high concentration polymer solution such as polyethylene glycol, a physical method using electrical stimulation, or the like can be used. Of these, specific examples of the chemical method are as follows.

That is, when polyethylene glycol is used as a high-concentration polymer solution, the antibody-producing cells at a temperature of 30 to 40°C, preferably 35 to 38°C, in a polyethylene glycol solution having a molecular weight of 1500 to 6000, preferably 2000 to 4000, Myeloma cells are mixed for 1 to 10 minutes, preferably 5 to 8 minutes.

(e) Selection of hybridoma groups

The method for selecting hybridomas obtained by the cell fusion is not particularly limited, but is typically a HAT (hypoxanthine, aminopterin, thymidine) selection method (Kohler et al., Nature (1975) 256, p.495; Milstein et al., Nature (1977) 266, p.550) are used.

This method is effective when hybridomas are obtained using myeloma cells of an HGPRT-deficient strain that cannot survive in aminopterin.

That is, by culturing unfused cells and hybridomas in HAT medium, only hybridomas having resistance to aminopterin can selectively remain and proliferate.

(f) Split into single cell clones (cloning)

As the cloning method of the hybridoma, known methods such as a methylcellulose method, a soft agarose method, and a limiting dilution method can be used (for example, Barbara, BM and Stanley, MS: Selected Methods). in Cellular Immunology, WH Freeman and Company, San Francisco (1980)). Among these methods, a three-dimensional culture method such as a methyl cellulose method is particularly preferred. For example, the hybridoma group formed by cell fusion is suspended and cultured in methylcellulose medium such as ClonaCell-HY Selection Medium D (manufactured by StemCell Technologies #03804), and the formed hybridoma colonies are recovered to recover the monoclonal hybrid. The cutting board can be acquired. Each recovered hybridoma colony was cultured, and the antibody titer observed stably in the obtained hybridoma culture supernatant was selected as a B7-H3 monoclonal antibody-producing hybridoma strain.

An example of the hybridoma strain thus established is B7-H3 hybridoma M30. In addition, in this specification, the antibody produced by the B7-H3 hybridoma M30 is described as "M30 antibody" or simply "M30".

The heavy chain of the M30 antibody has the amino acid sequence shown in sequence number 51 of the sequence table. Moreover, the light chain of M30 antibody has the amino acid sequence shown in sequence number 53 of the sequence table. Further, among the heavy chain amino acid sequences shown in SEQ ID NO: 51 in the sequence table, the amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence, and the amino acid sequence consisting of the 20th to 141th amino acid residues is a variable region, and 142 to 471 The amino acid sequence consisting of the first amino acid residue is the normal region. Further, among the light chain amino acid sequences shown in SEQ ID NO: 53 in the sequence table, the amino acid sequence consisting of the 1st to 22nd amino acid residues is a signal sequence, and the amino acid sequence consisting of the 23th to 130th amino acid residues is a variable region, and 131 to 235 The amino acid sequence consisting of the first amino acid residue is the normal region.

The heavy chain amino acid sequence shown in sequence number 51 of the sequence table is coded by the nucleotide sequence shown in sequence number 50 of the sequence table. The nucleotide sequence consisting of the 1st to 57th nucleotides of the nucleotide sequence shown in SEQ ID NO: 50 of the sequence table encodes the heavy chain signal sequence of the antibody, and the nucleotide sequence consisting of the 58th to 423th nucleotides represents the heavy chain variable region of the antibody. And the nucleotide sequence consisting of the 424-1413 nucleotides encodes the heavy chain constant region of the antibody.

The light chain amino acid sequence shown in sequence number 53 of the sequence table is coded by the nucleotide sequence shown in sequence number 52 of the sequence table. The nucleotide sequence consisting of 1 to 66 nucleotides of the nucleotide sequence shown in SEQ ID NO: 52 of the sequence table encodes the light chain signal sequence of the antibody, and the nucleotide sequence consisting of 67 to 390 nucleotides shows the light chain variable region of the antibody. And the nucleotide sequence consisting of 391 to 705 th nucleotides encodes the light chain constant region of the antibody.

(g) Preparation of monoclonal antibodies by culturing hybridomas

The monoclonal antibody can be efficiently obtained by culturing the hybridoma thus selected, but it is preferable to screen the hybridoma producing the desired monoclonal antibody prior to cultivation.

A method known per se may be employed for this screening.

The antibody titer in the present invention can be measured, for example, by the ELISA method described in the item (b) above.

The hybridoma obtained by the above method can be stored frozen in liquid nitrogen or in a freezer at -80°C or lower.

After completion of cloning, hybridomas are cultured by changing the medium from HT medium to normal medium.

Mass cultivation is performed by spin culture using a large culture bottle or spinner culture. The monoclonal antibody specifically binding to the protein of the present invention can be obtained from the supernatant in the mass culture by purification using methods well known to those skilled in the art, such as gel filtration.

Furthermore, by injecting a hybridoma into the abdominal cavity of a mouse of the same line (for example, BALB/c described above) or a Nu/Nu mouse, and propagating the hybridoma, the monoclonal antibody of the present invention is mass-produced. It is possible to obtain a plurality containing.

When administered intraperitoneally, 2,6,10,14-tetramethylpentadecane (2,6,10,14-tetramethyl pentadecane) (pristane) or other mineral oil is administered in advance (3-7 days before). , A larger amount of revenge is obtained.

For example, the immune cells are injected into the abdominal cavity of a hybridoma and a mouse of the same line in advance to inactivate T cells, and after 20 days, 10 ⁶ to 10 ⁷ hybridoma clone cells do not contain serum. It is suspended (0.5 ml) in an unused medium and administered into the abdominal cavity. Usually, the abdomen is swollen and ascites are collected from the mouse when the ascites are standing. By this method, a monoclonal antibody having a concentration of about 100 times or more compared to that in the culture medium is obtained.

The monoclonal antibody obtained by the above method can be purified, for example, by a method described in Weir, D. M.: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978).

The monoclonal antibody thus obtained has high antigen specificity for B7-H3.

(h) Assay of monoclonal antibodies

Determination of the isotype and subclass of the monoclonal antibody thus obtained can be carried out as follows.

First, the identification method includes the Ouchterlony method, ELISA method, or RIA method.

Although the octaloni method is simple, a concentration operation is required when the concentration of the monoclonal antibody is low.

On the other hand, in the case of using the ELISA method or the RIA method, the culture supernatant is reacted with the antigen-adsorbed solid phase as it is, and further, as a secondary antibody, various immunoglobulin isotypes and antibodies corresponding to subclasses are used to monoclonal. It is possible to identify the isotype and subclass of the antibody.

Further, as a more convenient method, a commercially available identification kit (eg, mouse typer kit; manufactured by BioRad) can be used.

In addition, the protein can be quantified by the polylinic method and a method calculated from absorbance at 280 nm [1.4 (OD280) = immunoglobulin 1 mg/ml].

In addition, even when the steps (a) to (h) of (2) are performed again to separately obtain monoclonal antibodies independently, it is possible to obtain antibodies having cytotoxic activity equivalent to M30 antibodies. An example of such an antibody is an antibody that binds to the same epitope as the M30 antibody. Since M30 recognizes an epitope in the IgC1 domain or IgC2 domain, which is a domain in the extracellular region of B7-H3, and binds to the IgC1 domain or the IgC2 domain or both, in particular, the epitope is the IgC1 domain or IgC2 domain of B7-H3 And epitopes present in. When the newly produced monoclonal antibody binds to the partial peptide or partial conformation to which the M30 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the M30 antibody. Further, by confirming that the monoclonal antibody competes with the binding of the M30 antibody to B7-H3 (that is, the monoclonal antibody interferes with the binding of the M30 antibody and B7-H3), specific epitope alignment Alternatively, even if the structure is not determined, it can be determined that the monoclonal antibody binds to the same epitope as the M30 antibody. When it is confirmed that the epitopes are identical, it is highly anticipated that the monoclonal antibody will have cytotoxic activity equivalent to that of the M30 antibody.

(3) Other antibodies

In the antibody of the present invention, in addition to the monoclonal antibody against B7-H3, a genetically modified antibody artificially modified for the purpose of lowering heterologous antigenicity to humans, for example, Chimeric Antibodies, humanized antibodies, human antibodies, and the like are also included. These antibodies can be prepared using known methods.

As a chimeric antibody, an antibody in which the variable region and the constant region of the antibody are heterogeneous to each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is conjugated to a human-derived constant region (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). The chimeric antibody derived from mouse anti-human B7-H3 antibody M30 comprises a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of the amino acid residues 20 to 141 of SEQ ID NO: 51 and amino acids 23 to 130 of SEQ ID NO: 53. It is an antibody consisting of a light chain comprising a light chain variable region consisting of an amino acid sequence consisting of residues, and may have any human-derived normal region. As an example of such a chimeric antibody, a heavy chain having an amino acid sequence consisting of amino acid residues 1 to 471 of SEQ ID NO: 63 in the sequence table, and a light chain having an amino acid sequence consisting of amino acid residues 1 to 233 of SEQ ID NO: 59 And antibodies. Further, among the heavy chain sequences shown in SEQ ID NO: 63 in the sequence table, the amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence, and the amino acid sequence consisting of the 20th to 141th amino acid residues is a variable region, and the 142th to 471th positions The amino acid sequence consisting of the amino acid residues of is the normal region. In addition, among the light chain arrangements shown in SEQ ID NO: 59 in the sequence table, the amino acid sequence consisting of the 1st to 20th amino acid residues is a signal sequence, and the amino acid sequence consisting of the 21st to 128th amino acid residues is a variable region, and the 129th to 233th positions The amino acid sequence consisting of the amino acid residues of is the normal region.

The heavy chain amino acid sequence shown in sequence number 63 of the sequence table is coded by the nucleotide sequence shown in sequence number 62 of the sequence table. The nucleotide sequence consisting of the 1st to 57th nucleotides of the nucleotide sequence shown in SEQ ID NO:62 of the sequence table encodes the heavy chain signal sequence of the antibody, and the nucleotide sequence consisting of the 58th to 423th nucleotides represents the heavy chain variable region of the antibody. And the nucleotide sequence consisting of the 424-1413 nucleotides encodes the heavy chain constant region of the antibody.

The light chain amino acid sequence shown in sequence number 59 of the sequence table is coded by the nucleotide sequence shown in sequence number 58 of the sequence table. The nucleotide sequence consisting of 1 to 60 th nucleotides of the nucleotide sequence shown in SEQ ID NO: 58 of the sequence table encodes the light chain signal sequence of the antibody, and the nucleotide sequence consisting of 61 to 384 nucleotides shows the light chain variable region of the antibody. And the nucleotide sequence consisting of 385-699 nucleotides encodes the light chain constant region of the antibody.

As a humanized antibody, an antibody in which only a complementarity determining region (CDR) is inserted into a human-derived antibody (see Nature (1986) 321, p.522-525), in addition to the arrangement of CDRs by CDR grafting, some Examples of the amino acid residues of the framework are grafted on human antibodies (International Publication Pamphlet WO90/07861).

However, humanized antibodies derived from M30 antibodies are not limited to specific humanized antibodies as long as the CDR sequences of all six types of M30 antibodies are maintained and have anti-tumor activity. In addition, the heavy chain variable region of the M30 antibody consists of CDRH1 (NYVMH) consisting of the amino acid sequence shown in SEQ ID NO: 92 in the sequence table, CDRH2 (YINPYNDDVKYNEKFKG) consisting of the amino acid sequence shown in SEQ ID NO: 93, and the amino acid sequence shown in SEQ ID NO: 94. CDRH3 (WGYYGSPLYYFDY). Further, the light chain variable region of the M30 antibody consists of CDRL1 (RASSRLIYMH) consisting of the amino acid sequence shown in SEQ ID NO: 95 in the Sequence Listing, CDRL2 (ATSNLAS) consisting of the amino acid sequence shown in SEQ ID NO: 96, and the amino acid sequence shown in SEQ ID NO: 97. CDRL3 (QQWNSNPPT).

Examples of the humanized antibody of mouse antibody M30 include (1) an amino acid sequence consisting of the 20th to 141st amino acid residues of SEQ ID NO: 85, 87, 89 or 91 of the sequence table, and (2) the amino acid sequence of (1) above. An amino acid sequence having at least 95% or more homology, and (3) a heavy chain variable region comprising any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above. And (4) an amino acid sequence consisting of the 21 to 128 amino acid residues of SEQ ID NOs: 71, 73, 75, 77, 79, 81 or 83, and (5) at least 95% or more of the amino acid sequence of (4) above. Any of the light chains comprising a light chain variable region composed of any one of amino acid sequences having homology, and (6) one or several amino acid deletions, substitutions or additions in the amino acid sequence of (4) above. And combinations.

In addition, "the number" in this specification means 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1- It means three or one or two.

In addition, a conservative amino acid substitution is preferable as the amino acid substitution in the present specification. Conservative amino acid substitutions are substitutions that occur within a group of amino acids that are related to the amino acid side chain. Preferred amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; Basic group = lysine, arginine, histidine; Non-polar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; And non-charged polar family = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are as follows: aliphatic hydroxy group = serine and threonine; Amide-containing group = asparagine and glutamine; Aliphatic group = alanine, valine, leucine and isoleucine; And aromatic groups = phenylalanine, tryptophan and tyrosine. It is preferable to perform such amino acid substitution within a range that does not degrade the properties of the substance having the original amino acid sequence.

Preferred combinations of the heavy and light chains include the heavy chain variable region consisting of the amino acid sequence of the 20 to 141 th amino acid residues of SEQ ID NO: 85 and the 21 to 128 amino acid residues of the SEQ ID NO: 71 The antibody consisting of a light chain comprising a light chain variable region consisting of an amino acid sequence consisting of, the heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of the 20th to 141st amino acid residues of SEQ ID NO: 85 and 21 of SEQ ID NO: 73 An antibody consisting of a light chain comprising a light chain variable region consisting of an amino acid sequence of the 128th amino acid residue, a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of the 20th to 141th amino acid residues of SEQ ID NO: 85 And a light chain variable region comprising a light chain variable region consisting of an amino acid sequence of 21 to 128 th amino acid residues of SEQ ID NO: 75, a heavy chain variable consisting of an amino acid sequence of 20 to 141 th amino acid residues of SEQ ID NO: 85 An antibody consisting of a light chain comprising a heavy chain comprising a region and a light chain variable region consisting of an amino acid sequence consisting of the 21-128th amino acid residues of SEQ ID NO: 77, an amino acid consisting of the 20-141th amino acid residues of SEQ ID NO: 85 Antibody consisting of a heavy chain comprising a heavy chain variable region consisting of an array and a light chain variable region consisting of an amino acid sequence consisting of the 21 to 128 amino acid residues of SEQ ID NO: 79, the 20 to 141 th of SEQ ID NO: 85 Consisting of a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues and a light chain comprising a light chain variable region consisting of an amino acid sequence consisting of the 21st to 128th amino acid residues of SEQ ID NO: 81 Antibody, heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of the 20th to 141st amino acid residues of SEQ ID NO: 85 and light chain variable region consisting of an amino acid sequence consisting of the 21st to 128th amino acid residues of SEQ ID NO: 83 An antibody consisting of a light chain comprising a, heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of the amino acid residues 20 to 141 of SEQ ID NO: 91 and an amino acid sequence consisting of the amino acid residues 21 to 128 of the SEQ ID NO: 71 The antibody consisting of a light chain comprising a light chain variable region consisting of, the heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of the amino acid residues 20 to 141 of SEQ ID NO: 91 and the 21 to 128 of the SEQ ID NO: 73 An antibody consisting of a light chain comprising a light chain variable region consisting of an amino acid sequence consisting of amino acid residues, a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of the 20th to 141st amino acid residues of SEQ ID NO: 91, and SEQ ID NO: 75 It comprises an antibody consisting of a light chain variable region consisting of an amino acid sequence consisting of the 21-128th amino acid residues of, and a heavy chain variable region consisting of an amino acid sequence consisting of the 20-141th amino acid residues of SEQ ID NO: 91 And an antibody consisting of a light chain comprising a light chain variable region consisting of a heavy chain and an amino acid sequence consisting of the 21 to 128 th amino acid residues of SEQ ID NO: 77.

More preferred combinations include antibodies consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 85 and a light chain having an amino acid sequence of SEQ ID NO: 71, a heavy chain having an amino acid sequence of SEQ ID NO: 85 and a gradient having an amino acid sequence of SEQ ID NO: 73. Antibody consisting of an antibody, consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 85 and a light chain having an amino acid sequence of SEQ ID NO: 85, an heavy chain having an amino acid sequence of SEQ ID NO: 85, and a light chain having an amino acid sequence of SEQ ID NO: 77 An antibody, an antibody consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 85 and a light chain having an amino acid sequence of SEQ ID NO: 79, an antibody consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 85 and a light chain having an amino acid sequence of SEQ ID NO: 81, An antibody consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 85 and a light chain having an amino acid sequence of SEQ ID NO: 83, an antibody consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 91 and a light chain having an amino acid sequence of SEQ ID NO: 71, SEQ ID NO: An antibody consisting of a heavy chain having an amino acid sequence of 91 and a light chain having an amino acid sequence of SEQ ID NO: 73, an antibody consisting of a heavy chain having an amino acid sequence of SEQ ID NO: 91 and a light chain having an amino acid sequence of SEQ ID NO: 75, and SEQ ID NO: 91 And antibodies consisting of a heavy chain having the amino acid sequence of and a light chain having the amino acid sequence of SEQ ID NO: 77.

By combining the above-described heavy chain amino acid sequence and light chain amino acid sequence with a sequence exhibiting high homology, it is possible to select an antibody having cytotoxic activity equivalent to each of the above antibodies. Such homology is generally at least 80% homology, preferably at least 90% homology, more preferably at least 95% homology, and most preferably at least 99% homology. In addition, by combining amino acid sequences in which 1 to several amino acid residues are substituted, deleted, or added to the heavy chain or light chain amino acid sequence, it is possible to select antibodies having cytotoxic activity equivalent to those of the above antibodies. Do.

The homology between the two amino acid sequences is Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), It can be determined by using the default parameters of ``Gapped BLAST and PSI-BLAST: a new generation of protein database search programs'', Nucleic Acids Res. 25: 3389-3402). Blast algorithm is

1334907992153_0

It can also be used by accessing.

Further, among the heavy chain amino acid sequences shown in SEQ ID NO: 85, 87, 89 or 91 of the sequence table, the amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence, and the amino acid sequence consisting of the 20th to 141st amino acid residues is variable. Region, and the amino acid sequence consisting of amino acid residues 142 to 471 is a normal region.

Further, among the light chain amino acid sequences shown in SEQ ID NOs: 71, 73, 75, 77, 79, 81, or 83 of the sequence table, the amino acid sequence consisting of the 1st to 20th amino acid residues is a signal sequence, and the 21st to 128th amino acid residues The amino acid sequence consisting of is a variable region, and the amino acid sequence consisting of 129th to 233th amino acid residues is a normal region.

The heavy chain amino acid sequence shown in sequence number 85, 87, 89 or 91 of the sequence table is coded by the nucleotide sequence shown in sequence number 84, 86, 88 or 90 of the sequence table, respectively. The arrangement of the arrangement numbers 84 and 85 is shown in Fig. 34, the arrangement of the arrangement numbers 86 and 87 is shown in Fig. 35, the arrangement of the arrangement numbers 88 and 89 is shown in Fig. 36, and the arrangement of the arrangement numbers 90 and 91 is shown in Fig. 37, respectively. It is described. The nucleotide sequence consisting of the 1st to 57th nucleotides of each nucleotide sequence encodes the heavy chain signal sequence of the antibody, and the nucleotide sequence consisting of the 58th to 423th nucleotides encodes the heavy chain variable region of the antibody, and 424 The nucleotide sequence consisting of 14-13 th nucleotides encodes the heavy chain constant region of the antibody.

The light chain amino acid sequence shown in sequence number 71, 73, 75, 77, 79, 81 or 83 of the sequence table is coded by the nucleotide sequence shown in sequence number 70, 72, 74, 76, 78, 80 or 82 of the sequence table, respectively. have. The arrangement of the arrangement numbers 70 and 71 is shown in Fig. 27, the arrangement of the arrangement numbers 72 and 73 is shown in Fig. 28, the arrangement of the arrangement numbers 74 and 75 is shown in Fig. 29, the arrangement of the arrangement numbers 76 and 77 is shown in Fig. 30, The arrangement of the arrangement numbers 78 and 79 is described in Fig. 31, the arrangement of the arrangement numbers 80 and 81 is shown in Fig. 32, and the arrangement of the arrangement numbers 82 and 83 is described in Fig. 33, respectively. The nucleotide sequence consisting of 1 to 60 th nucleotides of each nucleotide sequence codes for the light chain signal sequence of the antibody, and the nucleotide sequence consisting of 61 to 384 nucleotides encodes the light chain variable region of the antibody, and 385. The nucleotide sequence consisting of nucleotides 699 to 699 encodes the light chain constant region of the antibody.

The homology between these nucleotide sequences and the nucleotide sequence of other antibodies can also be determined by the Blast algorithm.

Examples of the antibody of the present invention include human antibodies that bind to the same epitope as the M30 antibody. The anti-B7-H3 human antibody means a human antibody having only the gene sequence of the antibody derived from the human chromosome. Anti-B7-H3 human antibody is a method using a human antibody-producing mouse having human chromosomal fragments containing the heavy and light chain genes of a human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143,; Kuroiwa, Y. et.al., Nucl. Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et.al., Animal Cell Technology: Basic and Applied Aspects vol. 10 , p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999.; Tomizuka, K. et.al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727, etc.).

Specifically, the human antibody-producing mouse specifically destroys the intrinsic immunoglobulin heavy chain and light chain loci, and instead substitutes the human immunoglobulin heavy chain and light chain via a yeast artificial chromosome (YAC) vector. Genetically modified animals introduced with the locus of can be produced by the production of knockout animals and transgenic animals, and crossing these animals.

In addition, by genetic recombination technology, eukaryotic cells are transformed with cDNA encoding each of the heavy and light chains of such human antibodies, preferably a vector containing the cDNA, and the genetically modified human monoclonal antibody This antibody can also be obtained in a culture supernatant by culturing the produced transformed cells.

Here, as a host, for example, eukaryotic cells, preferably CHO cells, lymphocytes, or mammalian cells such as myeloma can be used.

In addition, a method for obtaining a human antibody derived from a phage display selected from a human antibody library (Wormstone, IM et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p.2301-2308; Carmen, S. et.al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p.189-203; Siriwardena, D. et.al., Ophthalmology (2002) 109 (3), p.427-431, etc. See also).

For example, a phage display method in which a variable region of a human antibody is expressed on a phage surface as a one-chain antibody (scFv), and a phage that binds to an antigen is selected (Nature Biotechnology (2005), 23, (9), p. 1105-1116) can be used.

By analyzing the gene of the selected phage by binding to the antigen, it is possible to determine the DNA sequence encoding the variable region of the human antibody that binds the antigen.

When the DNA sequence of the scFv binding to the antigen is revealed, human antibodies can be obtained by preparing an expression vector having the sequence and introducing it into a suitable host (WO92/01047, WO92/20791, WO93/06213, WO93) /11236, WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) 23 (9), p.1105-1116).

When the newly produced human antibody binds to the partial peptide or partial conformation to which the M30 antibody binds, it can be determined that the human antibody binds to the same epitope as the M30 antibody. Further, by confirming that the human antibody competes for binding of the M30 antibody to B7-H3 (that is, the human antibody interferes with the binding of the M30 antibody and B7-H3), the specific epitope sequence or structure is determined. Even if it is not, it can be determined that the human antibody binds to the same epitope as the M30 antibody. When it is confirmed that the epitopes are the same, it is strongly expected that the human antibody will have cytotoxic activity equivalent to that of the M30 antibody.

A chimeric antibody, a humanized antibody, or a human antibody obtained by the above method can evaluate the binding to an antigen by the method shown in Example 3, etc., and select a preferable antibody.

An example of another index when comparing the properties of an antibody is stability of the antibody. Differential Scanning Calorimetry (DSC) is a device that can quickly and accurately measure the heat denaturation center (Tm), which is a good indicator of the relative structural stability of a protein. The difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values. It is known that the storage stability of the antibody shows a certain correlation with the thermal stability of the antibody (Lori Burton, et.al., Pharmaceutical Development and Technology (2007) 12, p.265-273), and the thermal stability as an index, Preferred antibodies can be selected. Other indicators for selecting an antibody include those having a high yield in a suitable host cell and those having low cohesiveness in an aqueous solution. For example, since it cannot be said that the antibody with the highest quantity shows the highest thermal stability, it is necessary to comprehensively judge on the basis of the indicators described above to select the antibody most suitable for administration to humans.

In addition, a method of linking the full-length arrangement of the heavy and light chains of the antibody using an appropriate linker and obtaining a single chain immunoglobulin is also known (Lee, HS, et. al., Molecular Immunology (1999) 36, p.61-71; Shirrmann, T. et.al., mAbs (2010), 2, (1) p.1-4). By dimerizing such one-chain immunoglobulin, it is possible to maintain the structure and activity similar to the antibody, which is a tetramer in nature. Further, the antibody of the present invention may be an antibody having a single heavy chain variable region and no light chain configuration. Such an antibody is called a single domain antibody (sdAb) or a nanobody, and is actually observed in camels or llamas, and it has been reported that antigen-binding ability is maintained (Muyldemans S. et. al. , Protein Eng. (1994) 7 (9), 1129-35, Hamers-Casterman C. et.al., Nature (1993) 363 (6428) 446-8). The said antibody can also be interpreted as a kind of functional fragment of the antibody in this invention.

The present invention also includes an antibody or a modifier of a functional fragment of the antibody. The modified body means that the antibody of the present invention or a functional fragment of the antibody is chemically or biologically modified. Chemical modifiers include binding of chemical moieties to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains, and the like. Biological modifiers include post-translational modifications (e.g., sugar chain addition to N- or O-links, processing of N or C words, deamidation, isomerization of aspartic acid, oxidation of methionine) , Adding a methionine residue to the N term by expression using a prokaryotic host cell. Also, in order to enable detection or isolation of the antibody or antigen of the present invention, a labeled one, for example, an enzyme label, a fluorescent label, and an affinity label are also included in the meaning of these modifications. Such an antibody of the present invention or a modification of the functional fragment of the antibody is useful for improving the stability and blood retention of the original antibody or functional fragment of the antibody, reducing antigenicity, detecting or isolating such an antibody or antigen Do.

In addition, it is possible to enhance the antibody-dependent cytotoxic activity by adjusting the sugar chain modification bound to the antibody of the present invention (glycosylation, defucosylation, etc.). WO99/54342, WO00/61739, WO02/31140, etc. are known as techniques for regulating sugar chain modification of antibodies, but are not limited thereto. The antibody of the present invention and the functional fragment of the antibody also include an antibody in which the sugar chain modification is regulated and a functional fragment of the antibody.

Once the antibody gene has been isolated and introduced into a suitable host to produce an antibody, a combination of a suitable host and expression vector can be used. Specific examples of the antibody gene include those in which the gene encoding the heavy chain sequence of the antibody described in this specification and the gene encoding the light chain sequence are combined. When transforming the host cell, the heavy chain sequence gene and the light chain sequence gene may be inserted into the same expression vector, or may be inserted into different expression vectors.

When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, as animal cells, mammalian cells, for example, COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650), which are cells of monkeys, mouse progenitor cells NIH3T3 (ATCC No. CRL-1658) or Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolic acid reductase deficiency strain (Urlaub, G. and Chasin, LA Proc. Natl. Acad. Sci. USA (1980) 77, 77) p.4126-4220).

When prokaryotic cells are used, for example, E. coli and Bacillus coli.

Antibodies are obtained by introducing target antibody genes or functional fragments of the antibodies into these cells by transformation, and culturing the transformed cells in vitro. In this culture, the quantity may be different depending on the arrangement of the antibodies, and it is possible to select, among the antibodies having equivalent binding activity, an easy production as a medicament based on the quantity. Therefore, the antibody of the present invention and the functional fragment of the antibody include a step of culturing the transformed host cell, and a step of collecting the target antibody or functional fragment of the antibody from the culture obtained in the step. The antibody obtained by the method for producing the antibody or the fragment characterized by the above-mentioned, or a functional fragment of the antibody is also included.

In addition, it is known that the lysine residue at the carboxyl terminus of the heavy chain of the antibody produced by mammalian culture cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and also the same at the heavy chain carboxyl terminus. It is known that a 2-amino acid residue of glycine and lysine is deleted, and a proline residue located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, deletion and modification of these heavy chain sequences does not affect the antibody's antigen-binding ability and effector function (activation of complement or antibody-dependent cell interference). Accordingly, the present invention also includes an antibody having the above modification and a functional fragment of the antibody, and a deletion complex in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and an amidated deletion (for example, And a heavy chain in which the proline residue at the carboxyl terminal site is amidated). However, as long as the antigen-binding ability and effector function are maintained, the deletion of the carboxyl terminal of the heavy chain of the antibody according to the present invention is not limited to the above-mentioned types. The two heavy chains constituting the antibody according to the present invention may be any one of the heavy chains selected from the group consisting of the full length and the above-described deletion group, or a combination of any two. The amount of each deletion may be influenced by the type and culture conditions of the mammalian cultured cells producing the antibody related to the present invention, but as the main component of the antibody related to the present invention, 1 of the carboxyl ends in both of the two heavy chains The case where the amino acid residues of dogs are deleted is mentioned. There is no restriction as an isotype of the antibody of the present invention, for example, IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD or IgE, and the like, preferably IgG or IgM , More preferably, IgG1 or IgG2 is mentioned.

Further, the antibody of the present invention may be a functional fragment of an antibody having an antigen-binding portion of the antibody or a modified product thereof. Fragments of the antibody can be obtained by treating the antibody with proteolytic enzymes such as papain or pepsin or by expressing the antibody gene in a suitable cultured cell by genetic engineering techniques. Of these antibody fragments, fragments that retain all or part of the function of the antibody can be referred to as functional fragments of the antibody.

Antibody functions include antigen binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody dependent cytotoxicity (ADCC) activity, and complement dependent cytotoxicity (CDC) activity. , The function of the antibody and functional fragments related to the present invention is a binding activity to B7-H3, preferably an antibody-dependent cell-mediated phagocytosis (ADCP) activity, more preferably cells through ADCP activity on tumor cells Shanghai activity (anti-tumor activity). In addition, the antibody of the present invention may have ADCC activity and/or CDC activity in addition to ADCP activity. In particular, for drugs containing existing anti-tumor antibodies, it acts directly on tumor cells to block proliferation signals, acts directly on tumor cells to induce cell death signals, inhibit angiogenesis, and NK cells It has been reported to induce ADCC activity through and inhibit the proliferation of tumor cells by inducing CDC activity through complement (J Clin Oncol 28: 4390-4399. (2010), Clin Cancer Res; 16 (1) ; 11-20. (2010)), at least the inventors do not know that the ADCP activity of the anti-B7-H3 antibody related to the present invention has been reported as the activity of a medicament containing an existing anti-tumor antibody.

Examples of fragments of the antibody include, for example, Fab, F(ab')2, Fv, or single chain Fv (scFv), diabody (diabodies), linear antibodies, which link the Fv of the heavy chain and light chain with a suitable linker, and And multispecific antibodies formed from antibody fragments. In addition, Fab', which is a monovalent fragment of the variable region of the antibody treated with F(ab')2 under reducing conditions, is also included in the fragment of the antibody.

Moreover, the antibody of the present invention may be a multispecific antibody having specificity for at least two different antigens. Typically, such molecules bind to two types of antigens (i.e., bispecific antibodies), but the "multispecific antibody" in the present invention is more than (e.g., three) antigens It contains an antibody having specificity for the antibody.

The multispecific antibody of the present invention may be an antibody of full length, or a fragment of such an antibody (eg, F(ab')2 bispecific antibody). Bispecific antibodies may be prepared by combining the heavy chain and light chain (HL pair) of two types of antibodies, or by fusion of hybridomas that produce different monoclonal antibodies, thereby producing bispecific antibody-producing fusion cells. It can also be prepared by (Millstein et al., Nature (1983) 305, p.537-539).

The antibody of the present invention may be a single chain antibody (also referred to as scFv). One chain antibodies are obtained by linking the heavy and light chain variable regions of an antibody with a linker of a polypeptide (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Rosenberg and Moore, Springer Verlag, New York, p.269) -315 (1994), Nature Biotechnology (2005), 23, p. 1126-1136) In addition, a BiscFv fragment prepared by binding two scFvs with a polypeptide linker can also be used as a bispecific antibody.

Methods for making one chain antibodies are well known in the art (see, e.g., U.S. Patent No. 4,946,778, U.S. Patent No. 5,260,203, U.S. Patent No. 5,091,513, U.S. Patent No. 5,455,030, etc.). In this scFv, the heavy chain variable region and the light chain variable region are linked via a linker that does not make a conjugate, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) ), 85, p.5879-5883). The heavy chain variable region and light chain variable region in scFv may be derived from the same antibody or may be derived from different antibodies.

As a polypeptide linker linking the variable region, any one chain peptide consisting of, for example, 12 to 19 residues is used.

The DNA encoding the scFv is the DNA encoding the heavy chain or heavy chain variable region of the antibody, and the DNA encoding the light chain or light chain variable region, all of these sequences, or a DNA portion encoding the desired amino acid sequence. A template, amplified by PCR using a primer pair defining both ends thereof, and then, a DNA pair encoding a polypeptide linker moiety, and a primer pair defining both ends of which are connected to a heavy chain and a light chain, respectively It is obtained by combining and amplifying.

In addition, once the DNA encoding the scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method, and by using the host, according to a conventional method You can get scFv. These antibody fragments can be produced by a host by obtaining and expressing a gene in the same manner as described above.

The antibody of the present invention may have a large amount to increase the affinity for the antigen. The antibody to be multimerized may be one type of antibody, or may be a plurality of antibodies that recognize multiple epitopes of the same antigen. Examples of the method for bulking the antibody include binding of an IgG CH3 domain to two scFvs, binding to streptavidin, introduction of a helix-turn-helix motif, and the like.

The antibody of the present invention may be a polyclonal antibody that is a mixture of multiple types of anti-B7-H3 antibodies having different amino acid sequences. As an example of a polyclonal antibody, a mixture of multiple types of antibodies having different CDRs can be mentioned. As such a polyclonal antibody, a mixture of cells producing different antibodies can be cultured, and antibodies purified from the culture can be used (see WO2004/061104).

As a modifier of the antibody, an antibody combined with various molecules such as polyethylene glycol (PEG) can also be used.

The antibody of the present invention may also be one in which these antibodies and other drugs form a conjugate (Immunoconjugate). Examples of such antibodies include those in which the antibody is combined with a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, p.1137-1146), for example, indium ( ¹¹¹ In) caproic MAB pende Tide ^{(indium (111 In) Capromab pendetide} ), technetium ^(99m Tc) nope Tomorrow MAB mer pentane ^{(technetium (99m Tc) Nofetumomab merpentan} ), indium ⁽¹¹¹ In) Ivry Tomorrow MAB (indium ⁽¹¹¹ In) Ibritumomab ) include yttrium ⁽⁹⁰ Y) Ivry Tomorrow MAB (yttrium ⁽⁹⁰ Y), Ibritumomab), iodine ⁽¹³¹ I) Toshio Tomorrow MAB (iodine ⁽¹³¹ I), Tositumomab) and the like.

The obtained antibody can be purified evenly. For the isolation and purification of antibodies, separation and purification methods used in conventional proteins can be used. For example, by properly selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparation polyacrylamide gel electrophoresis, isoelectric point electrophoresis, etc., antibodies can be isolated and purified (Strategies for Protein) Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), these It is not limited to.

Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography, and the like.

These chromatography can be performed using liquid chromatography such as HPLC or FPLC.

Protein A column and protein G column are mentioned as a column used for affinity chromatography. For example, Hyper D, POROS, Sepharose F. F. (Parmacia), etc. are mentioned as a column using a Protein A column.

In addition, the antibody may be purified by using an antigen-immobilized carrier and binding to the antigen.

3. Medicine containing anti-B7-H3 antibody

"2. The antibody obtained by the method described in the section "Preparation of anti-B7-H3 antibody" can be used as a medicament, particularly as a therapeutic agent and/or prophylactic agent for cancer, as it exhibits cytotoxic activity against cancer cells.

The killing activity by antibodies in vitro can be measured by the inhibitory activity of cell proliferation.

For example, cancer cell lines overexpressing B7-H3 can be cultured, and antibodies can be added to the culture system at various concentrations to measure inhibitory activity against focus formation, colony formation, and spheroid proliferation.

The therapeutic effect of the antibody against cancer using experimental animals in vivo is, for example, administering the antibody to a nude mouse transplanted with a tumor cell line expressing B7-H3 highly, and measuring the change in cancer cells. have.

Types of cancer include lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, polymorphic glioblastoma, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, esophageal cancer, etc. These cancer cells are not limited to these as long as they express B7-H3.

As a substance to be used in an acceptable agent in the pharmaceutical composition of the present invention, it is preferable that the pharmaceutical composition is non-toxic to the person to whom the pharmaceutical composition is administered in terms of dosage or administration concentration.

The pharmaceutical composition of the present invention may contain a substance for preparation for changing or maintaining pH, penetration pressure, viscosity, transparency, color, isotonic, sterility, stability, dissolution rate, sustained release rate, absorption rate, penetration rate. The following may be mentioned as preparation substances, but are not limited to these: amino acids such as glycine, alanine, glutamine, asparagine, arginine or lysine, antibacterial agents, antioxidants such as ascorbic acid, sodium sulfate or sodium hydrogen sulfite, phosphoric acid, Buffers such as citric acid, boric acid buffer, sodium hydrogen carbonate, Tris-HCl solution, fillers such as mannitol or glycine, chelating agents such as ethylenediaminetetraacetic acid (EDTA), caffeine, polyvinylpyrrolidine, β Complexing agents such as cyclodextrin or hydroxypropyl-β-cyclodextrin, bulking agents such as glucose, mannose or dextrin, other carbohydrates such as monosaccharides and disaccharides, coloring agents, flavoring agents, diluents, emulsifiers, polyvinylpyrrolidine, etc. Preservatives such as hydrophilic polymers, low molecular weight polypeptides, salt-forming counter ions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorexidine, sorbic acid or hydrogen peroxide, glycerin, propylene Solvents such as glycol or polyethylene glycol, sugar alcohols such as mannitol or sorbitol, suspending agents, sorbitan esters, polysorbates such as polysorbate 20 or polysorbate 80, triton, tromethamine , Surfactants such as lecithin or cholesterol, stabilization enhancers such as sucrose and sorbitol, elastic enhancers such as sodium chloride, potassium chloride or mannitol sorbitol, transport agents, excipients and/or pharmaceutical aids. It is preferable to add 0.001-100 times, especially 0.1-10 times, with respect to the weight of the anti-B7-H3 antibody. The composition of the preferred pharmaceutical composition in the formulation can be appropriately determined by a person skilled in the art according to the applied disease, the route of administration and the like.

The excipient or carrier in the pharmaceutical composition may be liquid or solid. Suitable excipients or carriers may be water for injection, saline, artificial cerebrospinal fluid, or other substances commonly used for parenteral administration. Neutral physiological saline or physiological saline containing serum albumin can also be used for the carrier. The pharmaceutical composition may contain a Tris buffer at pH 7.0-8.5, an acetic acid buffer at pH 4.0-5.5, and a citric acid buffer at pH 3.0-6.2. In addition, sorbitol and other compounds may be contained in these buffers.

The pharmaceutical composition of the present invention includes a pharmaceutical composition containing an anti-B7-H3 antibody, and a pharmaceutical composition containing an anti-B7-H3 antibody and at least one therapeutic agent for cancer, wherein the pharmaceutical composition of the present invention is selected composition and required purity. As a medicament having a lyophilized product or liquid. The pharmaceutical composition containing the anti-B7-H3 antibody, and the pharmaceutical composition containing the anti-B7-H3 antibody and at least one cancer therapeutic agent may be molded as a freeze-dried product using a suitable excipient such as sucrose.

In the above-mentioned pharmaceutical composition, the cancer therapeutic agent contained together with the anti-B7-H3 antibody may be administered to the individual simultaneously, separately or sequentially with the anti-B7-H3 antibody, or may be administered by changing the respective administration intervals. As such a cancer treatment agent, abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin or a drug described in the international publication WO2003/038043 pamphlet, also LH-RH analog (leuproline, Goserelin, etc.), estradiol phosphate, estrogen antagonists (tamoxifen, raloxifen, etc.), aromatase inhibitors (anastrosol, letrozole, exemestane, etc.) and the like, but drugs having anti-tumor activity If it is, it is not limited to the said drug.

The subject to be administered is not particularly limited, but is preferably a mammal, more preferably a human.

The pharmaceutical composition of the present invention may be prepared for parenteral administration or may be prepared for oral absorption by the digestive tract. The composition and concentration of the formulation can be determined according to the method of administration, and the affinity of the anti-B7-H3 antibody contained in the pharmaceutical composition of the present invention for B7-H3, that is, the dissociation constant for B7-H3 ( For the Kd value), the higher the affinity (lower the Kd value), the lower the dose to humans can exert, so that the pharmaceutical composition of the present invention can be determined for humans based on these results. have. When the human anti-B7-H3 antibody is administered to humans, the dose may be administered about 0.001 to 100 mg/kg once or multiple times at intervals of 1 to 180 days. Examples of the form of the pharmaceutical composition of the present invention include injections containing drops, suppositories, nasal drops, sublinguals, and transdermal absorbers.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples. In addition, in the following Examples, each manipulation related to genetic manipulation is "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press, unless otherwise specified) (Published in 1989), or when a commercially available reagent or kit was used, it was used according to the instructions of the commercial product.

Example

Hereinafter, the present invention will be described in more detail in Examples, but the present invention is not limited to these.

In addition, in the following Examples, each manipulation related to genetic manipulation was "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T. Low, Cold SpringHarbor Laboratory Press, unless otherwise specified. It was carried out by the method described in 1989) and other experiments used by those skilled in the art, or when using a commercially available reagent or kit, according to the instructions of the commercial product.

Example 1. Plasmid preparation

1)-1 Preparation of human B7-H3 expression vector

1) Preparation of 1-1 full-length human B7-H3 variant 1 expression vector

Primer set using cDNA synthesized from LNCaP cells (American Type Culture Collection: ATCC) total RNA as a template:

And,

PCR reaction was performed using to amplify cDNA encoding human B7-H3 variant 1.

Next, the obtained PCR product was purified by MagExtractor PCR & Gel cleanup (TOYOBO). In addition, after digestion with a restriction enzyme (NheI/NotI), it was purified by MagExtractor PCR & Gel cleanup (TOYOBO). pcDNA3.1 (+) plasmid DNA was digested with the same restriction enzymes (NheI/NotI), followed by purification with MagExtractor PCR & Gel cleanup (TOYOBO).

The purified DNA solution was mixed, further added Ligation high (TOYOBO), incubated at 16° C. for 8 hours, and ligated.

The reaction was transformed by adding it to E. coli DH5α competent cells (Invitrogen).

The colonies obtained above were subjected to colony direct PCR using PCR primers and BGH reverse primers to select candidate clones.

The obtained candidate clone was cultured in a liquid medium (LB/Amp), and plasmid DNA was extracted with MagExtractor-Plasmid- (TOYOBO).

Plasmid DNA obtained as a template

And

Sequence analysis was performed between the obtained clones and the provided CDS sequence.

After confirming the arrangement, the obtained clones were cultured in 200 ml of LB/Amp medium, and plasmid DNA was extracted using the VioGene Plasmid Midi V-100 kit.

This vector was named pcDNA3.1-B7-H3. The sequence of the ORF portion of the B7-H3 variant 1 gene cloned into this vector is shown in nucleotide numbers 1 to 1602 of sequence number 5 in the sequence table. In addition, the amino acid sequence of B7-H3 variant 1 is shown in sequence number 6 of the sequence table.

1)-1-2 Preparation of full length human B7-H3 variant 2 expression vector

The primer set described below using cDNA synthesized from total RNA of LNCaP cells as a template:

PCR was performed to amplify cDNA encoding human B7-H3 variant 2.

Example 1) Purification was carried out in the same manner as -1-1, and the purified PCR product was inserted into the pDONR221 vector (Invitrogen) by the Gateway BP reaction to transform E. coli TOP10 (Invitrogen). .

Clones obtained after transformation were confirmed by colony PCR for insert size. Eight clones, each of which confirmed the length of the insert, were subjected to a sequence reaction of one reaction in the direction of the insert from the vector side, and the 3'and 5'terminal DNA sequences of the insert were confirmed. Gateway LR reaction was performed on the entry clone and the Gateway destination vector pcDNA-DEST40 (Invitrogen) confirming the arrangement. Clones obtained after transformation of E. coli TOP10 were confirmed for insert size by colony PCR. The clones having confirmed the length of the insert were analyzed for the 3'and 5'end DNA sequences of the insert to confirm that the target insert was correctly inserted. The cloned plasmid was purified to 1 mg or more using the Invitrogen PureLink HiPure Plasmid Megaprep Kit.

This vector was named pcDNA-DEST40-B7-H3 variant 2. The sequence of the ORF portion of the B7-H3 variant 2 gene cloned into this vector is shown in nucleotide numbers 1 to 948 of sequence number 9 in the sequence table. In addition, the amino acid sequence of B7-H3 variant 2 is shown in sequence number 10 of the sequence table.

1)-2 Preparation of B7-H3 partial protein expression vector

Using the B7-H3 full-length plasmid related to B7-H3 variant 1 of Example 1)-1-1 as a template, the regions described below were amplified by PCR, respectively. The target region number corresponds to the nucleotide number of B7-H3 represented by SEQ ID NO:5. The primer was added to the Gateway att array, and the 3'side was designed to contain a stop codon.

The following 1), 2), and 3) amplified 2 regions, and then connected by PCR to obtain 1 fragment. That is, 1) was amplified in primers 7 and 12 and primers 15 and 11, and the PCR product was also amplified in primers 7 and 11. 2) was amplified in primers 8 and 13 and primers 15 and 11, and the PCR product was also amplified in primers 8 and 11. 3) was amplified in primers 9 and 14 and primers 15 and 11, and the PCR product was also amplified in primers 9 and 11. 4) was amplified in primers 10 and 11. 5) was amplified in primers 8 and 11. 6) was amplified in primers 9 and 11.

·Purpose area

1) B7-H3 Variant 1 ORF: 79 to 417 and 1369 to 1602 (573 bp)

2) B7-H3 variant 1 ORF: 418 to 732 and 1369 to 1602 (549 bp)

3) B7-H3 Variant 1 ORF: 733 ∼ 1071 and 1369 ∼ 1602 (573 bp)

4) B7-H3 Variant 1 ORF: 1072 ∼ 1602 (531 bp)

5) B7-H3 Variant 1 ORF: 418 ∼ 1602 (1185 bp)

6) B7-H3 Variant 1 ORF: 733 ∼ 1602 (870 bp)

· Primer number and base sequence

Purification was carried out in the same manner as in Example 1)-1-1, and each amplified product after purification was inserted into the pDONR221 vector by a Gateway BP reaction to transform E. coli TOP10. Clones obtained after transformation were confirmed by colony PCR for insert size.

For each clone that confirmed the length of the insert, a sequence reaction of 1 reaction was performed in the direction of the insert from the vector side, and the 3'and 5'end DNA sequences of the insert were confirmed.

For the clones in which the target insert was identified, the entire DNA sequence of the insert was also confirmed using the following primers. As a result of the array analysis, it was confirmed that all of them were in perfect agreement with the target array information.

Gateway LR reaction was performed in each entry clone and pFLAG-myc-CMV-19-DEST (Invitrogen) confirming the sequence. Clones obtained after transformation of E. coli DH10B (Invitrogen) were confirmed by colony PCR for insert size.

For each clone that confirmed the length of the insert, the 3'and 5'end DNA sequences of the insert were analyzed to confirm that the target insert was correctly inserted. Hereinafter, the expression vectors into which 1) to 6) were inserted are B7-H3 IgV1, B7-H3 IgC1, B7-H3 IgV2, B7-H3 IgC2, B7-H3 IgC1-V2-C2, B7-H3 IgV2-C2, respectively. Is written.

The nucleotide sequence of the ORF portion of the B7-H3 IgV1, B7-H3 IgC1, B7-H3 IgV2, B7-H3 IgC2, B7-H3 IgC1-V2-C2, and B7-H3 IgV2-C2 genes cloned into this vector is arrayed, respectively. It is shown in table numbers 20, 22, 24, 26, 28 and 30. In addition, the amino acid sequence of B7-H3 IgV1, B7-H3 IgC1, B7-H3 IgV2, B7-H3 IgC2, B7-H3 IgC1-V2-C2, B7-H3 IgV2-C2 is shown in SEQ ID NO: 21, 23, 25 , 27, 29, 31. The arrangement of the arrangement numbers 20 and 21 is shown in Fig. 15, the arrangement of the arrangement numbers 22 and 23 is shown in Fig. 16, the arrangement of the arrangement numbers 24 and 25 is shown in Fig. 17, the arrangement of the arrangement numbers 26 and 27 is shown in Fig. 18, The arrangement of the arrangement numbers 28 and 29 is described in FIG. 19, and the arrangement of the arrangement numbers 30 and 31 is described in FIG. 20, respectively.

1)-3 Preparation of B7 family gene expression vector

The gene expression vector, pCMV6-XL-4-B7RP-1, pCMV6-XL-4-B7-H1, into which the B7 family genes B7RP-1, B7-H1, and B7-DC were inserted into the expression vector pCMV6-XL-4, pCMV6-XL-4-B7-DC were all purchased from Origene.

Vectors expressing the B7 family genes CD80, CD86, and B7-H4 were prepared as follows.

The clones in which CD80, CD86, and B7-H4 were inserted into the entry vector pENTR/221, pENTR/221-CD80, pENTR/221-CD86, and pENTR/221-B7-H4 were purchased from Invitrogen.

Gateway LR reaction was performed in each entry clone and pcDNA3.1-DEST (Invitrogen) confirming the arrangement. Clones obtained after transformation of E. coli DH10B were confirmed for insert size by colony PCR. Analysis of the 3'and 5'end DNA sequences of the insert was performed for each clone that confirmed the insert length, and it was confirmed that the target insert was correctly inserted.

The nucleotide sequence of the ORF portion of the B7RP-1, B7-H1, B7-DC, CD80, CD86, and B7-H4 genes cloned into this vector is shown in SEQ ID NOs: 32, 34, 36, 38, 40 and 42, respectively. have. In addition, the amino acid sequence of B7RP-1, B7-H1, B7-DC, CD80, CD86, B7-H4 is shown in SEQ ID NO: 33, 35, 37, 39, 41, 43 of the sequence table.

Example 2. Monoclonal antibody preparation and antibody screening

2)-1 immunity

BALB/cAnNCrlCrlj mice (Nippon Charles River) of 4 to 6 weeks of age, FcgRII KO mice (Taconic Corporation, Institute of Immunology and Biology) or GANP mice (Transgenic) were used. LNCaP cells, MCF7 cells (ATCC) or AsPC1 cells (ATCC) peeled with Verssen (Invitrogen) on day 0, day 7, 15, and 24 were administered 5×10 ⁶ cells/mouse subcutaneously to the mouse dorsal cavity. . On the 31st day, the same cells were intravenously administered to 5 x 10 ⁶ cells of each mouse, and on the 34th day, the mouse spleen was collected and used for hybridoma production.

2)-2 Preparation of hybridomas

Spleen cells and mouse myeloma P3X63Ag8U. Hybridomas were prepared by fusion of 1 cells (ATCC) using PEG4000 (manufactured by IBL).

As a result, hybridomas of 9639 clones from LNCaP cell immune mice, 4043 clones from MCF7 cell immune mice, and 3617 clones from AsPC1 cell immune mice were established. The resulting hybridoma culture supernatant was used to screen for antibody-producing hybridomas by CDC assay.

2)-3 Antibody Screening: CDC Assay

On day 0, LNCaP cells or MCF7 cells were diluted to 5000 cells/80 μl, and 80 μl/well was added to a 96 well plate and cultured overnight. 20 μl/well of hybridoma culture supernatant was added to the plate from which the cells were seeded, and stopped at 4° C. for 1 hour. To the diluted freeze-dried product of rabbit complement (Cedarlane), 1 ml of sterile water per vial was added on ice. After standing still for 1 minute and mixing, it was mixed with 19 ml of 0.1% BSA/RPMI 1640 medium (BSA Sigma). The reaction was carried out at 37°C for 1 hour.

The plate was left at room temperature for 30 minutes to return to room temperature. CellTiter-Glo reagent (promega) was added to each well of 120 µl and reacted at room temperature for 10 minutes. The amount of light emission was measured by a plate reader (ARVO HTS Prekin Elmer). It was judged that the well with low luminescence induced complement-dependent cell death. Hybridomas that produced culture supernatants that induced such complement-dependent cell death were selected.

As a result, screening positive clones of 24 clones from LNCaP immune-derived clones, 36 clones from MCF7 immune-derived clones, and 3 clones from AsPC1 immune-derived clones were obtained, respectively.

Example 3. Antigen identification

3)-1 Identification of immunoprecipitates

3)-1-1 immunoprecipitation

MCF7 cells were cultured in 5-10 x 10 ⁸ cells. These cells were separated and recovered with a cell scraper, and then cryopreserved at -80 degrees. 10 mL of Lysis buffer containing 1% NP-40 (Sigma Aldrich) and Protease inhibitor (Roche) cooled to 4° C. were added to the frozen cells, and the pellet was dissolved with a pipette to prevent foaming on ice. When the pellet was completely dissolved, it was left on the ice for 30 minutes. The solubilized samples were centrifuged at 4° C., 10000-15000 revolutions, 20 minutes to transfer the supernatant to a 15 ml Falcon tube.

500 μl of Protein G sepharose 4FF beads (Amersham Pharmacia) was washed 3 times and replaced with Lysis buffer. 500 μl of Protein G sepharose 4FF beads on ice was added to the solubilized sample supernatant, and the mixture was stirred overnight at 4°C.

The sample was passed through Polyprep column chromatography columns (Biorad), and flow-through was used as an immunoprecipitation sample.

3 µg of the antibody solution used for immunoprecipitation was added to 50 µl of Protein G sepharose 4FF beads substituted with phosphate buffered physiological saline (PBS) (1.5 ml tube), and stirred at 4°C for 1 to 16 hours to rotate the antibody. It was bound to beads. The antibody-bound beads were added to the immunoprecipitation sample, and stirred at 4°C for 3 hours.

The column was transferred to an empty 15 ml Falcon tube and 6.5 ml Lysis buffer was added. This operation was repeated 4 times.

The column was covered with a lid, pipetted with 500 μl Lysis buffer, and beads were collected in a 1.5 mL tube. This operation was repeated twice.

After centrifugation at 5000 revolutions for 1 minute at 4° C., the supernatant was slowly removed, and 90 μl of Elution buffer (10 mM Glycine-HCl pH 2.0) was added, followed by centrifugation after voltex. The column of 1.5 ml spin culmn was removed, and 10 µl 1 M Tris-HCL, pH 8.5 was added. The column was returned to its original position, the elution fraction was transferred, and centrifuged for 1 minute at 10000 revolutions. 100 μl of sample was obtained.

This sample was subjected to MS analysis by a liquid layer digestion method as shown in 3)-1-2 below.

3)-1-2 Antigen identification by mass spectrometry

The fraction obtained by the immunoprecipitation method was added with trypsin (modifitrypsin, promega) by a liquid layer digestion method according to a conventional method, and a digestion reaction was performed at 37°C for 16 hours. The resulting digested peptide was subjected to liquid chromatography (LC)/tandem mass spectrometry (MS/MS) (Thermo Fisher Science). The obtained mass spectrum data were analyzed by database search software (Mascot, Matrix Science). The International Protein Index (IPI) was used as the database. As a result, 34 types of antigens were identified.

Among the identified antigen information, a literature information search considering a cell membrane protein was performed, and the following experiments of 3)-2 and 3)-3 were carried out with attention to the B7-H3 (CD296) antigen (B7-H3 variant 1). Did.

3) Preparation of antigen gene expressing cells

NIH-3T3 cells (ATCC) were seeded in a collagen type I coat flask (manufactured by IWAKI) so as to be 5×10 ⁴ cells/cm 2, and 37 in DMEM medium (Invitrogen) containing 10% fetal bovine serum (FBS). The cells were cultured overnight at a temperature of 5% CO ₂ .

The next day, each of pcDNA3.1-B7-H3 prepared in Example 1)-1-1, pcDNA-DEST40-B7-H3 variant 2 prepared in 1)-1-2, and the empty vector pcDNA-DEST40 respectively NIH-3T3 cells were transfected with Lipofectamine 2000 (manufactured by Invitrogen), and further cultured overnight at 37°C under 5% CO ₂ conditions.

The next day, transfected NIH-3T3 cells were trypsinized, washed with 10% FBS-containing DMEM, and then suspended in PBS containing 5% FBS. The obtained cell suspension was used for flow cytometry analysis.

3)-3 Flow cytometry analysis

The binding specificity of B7-H3 variant 1 to B7-H3 of the antibody produced by hybridoma immunoprecipitated with MS was confirmed by flow cytometry. After removing the supernatant by centrifuging the cell suspension prepared in Example 3)-2, each vector was transfected with NIH-3T3 cells and suspended by adding a hybridoma culture supernatant and stopped at 4°C for 1 hour.

After washing twice with PBS containing 5% FBS, Fluorescein-conjugated goat IgG fraction to mouse IgG (Whole Molecule) (#55493 manufactured by ICN Pharmaceuticals) diluted 1000 times with PBS containing 5% FBS was added and suspended, 4 The temperature was allowed to stand still for 1 hour.

After washing twice with PBS containing 5% FBS, it was resuspended in PBS containing 5% FBS containing 2 µg/ml 7-aminoactinomycin D (manufactured by Molecular Probes) and flow cytometer (FC500: Detection was carried out with BeckmanCoulter). Data analysis was performed by Flowjo (TreeStar).

After excluding 7-aminoactinomycin D-positive dead cells as a gate, a histogram of FITC fluorescence intensity of living cells was prepared.

The histograms of B7-H3 variant 1 expressing NIH-3T3 cells and B7-H3 variant 2 expressing NIH-3T3 cells are shifted toward the intensity of fluorescence intensity against the fluorescence intensity histogram of NIH-3T3 cells into which a control vector is introduced. Hybridomas producing samples were obtained as anti-B7-H3 antibody-producing hybridomas.

As a result, it was evident that the anti-B7-H3 antibody-producing hybridoma-derived antibodies from 5 clones of L7, L8, L11, M30, and M31 exhibited cross-reactivity with B7-H3 variant 1 and B7-H3 variant 2. .

3)-4 Monoclonal antibody binding to cancer cell line

Cancer cells in which the binding of B7-H3 variant 1 and B7-H3 variant 2 in Example 3)-3 confirmed that the monoclonal antibody highly expresses B7-H3 variant 1 and B7-H3 variant 2 Whether or not to be bound to was examined by the same flow cytometry method as in Example 3)-3.

Human breast cancer cell line (MDA-MB-231) (ATCC) and human lung cancer cell line (NCI-H322) (ATCC) were used instead of transfected NIH-3T3 cells. As a result, it was confirmed that all the established monoclonal antibodies bind to these cancer cell lines.

3)-5 Monoclonal antibody isotype determination

The isotype of the monoclonal antibody was determined by the Mouse monoclonal isotyping kit (manufactured by Serotec). As a result, all of the isotypes of the antibodies derived from the anti-B7-H3 antibody-producing hybridomas (L7, L8, L11, M30, M31) were IgG2a.

3) Preparation of 6 monoclonal antibody

The monoclonal antibody was purified from ascites or hybridoma culture supernatant (hereinafter referred to as "antibody purification raw material") of hybridoma-grafted mice.

Mouse ascites were prepared as follows. First, 7 to 8 weeks old BALB/cAJcl-nu/nu (Nippon Clea) was treated with Pristan (manufactured by Sigma), and hybridomas washed with physiological saline after about 3 weeks were 1 x 10 ⁷ cells per mouse. Transplanted into the abdominal cavity. After 1-2 weeks, ascites stored in the abdominal cavity were collected, passed through a 0.22 µm filter, and sterilized to use as an antibody purification raw material.

The hybridoma culture supernatant was prepared using CELLine (manufactured by BD Biosciences). The medium was cultured according to the manufacturer's instructions except that ClonaCell-HY Growth Medium E (StemCell Technologies, Inc. #03805) was used. The recovered culture supernatant was filtered through a 0.45 μm filter, and used as a raw material for antibody purification.

For the antibody, Recombinat Protein A rPA50 (manufactured by RepliGen) is immobilized on formyl-cellulofine (manufactured by Biochemical Industry), an affinity column (hereinafter abbreviated as "formylcellulopine Protein A") or Hitrap MabSelect SuRe (GE Healthcare Bio-Sciences). In formylcellopine Protein A, the antibody purified raw material was diluted three times with binding buffer (3 M NaCl, 1.5 M Glycine pH 8.9) to the column, washed with binding buffer, and eluted with 0.1 M citric acid pH 4.0. .

On the other hand, in Hitrap MabSelect SuRe (GE Healthcare), antibody purification raw materials were added to the column, washed with PBS, and eluted with 2 M Arginine-HCl pH 4.0.

After neutralization, the eluted antibody solution was buffer-substituted in PBS.

The concentration of the antibody was determined by eluting the antibody bound to POROS G 20 μm Column, PEEK, 4.6 mm×100 mm, 1.7 ml (Applied Biosystems), and measuring the absorbance (O.D. 280 nm) of the eluate. Specifically, an antibody sample diluted with PBS was added to POROS G 20 μm equilibrated with an equilibration buffer (30.6 mM sodium dihydrogenphosphate 12 water, 19.5 mM monopotassium phosphate, 0.15 M NaCl, pH 7.0), and the equilibration buffer After washing with a column, the antibody bound to the column was eluted with eluent (0.1% (v/v) HCl, 0.15 M NaCl). The peak area of the absorbance (O.D. 280 nm) of the eluate was measured, and the concentration was calculated by the following equation.

Antibody sample concentration (mg/ml) = (peak area of antibody sample)/(peak area of standard product (human IgG1)) × concentration of standard product (mg/ml) × dilution factor of sample

In addition, the concentration of endotoxin contained in the obtained antibody was measured using an endospec ES-50M set (Biochemical Industry #020150) and an endotoxin standard product CSE-L set (Biochemical Industry #020055), confirming that it was 1 EU/mg or less. It was used for the following experiment.

Example 4. Properties of anti-B7-H3 antibodies

4)-1 ADCP activity

Balb/c-nu/nu mice (♀, 6-10 wk) (Nippon Charles River Corporation) were administered intraperitoneally with 1.5 ml of thio glycolate. After 5 days, macrophage cells in the abdominal cavity were recovered. 500 μl/well (1×10 ⁵ cell/well) was added to a 24 well plate and incubated overnight at 37°C. This cell was used as an effector cell.

Labeling of NCI-H322 cells to be target cells was performed using a PKH26 dye labeling kit (Sigma). Target cells were peeled off with TrypLE (Invitrogen), and washed twice with PBS. Cells were suspended with Diluent C to 1 x 10 ⁷ cells/ml. PKH26 dye stock (1 mM) was diluted with Diluent C to 8 μM, and a cell suspension and an equal amount of dye dilution were added. It was left at room temperature for 5 minutes. Serum 1 ml was added, additional serum-containing medium was added, and washing was performed twice. This cell was used as a target cell.

The antibody obtained in Example 3)-6 was diluted with culture solution to 20 µg/ml. Next, the target cells obtained in Example 4)-1-1 were dispensed into 2×10 ⁶ /100 μl/tube and mixed. It was allowed to stand still for 30 minutes on the ice. The supernatant was discarded and washed twice with the culture medium. It was suspended in 500 µl of the culture. The supernatant was removed from the effector cells, the antibody was treated, and suspended cells were added to the culture medium and mixed. Incubated for 3 hours in a CO ₂ incubator. The cells were detached with Trypsin-EDTA, and the cells were recovered. FITC-labeled anti-mouse CD11b antibody (Becton Dickinson) was added to the recovered cells, and allowed to stand still for 30 minutes on ice. The supernatant was discarded and washed twice with the culture medium. The recovered cells were suspended in a culture solution of 300 µl, and measured by FACS Calibur (Becton Dickinson). In the CD11b-positive macrophage cells, evaluation was performed using a PKH26-positive fraction as a phagocytic positive cell (n = 3).

As a result, as shown in FIG. 1, L7, L8, L11, M30, and M31 are respectively 48.0 ± 0.9%, 52.3 ± 1.1%, 57.1 ± 2.5%, 61.9 ± 2.1 of the phagocytic rate of NCI-H322 cells by macrophages. %, 57.7 ± 3.0%, L7, L8, L11, M30, M31 antibodies were found to have ADCP activity against NCI-H322 cells.

Similarly, commercially available anti-B7-H3 antibody was obtained and its ADCP activity was measured. Rat anti-human B7-H3 antibody MIH35 (eBioscience), mouse anti-human B7-H3 antibody 185504 (R & D Systems), MIH42 (Serotec), and DCN70 (Biolegend) were obtained, respectively. It was confirmed that these antibodies bind to B7-H3 in the same manner as in Example 3)-3. ADCP activity was measured by the above method using these antibodies.

As a result, as shown in Fig. 2, when MIH35, MIH42, and DCN70 were added at 1 µg/ml, the phagocytosis of NCI-H322 cells by macrophages was induced by 4.2%, 8.2%, and 10.8%, respectively. In this regard, it became clear that MIH35, MIH42, and DCN70 showed little ADCP activity.

In this regard, it was shown that the B7-H3 recognition clone M30 obtained by this screening has a particularly strong ADCP activity compared to a commercially available B7-H3 antibody.

4)-2 ADCC activity

4) Preparation of 2-1 effector cells

Spleens were collected aseptically from nude mice CAnN.Cg-Foxn1 ^nu /CrlCrlj (Nippon Charles River). The collected spleen was homogenized with two slide glasses, and hemolysis treatment was performed using BD Pharm Lyse (#555899 manufactured by BD Biosciences). The obtained spleen cells were suspended in phenol red-free RPMI1640 (Invitrogen) (hereinafter abbreviated as "ADCC medium") containing 10% of Fetal Bovine Serum, Ultra-low IgG (Invitrogen). After passing through a cell strainer (pore size 40 µm: manufactured by BD Biosciences), the number of viable cells was measured by a trypan blue pigment exclusion test. After centrifugation of the spleen cell suspension, the medium was removed, and resuspended in ADCC medium to obtain a cell density of 1.5 x 10 ⁷ cells/ml with a viable cell density to effector cells.

4)-2-2 Preparation of target cells

Example 3) B7-H3 expressing 293 cells (ATCC) prepared in the same manner as -3 and 293 cells expressing the empty vector were trypsinized, and after washing the cells with RPMI1640 (Invitrogen) containing 10% FBS , Resuspended in 10% FBS-containing RPMI1640, each cell 4×10 ⁶ was mixed with Chromium-51 (5550 kBq) sterilized with a 0.22 μm filter, and labeled for 1 hour under conditions of 37° C. and 5% CO ₂ . . The labeled cells were washed 3 times with 10% FBS-containing RPMI1640 (Invitrogen), and resuspended to 2 x 10 ⁵ cells/ml with ADCC medium to obtain target cells.

4)-2-3 ⁵¹ Cr release assay

Target cells at 2 x 10 ⁵ cells/ml were dispensed into a 96 hole U bottom microplate at 50 μl/well. To this, 50 µl of M30, an isotype control antibody (mIgG2a) (eBioscience) diluted with ADCC medium was added to a final concentration after effector cell addition to 2.5 µg/ml, and the mixture was stopped at 4°C for 1 hour. To this, 100 µl of 1.5×10 ⁷ cells/ml effector cells were added, and cultured overnight at 37° C. under 5% CO ₂ conditions. The next day, the supernatant was collected in LumaPlate (manufactured by PerkinElmer), and the gamma dose emitted by the gamma counter was measured. Cell lysis rate by ADCC activity was calculated by the following equation.

Cell lysis rate (%) = (A-B)/(C-B) × 100

A: Sample well count

B: Average value of natural release (antibody/effector cell-free well) count (n = 3). At the time of antibody addition and effector cell addition, 50 µl and 100 µl of ADCC medium were added, respectively. Other than that, the same operation as the sample well was performed.

C: Average of the maximum release (wells in which target cells were lysed with surfactant) count (n=3). When the antibody was added, 50 µl of ADCC medium and 100 µl of ADCC medium containing 2% (v/v) of Triton-X100 when effector cells were added were added. Other than that, the same operation as the sample well was performed.

Average of 3 experiments, error bar indicates standard deviation, and P value was calculated by Student's t-test. The measurement results are shown in Fig. 3.

As a result, M30 showed a cell lytic activity of 31.6 ± 3.3% for B7-H3 expressing 293 cells, and thus the M30 antibody had ADCC activity for B93-H3 expressing 293 cells.

4)-3 CDC activity

The experiment was conducted by the same method as in Example 2)-3-1. NCI-H322 cells were used as the evaluation cells. Anti-B7-H3 antibody (L7, L8, L11, obtained with 6) diluted with 10% FBS-containing RPMI1640 (antibiotic-containing: penicillin, streptomycin) to a final concentration of 25 µg/ml after addition of complement M30, M31) and isotype control antibody (mIgG2a) were added, and stopped at 4°C for 1 hour. To this, rabbit complement diluted by 30% with RPMI1640 (#CL3051 manufactured by CEDARLANE) was added to a final concentration of 5%, incubated for 1 hour under conditions of 37°C and 5% CO ₂ , and further 30 at room temperature. It was stopped for a minute. In order to measure the cell viability, the same amount of CellTiter-Glo Luminescent Cell Viability Assay (manufactured by Promega) was added to the culture medium, and the mixture was stirred at room temperature for 10 minutes, and then the amount of luminescence was measured with a plate reader. Cell viability was calculated by the following equation.

Cell viability (%) = (a-b)/(c-b) × 100

a: Light emission amount of sample well

b: Average value of background (cell/antibody non-addition well) light emission (n = 3). When seeding cells, equivalent amounts of 10% FBS-containing RPMI1640 (antibiotic containing: penicillin, streptomycin) are added instead of the cell suspension, and when adding antibodies, equivalent amounts of 10% FBS with antibody-diluted RPMI1640 (containing antibiotic: penicillin, Streptomycin). Other than that, the same operation as the sample well was performed.

c: Average value of the light emission amount of the antibody-free well (n = 3). At the time of antibody addition, RPMI1640 (antibiotic containing: penicillin, streptomycin) containing 10% FBS in the same amount as the antibody dilution was added. Other than that, the same operation as the sample well was performed.

The measurement results are shown in Fig. 4. The average of three experiments, and the error bar indicates the standard deviation. As a result, the control antibodies, L7, L8, L11, M30, M31, respectively, were 101.5±3.3%, 6.3±4.2%, 13.6±9.1%, 7.2±1.4%, 7.5±1.8% for NCI-H322 cells in the presence of complement. , 12.8 ± 2.0% in that the cell viability was lowered, L7, L8, L11, M30, M31 antibodies were found to have CDC activity against NCI-H322 cells.

4)-4 Determination of binding domain

Which domain of B7-H3 is bound to M30 was examined by the same flow cytometry method as in Example 3)-3. NIH-3T3 cells transfected with each B7-H3 partial protein expression vector adjusted in Example 1)-1-3 were used.

As a result, it was confirmed that M30 binds to B7-H3 IgC1, B7-H3 IgC2, B7-H3 IgC1-V2-C2, and B7-H3 IgV2-C2 as shown in FIG. 5. It did not bind to B7-H3 IgV1, B7-H3 IgV2.

In this regard, it was shown that M30 binds to the C1 domain of B7-H3 (the amino acid sequence shown at amino acid numbers 140 to 244 of SEQ ID NO: 6) and the C2 domain (the amino acid sequence shown at amino acid numbers 358 to 456 of SEQ ID NO: 6). . Similarly, L8, L11 and M31 are also shown to bind to the C1 domain and the C2 domain, and L7 is the V1 domain (the amino acid sequence shown in amino acid numbers 27 to 139 of SEQ ID NO: 6) and the V2 domain (the amino acid number of array number 6) 245 to 357).

Therefore, it was shown that M30 recognizes epitopes in the IgC1 domain and/or IgC2 domain, which are domains in the extracellular region of B7-H3, and binds to the IgC1 domain, the IgC2 domain, or both.

4)-5 antigen specificity

The antigen specificity of M30 was examined by the same flow cytometry method as in Example 3)-3.

Example 1) 293T cells transfected with B7 family proteins CD80, CD86, B7-RP-1, B7-H1, B7-DC, and B7-H4 protein expression vectors, respectively, adjusted to-1-4 were used. .

As a result, it was shown that M30 does not bind to the B7 family molecules CD80, CD86, B7-RP-1, B7-H1, B7-DC, and B7-H4.

Example 5. Anti-tumor effect in vivo

5)-1 Anti-tumor effect in vivo of anti-B7-H3 antibody

After the NCI-H322 cells were trypsinized and removed from the culture flask, they were suspended in 10% FBS-containing RPMI1640 (Invitrogen) and centrifuged to remove the supernatant. After washing the cells twice in the same medium, the cells were suspended in physiological saline (manufactured by Otsuka Pharmaceutical Factory), and subcutaneously in the axillary subcutaneously at 1×10 ⁷ cells/mouse in 6-week-old BALB/cAJcl-nu/nu (Nippon Clea). Transplanted. L7, L8, L11, M30, and M31 antibodies were administered intraperitoneally at 500 µg/mouse (about 25 mg/kg) on days 10, 17, 24, 31, and 38 on the day of transplantation. The control was administered intraperitoneally with an antibody and the same volume (500 μl) of PBS. Tumor volume was measured on days 10, 17, 24, 31, 38 and 45, and the antitumor effect by antibody administration was examined.

As a result, the tumor proliferation was significantly suppressed in the M30 and M31 administration groups compared to the PBS administration group (P values of M30 and M31 were P <0.05 and P <0.01, respectively, in comparison with the PBS administration group for the tumor volume at day 45. P value is calculated by Student's t-test). In addition, the rate of tumor proliferation inhibition (= 100-(average of tumor volume in the antibody-administered group)/(average of tumor volume in the PBS-administered group) x 100) at day 45 was -16.1 in L7, L8, L11, M30, and M31, respectively. %, 0.2%, 25.5%, 47.2%, and 58.2%. Very strong anti-tumor effect was observed in vivo in M30 and M31 antibodies (Fig. 6).

From the above results, it became clear that the M30 and M31 antibodies were antibodies that recognized the anti-tumor effect by recognizing the B7-H3 antigen.

5)-2 Anti-tumor effect in vivo when phagocytic cells are removed

To remove phagocytic cells in vivo, clodronate-encapsulated liposomes were prepared. That is, it has been reported that phagocytic cells (macrophages) are removed in vivo by administering clodronate-encapsulated liposomes in vivo (Journal of immunological methods 1994, Vol. 174, p. 83-93), and the method of the report. Clodronate-encapsulated liposomes were prepared according to the following experiments.

After the NCI-H322 cells were trypsinized and removed from the culture flask, they were suspended in 10% FBS-containing RPMI1640 (Invitrogen) and centrifuged to remove the supernatant. After the cells were washed twice in the same medium, they were suspended in physiological saline (PBS, manufactured by Otsuka Pharmaceutical Factory), and 1×10 ⁷ cells/mouse in 6-week-old BALB/cAJcl-nu/nu (Nippon Clea). They were implanted subcutaneously. Grouping was performed on day 0 with the day of transplantation being -14 days.

In the group to remove macrophages in the living body of mice, clodronate-encapsulated liposomes were intravenously injected at 0.2 ml/mouse on days 0, 4, 7, 11, 14, 18, 21, 25, 28, and 32 days. In addition, the negative control group was injected intravenously with PBS at 0.2 ml/mouse on the same day (day 0, 4, 7, 11, 14, 18, 21, 25, 28, 32).

Next, M30 antibody was administered to both groups intraperitoneally at 500 μg/mouse (about 25 mg/kg) on days 1, 8, 15, 22 and 29. In addition, as a negative control, the M30 antibody and the same volume (500 μl) of PBS were administered intraperitoneally to both groups on the same day (Days 1, 8, 15, 22 and 29).

Tumor volume was measured on days 0, 8, 15, 22, 29 and 36, and the antitumor effect by antibody administration was examined (n=8).

The results are shown in Table 1, Table 2 and FIG. 7.

In the PBS intravenous administration + M30 antibody intraperitoneal administration group (PBS + M30 administration group), tumor proliferation was significantly inhibited compared to the negative PBS intravenous administration + PBS intraperitoneal administration group (PBS + PBS administration group). That is, in comparison with the PBS + PBS administration group for the tumor volume at the 36th day time point, the P value of the PBS + M30 administration group was P <0.05 (P value calculated by Student's t-test). In addition, the tumor growth inhibition rate (= 100-(average value of tumor volume in PBS + M30 administration group)/(average value of tumor volume in PBS + PBS administration group) x 100) at day 36 was 49.8% (Table 2).

On the other hand, inhibition of tumor proliferation was not observed in intravenous administration of clodronate-encapsulated liposomes + PBS intraperitoneal administration group (Clod lip + PBS administration group) and intravenous administration of clodronate-encapsulated liposomes + M30 antibody intraperitoneal administration group (Clod lip + M30 administration group). Did. That is, in comparison with the PBS + PBS administration group for the tumor volume at the 36th day time point, the P values of the Clod lip + PBS administration group and the Clod lip + M30 administration group were P = 0.52 and P = 1, respectively (P value in Student's t-test. Calculated by). In addition, the rate of tumor proliferation inhibition at the 36th day time point (= 100-(mean value of tumor volume of the Cl lip + PBS-treated group or Clod lip + M30-treated group)/(average of the tumor volume of the PBS + PBS-treated group) × 100) was each- It was 21.2% and -1.4% (Table 2).

From the above results, it was found that the anti-tumor effect of the M30 antibody was suppressed by administration of clodronate-encapsulated liposomes, and the anti-tumor effect of the M30 antibody was mainly an effect through macrophages.

Example 6. Cloning and alignment of cDNA of mouse antibody M30

6) Determination of N-terminal amino acid sequence of heavy chain and light chain of mouse antibody M30

To determine the N-terminal amino acid sequence of the heavy and light chains of mouse antibody M30, mouse antibody M30 purified in Example 3)-6 was isolated by SDS-PAGE. The protein in the gel is transferred from the gel after separation to a PVDF membrane (pore size 0.45 µm; manufactured by Invitrogen), washed with a washing buffer (25 mM NaCl, 10 mM sodium borate buffer pH 8.0), and then stained (50 % Methanol, 20% acetic acid, and 0.05% Coomassie Brilliant Blue) for 5 minutes, dyed, and then decolorized with 90% methanol.

The band portions corresponding to the heavy chain (the band with the small mobility) and the light chain (the band with the large mobility) visualized on the PVDF membrane were cut out.

For the band corresponding to the light chain, incubated in a small amount of 0.5% polyvinylpyrrolidone/100 mM acetic acid solution at 37°C for 30 minutes, washed well with water, and then Pfu pyroglutamic acid aminopeptidase kit (Takara Bio Co., Ltd.) ) To remove the modified N-terminal residue, washed with water and then air dried. Identification of each N-terminal amino acid sequence by automated Edman method (see Edman et al. (1967) Eur. J. Biochem. 1, 80) using Procise® cLC protein sequencer Model492cLC (Applied Biosystems) Tried.

As a result, the amino acid sequence of the N-terminal of the band corresponding to the heavy chain of the mouse antibody M30 is

EVQLQQSGPE (arrangement number 44 in the array table)

And the N-terminal amino acid sequence of the band corresponding to the light chain is

IVLSQSPTILSASP (arrangement number 45 in the array table)

It was.

6-2) Preparation of mRNA from mouse antibody M30-producing hybridoma

To clone the cDNA encoding the heavy and light chains of mouse antibody M30, mRNA was prepared using a Quick Prep mRNA Purification Kit (GE Healthcare) from mouse antibody M30 produced hybridomas.

6-3) Determination of cloning and alignment of cDNA of mouse antibody M30

The heavy and light chain isotypes of the mouse antibody M30 are γ2a and κ (Example 3)-5), the N-terminal amino acid sequence of the heavy chain and light chain as determined in Example 1-1), and the amino acid of the antibody With reference to the array database (Kabat, EA et al., (1991) in Sequences of Proteins of Immunological Interest Vol. I and II, US Department of Health and Human Services), the 5'end side of the antibody gene translation region and Several oligonucleotide primers each hybridizing to the 3'end portion containing the end codon were synthesized, and the mRNA prepared in Example 6-2) and TaKaRa One Step RNA PCR Kit (AMV) (Takara Bio) were used. As a result, the cDNA encoding the heavy chain and light chain was amplified, and the following primer set was able to amplify the heavy chain of the antibody and the cDNA encoding the light chain.

Primer set for heavy chain

Primer set for light chains

The cDNA of the heavy chain and the light chain amplified by PCR were cloned using pEF6/V5-His TOPO TA Expression Kit (Invitrogen), respectively, and gene sequence analysis of each nucleotide sequence of the cloned heavy chain and light chain was performed. It was determined using an apparatus ("ABI PRISM 3700 DNA Analyzer; Applied Biosystems" or "Applied Biosystems 3730 x l Analyzer; Applied Biosystems"). GeneAmp 9700 (Applied Biosystems) was used for the reaction of the sequence.

The nucleotide sequence of cDNA encoding the heavy chain of the determined mouse antibody M30 is shown in SEQ ID NO: 50, and the amino acid sequence is shown in SEQ ID NO: 51. In addition, the arrangement of the arrangement numbers 50 and 51 is described in FIG.

The nucleotide sequence of cDNA encoding the light chain of the determined mouse antibody M30 is shown in SEQ ID NO: 52 of the sequence table, and the amino acid sequence is shown in sequence number 53 of the sequence table. In addition, the arrangement of the arrangement numbers 52 and 53 is described in FIG.

In addition, as a result of comparing and examining the amino acid sequence of the heavy chain and light chain using KabatMan (PROTEINS: Structure, Function and Genetics, 25 (1996), 130-133.), which is a database of the amino acid sequence of the antibody, the mouse antibody M30 For the heavy chain of, it became clear that the amino acid sequence shown in amino acid numbers 20 to 141 of sequence number 51 in the sequence table was a variable region. In addition, for the light chain of the mouse antibody M30, it became clear that the amino acid sequence shown in amino acid numbers 23 to 130 of SEQ ID NO: 53 in the sequence table was a variable region.

Example 7. Preparation of chimeric antibody M30 (cM30 antibody)

7)-1 Preparation of universal expression vectors pEF6KCL and pEF1FCCU

7) Construction of the 1-1 chimera and humanized light chain expression vector pEF6KCL

DNA fragment (f1 origin) from immediately after (Sequence Position 2174) to Sma I (Sequence Position 2958) by BGHpA by PCR using plasmid pEF6/V5-HisB (Invitrogen) as a template and PCR using the following primers. DNA fragments containing of replication and SV40 promotor and origin, hereinafter referred to as "fragment A") were obtained.

A DNA fragment comprising the obtained fragment A, a DNA sequence encoding a human κ chain secretion signal, a human κ chain normal region, and a human polyA addition signal (arrangement number 56: hereinafter referred to as "fragment B". Arrangement of SEQ ID NO: 56) Is described in Figure 23) was combined by Overlap Extension PCR. The obtained fragment A and fragment B bound DNA fragments were digested with restriction enzymes KpnI and SmaI, ligated with restriction enzymes KpnI and SmaI and plasmids pEF6/V5-HisB (Invitrogen), downstream of the EF1 promoter. The chimeric and humanized light chain expression vector pEF6KCL was constructed with signal sequence, cloning site, human κ chain constant region, and human polyA addition signal sequence.

7)-2 Construction of pEF1KCL

The DNA fragments cut with restriction enzymes KpnI and SmaI from pEF6KCL obtained by the above method were ligated with pEF1/myc-HisB (Invitrogen) digested with KpnI and SmaI to construct plasmid pEF1KCL.

7) Construction of chimeric and humanized heavy chain expression vector pEF1FCCU

A DNA fragment comprising the human IgG1 signal sequence and the DNA sequence encoding the amino acid of the normal region (SEQ ID NO: 57: the sequence of SEQ ID NO: 57 is described in Figure 24) is digested with restriction enzymes NheI and PmeI, NheI and PmeI In combination with the plasmid pEF1KCL digested with, a chimeric and humanized heavy chain expression vector pEF1FCCU with a signal sequence, cloning site, human heavy chain constant region, and human polyA addition signal sequence was constructed downstream of the EF1 promoter.

7)-4 Construction of M30 chimeric type light chain expression vector

Using cDNA encoding the light chain of mouse antibody M30 as a template, a portion containing cDNA encoding the variable region of the light chain is amplified with KOD-Plus- (TOYOBO) and the following primer set, restriction enzyme NdeI and The M30 chimeric type light chain expression vector was constructed by inserting the DNA fragment cut by BsiWI at the point where the chimeric and humanized antibody light chain expression universal vector (pEF6KCL) was cut with restriction enzymes NdeI and BsiWI. The resulting expression vector was termed "pEF6KCL/M30L". The nucleotide sequence of the M30 chimeric type light chain is shown in SEQ ID NO: 58 in the sequence table, and the amino acid sequence is shown in SEQ ID NO: 59. In addition, the arrangement of the arrangement numbers 58 and 59 is described in FIG. In addition, in the amino acid sequence of the cM30 antibody light chain described in SEQ ID NO: 59 of the sequence table, the 128 th threonine residue is the carboxyl terminal of the light chain variable region, and in the amino acid sequence of the M30 antibody light chain of SEQ ID NO: 53 of the sequence table. Although it corresponds to the 130th alanine residue, in the amino acid sequence of SEQ ID NO: 59, it has already been substituted with a threonine residue derived from the human antibody light chain.

Primer set for light chains

7) Construction of 5 M30 chimeric type heavy chain expression vector

Using cDNA encoding the heavy chain of mouse antibody M30 as a template, PCR was performed using the DNA fragment obtained by PCR using KOD-Plus- (TOYOBO) and primer set A described below and primer set B described below. By binding the DNA fragment obtained by the execution by Overlap Extension PCR using the following primer set C, BlpI in the variable region was removed and a portion containing cDNA encoding the heavy chain variable region was amplified. The M30 chimeric type heavy chain expression vector was constructed by inserting the DNA fragment cut by the restriction enzyme BlpI at the point where the chimeric and humanized antibody heavy chain expression universal vector (pEF1FCCU) was cut with the restriction enzyme BlpI. The resulting expression vector was termed "pEF1FCCU/M30H".

The nucleotide sequence of the M30 chimeric type heavy chain is shown in SEQ ID NO: 62 in the sequence table, and the amino acid sequence is shown in SEQ ID NO: 63. In addition, the arrangement of the arrangement numbers 62 and 63 is described in FIG.

Primer set A

Primer set B

Primer set C

7)-6 chimeric antibody M30 preparation

7) Production of 6-1 chimeric antibody M30

Freestyle 293F cells of 1.2 × 10 ⁹ logarithmic growth phase (Invitrogen) were seeded in fresh 1.2 L of FreeStyle 293 expression medium (Invitrogen), and at 90 rpm in an incubator at 37°C and 8% CO ₂ at 90 rpm. Cultured with shaking for an hour. 3.6 mg of Polyethyleneimine (Polyscience #24765) was dissolved in 20 ml of Opti-Pro SFM medium (Invitrogen), followed by pEF1FCCU/M30H (0.4 mg) prepared using PureLink HiPure Plasmid kit (Invitrogen). pEF6KCL/M30L (0.8 mg) was suspended in 20 ml of Opti-Pro SFM medium. To 20 ml of the polyethyleneethylene/Opti-Pro SFM mixture, 20 ml of the expression vector/Opti-Pro SFM mixture was added and stirred carefully, and after standing for an additional 5 minutes, it was added to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 37 rpm for 8 days at 90 rpm in an 8% CO ₂ incubator was filtered with a Disposable Capsule Filter (Advantec #CCS-045-E1H).

The chimeric antibody M30 obtained by the combination of pEF1FCCU/M30H and pEF6KCL/M30L was designated as "cM30" or "cM30 antibody".

7) Purification of 6-2 cM30

The culture supernatant obtained in Example 7-1) was purified by a two-step process of rProteinA affinity chromatography (under 4-6°C) (GE Healthcare Japan) and ceramic hydroxylapatite (under room temperature). The buffer substitution process after rProteinA affinity chromatography purification and after ceramic hydroxylapatite purification was performed at room temperature.

First, the culture supernatant 1100-1200 ml was applied to MabSelectSuRe (HiTrap column manufactured by GE Healthcare Bioscience, volume: 1 ml x 2 connection) equilibrated with PBS. After all of the culture solution entered the column, the column was washed with 15-30 ml of PBS. Next, eluted with a 2 M arginine hydrochloride solution (pH 4.0), the fractions containing the antibody were collected. The fraction was replaced with a buffer of 5 mM sodium phosphate/50 mM MES/20 mM NaCl/pH 6.5 using a desalting column (GE Healthcare Bioscience, HiTrap Desalting column: volume 5 mL×2 connection).

Further, the substituted antibody solution was equilibrated with a buffer of 5 mM NaPi/50 mM MES/20 mM NaCl/pH 6.5, a ceramic hydroxylapatite column (Nippon Biorad, Bio-Scale CHT2-1 Hydroxyapatite Column: 2 ml in volume) It was applied. A linear concentration gradient elution with sodium chloride was performed to collect fractions containing the antibody. This fraction was subjected to liquid substitution with CBS (10 mM citric acid buffer/140 mM sodium chloride, pH 6.0) with a desalting column (GE Healthcare Bioscience, HiTrap Desalting column: volume 5 mL×2 connection).

Finally, it was concentrated to Centrifugal UF Filter Device VIVASPIN20 (fraction molecular weight 30 K, Sartorius, 4° C.), and the IgG concentration was adjusted to 1.0 mg/ml or more to prepare a purified sample.

Example 8. Activity of cM30 antibody

8)-1 cM30 antibody binding activity to B7-H3

The affinity of the M30 antibody and cM30 antibody with the B7-H3 antigen was measured by a surface plasmon resonance (SPR) device (GE Healthcare). By routine method, an anti-mouse IgG or anti-human IgG antibody is immobilized on a sensor chip, and after binding the M30 antibody sample or cM30 antibody there, an extracellular region polypeptide of the recombinant B7-H3 variant 2 antigen at each concentration ( R&D Systems Inc.: #2318-B3-050/CF) was added, and the amount of binding to the antibody in a running buffer (phosphate buffer, 0.05% SP20) was measured over time. The measured binding amount was analyzed by a dedicated software (BIAevaluation Version 4.1, GE Healthcare) to calculate the dissociation constant.

As a result, the M30 antibody and cM30 antibody bound to the recombinant B7-H3 antigen with dissociation constants of 5.89 nM and 3.43 nM, respectively. In this regard, it was confirmed that the M30 antibody and the cM30 antibody bind to the B7-H3 antigen, and the binding strengths thereof are almost equal.

8)-2 cM30 antibody ADCP activity

Peripheral blood mononuclear cells (PBMC) of normal humans were isolated according to the normal method. It was suspended in RPMI-10% FCS (Invitrogen) and seeded in a flask. Incubation was carried out overnight in a CO ₂ incubator. The culture supernatant was discarded, RPMI-10% FCS with M-CSF and GM-CSF (PeproTech) added to the cells attached to the flask, and cultured for 2 weeks. Cells were recovered by detaching with TrypLE. 500 μl/well (1×10 ⁵ cell/well) was added to a 24 well plate and incubated overnight at 37°C. This cell was used as an effector cell.

Labeling of NCI-H322 cells to be target cells was performed using a PKH26 dye labeling kit (Sigma). Target cells were peeled off with TrypLE and washed twice with PBS. Cells were suspended with Diluent C to 1 x 10 ⁷ cells/ml. PKH26 dye stock (1 mM) was diluted with Diluent C to 8 μM, and a cell suspension and an equal amount of dye dilution were added. It was left at room temperature for 5 minutes. Serum 1 ml was added, additional serum-containing medium was added, and washing was performed twice.

The M30 and cM30 antibodies were diluted to 20 μg/ml with the culture medium. Target cells were mixed by 2×10 ⁶ /100 μl/tube dispensing. It was allowed to stand still for 30 minutes on the ice. The supernatant was discarded and washed twice with the culture medium. It was suspended in 500 µl of the culture. The supernatant was removed from the effector cells, the antibody was treated, and suspended cells were added to the culture medium and mixed. Incubated for 3 hours in a CO ₂ incubator. The cells were detached with Trypsin-EDTA, and the cells were recovered. FITC-labeled anti-mouse CD11b antibody (Becton Dickinson) was added to the recovered cells, and allowed to stand still for 30 minutes on ice. The supernatant was discarded and washed twice with the culture medium. The recovered cells were suspended in 300 µl medium, and measured by FACS Calibur (Becton Dickinson). In the CD11b-positive macrophage cells, evaluation was performed using a PKH26-positive fraction as a phagocytic positive cell.

As a result, when 10 µg/ml of M30 and cM30 antibodies were added as shown in Fig. 8, phagocytosis of NCI-H322 cells by macrophage was induced by 33±1% and 35±2%, respectively. In this regard, it was shown that the cM30 antibody has ADCP activity against NCI-H322 cells in the same way as the M30 antibody. The same experimental results were also obtained for ADCP activity against MDA-MB-231 cells (ATCC).

8)-3 in vivo anti-tumor effect of cM30 antibody

MDA-MB-231 cells were trypsinized and removed from the culture flask, suspended in RPMI1640 medium containing 10% FBS (Invitrogen) and centrifuged to remove the supernatant. After washing the cells twice in the same medium, they were suspended in a BD Matrigel basement membrane matrix (manufactured by BD Biosciences) and 5 weeks old in mice (CB17/Icr-Prkdc[scid]/CrlCrlj, Nippon Charles River). The cells were implanted subcutaneously with x 10 ⁶ cells/mouse. On the day of transplantation, day 0, M30 antibody and cM30 antibody were administered intraperitoneally at days of 14, 21, 28, 35 and 42 at 500 µg/mouse (about 25 mg/kg). Tumor volumes were measured on days 14, 18, 21, 25, 28, 32, 35, 39, 42, 46, 49 and 52, and the antitumor effect by antibody administration was examined.

As a result, tumor proliferation was significantly inhibited in the M30 antibody and cM30 antibody administration groups compared to the untreated group without administration of the antibody. In comparison with the untreated group, the P values of the M30 antibody and the cM30 antibody were P<0.001 for tumor weight at day 52. The P value was calculated by Dunnett's multiple comparison.

In addition, the rate of tumor proliferation inhibition (= 100-(average of tumor volume in the antibody-administered group)/(average of tumor volume in the untreated group) x 100) at day 52 was 71.3% for the M30 antibody and 71.7% for the cM30 antibody. , cM30 antibody was observed in the same strong anti-tumor effect in vivo as the M30 antibody (Fig. 9).

Example 9. Design of humanized antibody of mouse anti-human B7-H3 antibody #M30

9)-1 Design of humanized version of M30

9)-1-1 Molecular modeling of the variable region of M30

Molecular modeling of the variable region of M30 was performed by methods commonly known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). Example 6 of the primary arrangement of variable regions of human immunoglobulins (a three-dimensional structure derived from an X-ray crystal structure is available) registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) -3) was compared with the variable region of M30.

As a result, 3BKY was selected as the one with the highest sequence homology among antibodies with deletions in the framework equally to the variable region of the light chain of M30. In addition, 3DGG was selected as having the highest alignment homology to the variable region of the heavy chain of M30.

The three-dimensional structure of the framework region was produced by combining the coordinates of 3BKY and 3DGG corresponding to the light and heavy chains of M30 to obtain a "framework model". The CDR of M30 is Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)), and according to H3 rules (FEBS letter 399, 1-8 (1996)), CDRH1 (SEQ ID NO: 92), CDRH2 (SEQ ID NO: 93) and the coordinates with the closest conformation to CDRH3 (array number 94), CDRL1 (array number 95), CDRL2 (array number 96), CDRL3 (array number 97), respectively 2HOJ, 1BBD, 1Q9O, 2FBJ, 1LNK , 1TET was selected and inserted into the framework model.

Finally, in order to obtain a molecular model with the potential of the variable region of M30 in terms of energy, energy calculations were performed to eliminate adverse interatomic contact. The above procedure was carried out using a commercial protein conformational structure prediction program Prime and a coordinate search program MacroModel (Schrodinger, LLC).

9)-1-2 Design of amino acid sequence for humanized M30

Construction of the humanized M30 antibody was carried out by a method generally known as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)).

Acceptor antibodies were selected based on amino acid homology within the framework region. The arrangement of the framework region of M30 was compared to all human frameworks of the Kabat database of amino acid sequences of antibodies (Nuc. Acid Res. 29, 205-206 (2001)), and as a result, mAb49' CL antibody (NCBI's GenBank : D16838.1 and D16837.1) were selected as acceptors due to 70% alignment homology to the framework regions.

The amino acid residues of the framework region for mAb49' CL were aligned with the amino acid residues for M30, and positions where different amino acids were used were identified. The positions of these residues were analyzed using the three-dimensional model of M30 constructed above, and the donor residues to be grafted onto the acceptor were described in Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). By introducing several selected donor residues into the acceptor antibody, the humanized #32A1 sequence was constructed as described in the Examples below.

9)-2 M30 heavy chain humanization

9)-2-1 M30-H1 type heavy chain:

CM30 heavy chain amino acid numbers 20 (glutamic acid), 24 (glutamine), 28 (proline), 30 (leucine), 31 (valine), 35 (alanine), 39 (methionine), 57 (shown in SEQ ID NO: 63 of the Sequence Listing) Lysine), 59 (lysine), 67 (isoleucine), 86 (lysine), 87 (alanine), 89 (glutamine), 91 (serine), 93 (lysine), 95 (serine), 106 (threonine), 110 ( Serine), 136 (Threonine), 137 (Leucine) glutamine, valine, alanine, valine, lysine, serine, valine, arginine, alanine, methionine, arginine, valine, isoleucine, alanine, glutamic acid, threonine, arginine, threonine, The humanized M30 heavy chain designed by substituting with leucine and valine was named "M30-H1 type heavy chain".

The amino acid sequence of the M30-H1 type heavy chain is shown in SEQ ID NO: 85.

9)-2-2 M30-H2 type heavy chain:

CM30 heavy chain amino acid numbers 20 (glutamic acid), 24 (glutamine), 28 (proline), 30 (leucine), 31 (valine), 35 (alanine), 39 (methionine), 57 (shown in SEQ ID NO: 63 of the Sequence Listing) Lysine), 59 (lysine), 86 (lysine), 87 (alanine), 89 (glutamine), 91 (serine), 93 (lysine), 95 (serine), 106 (threonine), 110 (serine), 136 ( Threonine) and 137 (Leucine) are replaced by glutamine, valine, alanine, valine, lysine, serine, valine, arginine, alanine, arginine, valine, isoleucine, alanine, glutamic acid, threonine, arginine, threonine, leucine, valine, respectively. The humanized M30 heavy chain designed accordingly was named "M30-H2 type heavy chain".

The amino acid sequence of the M30-H2 type heavy chain is shown in SEQ ID NO: 87.

9)-2-3 M30-H3 type heavy chain:

CM30 heavy chain amino acid numbers 24 (glutamine), 28 (proline), 30 (leucine), 31 (valine), 35 (alanine), 39 (methionine), 59 (lysine), 89 (shown in SEQ ID NO: 63 of the Sequence Listing) Glutamine), 91 (serine), 93 (lysine), 95 (serine), 106 (threonine), 110 (serine), 136 (threonine), 137 (leucine), respectively, valine, alanine, valine, lysine, serine, valine , Humanized M30 heavy chain designed by substituting with alanine, isoleucine, alanine, glutamic acid, threonine, arginine, threonine, leucine, valine was named "M30-H3 type heavy chain".

The amino acid sequence of the M30-H3 type heavy chain is shown in SEQ ID NO: 89.

9)-2-4 M30-H4 type heavy chain:

CM30 heavy chain amino acid numbers 24 (glutamine), 28 (proline), 30 (leucine), 31 (valine), 35 (alanine), 39 (methionine), 59 (lysine), 95 (shown in SEQ ID NO: 63 of the Sequence Listing) Serine), 106 (threonine), 110 (serine), 136 (threonine), and 137 (leucine) to valine, alanine, valine, lysine, serine, valine, alanine, threonine, arginine, threonine, leucine, valine, respectively. The humanized M30 heavy chain designed along the line was named "M30-H4 type heavy chain".

The amino acid sequence of the M30-H4 type heavy chain is shown in SEQ ID NO: 91.

9)-3 Humanization of M30 light chain

9)-3-1 M30-L1 type light chain:

CM30 light chain amino acid numbers 21 (glutamine), 25 (serine), 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (shown in SEQ ID NO: 59 in the Sequence Listing) Methionine), 42 (threonine), 61 (serine), 62 (serine), 64 (lysine), 65 (proline), 66 (tryptophan), 77 (valine), 89 (serine), 90 (tyrosine), 91 ( Serine), 97 (valine), 99 (alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, glutamic acid, threonine, alanine, threonine, leucine, arginine Humanized M30 light chain designed to replace with alanine, leucine, serine, glutamine, alanine, arginine, leucine, leucine, isoleucine, aspartic acid, phenylalanine, threonine, leucine, proline, phenylalanine, valine, glutamine, valine, isoleucine It was named "M30-L1 type light chain".

The amino acid sequence of the M30-L1 type light chain is shown in SEQ ID NO: 71.

9)-3-2 M30-L2 type light chain:

CM30 light chain amino acid numbers 21 (glutamine), 25 (serine), 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (shown in SEQ ID NO: 59 in the Sequence Listing) Methionine), 42 (threonine), 61 (serine), 62 (serine), 64 (lysine), 65 (proline), 77 (valine), 89 (serine), 91 (serine), 97 (valine), 99 ( Alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, glutamic acid, threonine, alanine, threonine, leucine, arginine, alanine, leucine, serine, glutamine, alanine , Humanized M30 light chain designed by substituting with arginine, leucine, isoleucine, aspartic acid, threonine, leucine, proline, phenylalanine, valine, glutamine, valine, isoleucine was named "M30-L2 type light chain".

The amino acid sequence of the M30-L2 type light chain is shown in SEQ ID NO: 73.

9)-3-3 M30-L3 type light chain:

CM30 light chain amino acid numbers 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (methionine), 62 (serine), 65 (shown in SEQ ID NO: 59 in the Sequence Listing) Proline), 77 (valine), 91 (serine), 97 (valine), 99 (alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, alanine The humanized M30 light chain designed by substituting threonine, leucine, arginine, alanine, leucine, alanine, leucine, isoleucine, threonine, leucine, proline, phenylalanine, valine, glutamine, valine, isoleucine ``M30-L3 type light chain It was named.

The amino acid sequence of the M30-L3 type light chain is shown in SEQ ID NO: 75.

9)-3-4 M30-L4 type light chain:

CM30 light chain amino acid numbers 21 (glutamine), 25 (serine), 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (shown in SEQ ID NO: 59 in the Sequence Listing) Methionine), 42 (threonine), 61 (serine), 62 (serine), 64 (lysine), 66 (tryptophan), 77 (valine), 89 (serine), 90 (tyrosine), 91 (serine), 96 ( Arginine), 97 (valine), 99 (alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, glutamic acid, threonine, alanine, threonine, leucine, arginine Humanized M30 light chain designed to replace with alanine, leucine, serine, glutamine, alanine, arginine, leucine, isoleucine, aspartic acid, phenylalanine, threonine, serine, leucine, proline, phenylalanine, valine, glutamine, valine, isoleucine It was named "M30-L4 type light chain".

The amino acid sequence of the M30-L4 type light chain is shown in SEQ ID NO: 77.

9)-3-5 M30-L5 type light chain:

CM30 light chain amino acid numbers 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (methionine), 62 (serine), 77 (shown in SEQ ID NO: 59 in the Sequence Listing) Valine), 91 (serine), 97 (valine), 99 (alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, alanine, threonine, leucine, The humanized M30 light chain designed by substituting arginine, alanine, leucine, alanine, isoleucine, threonine, leucine, proline, phenylalanine, valine, glutamine, valine, and isoleucine was named "M30-L5 type light chain".

The amino acid sequence of the M30-L5 type light chain is shown in SEQ ID NO: 79.

9)-3-6 M30-L6 type light chain:

CM30 light chain amino acid numbers 21 (glutamine), 25 (serine), 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (shown in SEQ ID NO: 59 in the Sequence Listing) Methionine), 42 (threonine), 61 (serine), 62 (serine), 64 (lysine), 66 (tryptophan), 77 (valine), 89 (serine), 90 (tyrosine), 91 (serine), 97 ( Valine), 99 (alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, glutamic acid, threonine, alanine, threonine, leucine, arginine, alanine, leucine, The humanized M30 light chain designed to replace serine, glutamine, alanine, arginine, leucine, isoleucine, aspartic acid, phenylalanine, threonine, leucine, proline, phenylalanine, valine, glutamine, valine, and isoleucine is called ``M30-L6 type light chain. It was named.

The amino acid sequence of the M30-L6 type light chain is shown in SEQ ID NO: 81.

9)-3-7 M30-L7 type light chain:

CM30 light chain amino acid numbers 21 (glutamine), 25 (serine), 29 (threonine), 30 (isoleucine), 33 (alanine), 38 (lysine), 39 (valine), 41 (shown in SEQ ID NO: 59 in the Sequence Listing) Methionine), 42 (threonine), 61 (serine), 62 (serine), 64 (lysine), 66 (tryptophan), 77 (valine), 89 (serine), 91 (serine), 97 (valine), 99 ( Alanine), 102 (alanine), 104 (threonine), 119 (threonine), 123 (leucine), 125 (leucine), respectively, glutamic acid, threonine, alanine, threonine, leucine, arginine, alanine, leucine, serine, glutamine, alanine , Humanized M30 light chain designed by substituting with arginine, leucine, isoleucine, aspartic acid, threonine, leucine, proline, phenylalanine, valine, glutamine, valine, isoleucine was named "M30-L7 type light chain".

The amino acid sequence of the M30-L7 type light chain is shown in SEQ ID NO: 83.

(Example 10) Preparation of humanized antibody

10)-1 Construction of M30-L1, M30-L2, M30-L3, M30-L4, M30-L5, M30-L6, and M30-L7 type light chain expression vectors

Amino acid number 21 to 128 of sequence number 71 of the sequence table, amino acid number 21 to 128 of sequence number 73, amino acid number 21 to 128 of sequence number 75, amino acid number 21 to 128 of sequence number 77, amino acid number 21 to 128 of sequence number 79 M30-L1, M30-L2, M30-L3, M30-L4, M30-L5, M30-L6, and the amino acid numbers 21 to 128 of SEQ ID NO: 81 and amino acid numbers 21 to 128 of SEQ ID NO: 83, respectively. DNA comprising a gene encoding the M30-L7 type light chain variable region is synthesized based on SEQ ID NOs: 70, 72, 74, 76, 78, 80 and 82 related to the nucleotide sequence corresponding to the sequence number related to the amino acid sequence. (GENEART, artificial gene synthesis service), and inserting the DNA fragments cut with restriction enzymes NdeI and BsiWI into the point where the chimeric and humanized antibody light chain expression universal vector (pEF6KCL) was cleaved with restriction enzymes NdeI and BsiWI, and , M30-L2, M30-L3, M30-L4, M30-L5, M30-L6, and M30-L7 type light chain expression vectors were constructed.

"PEF6KCL/M30-L1", "pEF6KCL/M30-L2", "pEF6KCL/M30-L3", "pEF6KCL/M30-L4", "pEF6KCL/M30-L5", and "pEF6KCL/M30-" respectively. L6” and “pEF6KCL/M30-L7”.

10) Construction of M30-H1, M30-H2, M30-H3, and M30-H4 type heavy chain expression vectors

M30-H1, M30 shown to amino acid number 20-141 of sequence number 85 of the sequence table, amino acid number 20-141 of sequence number 87, amino acid number 20-141 of sequence number 89, and amino acid number 20-141 of sequence number 91, respectively. DNA comprising a gene encoding a -H2, M30-H3, and M30-H4 type heavy chain variable region is based on SEQ ID NOs: 84, 86, 88 and 90 associated with a nucleotide sequence corresponding to the sequence number associated with the amino acid sequence. By synthesizing (GENEART artificial gene synthesis service), and inserting the DNA fragment cut by the restriction enzyme BlpI into the point where the humanized antibody heavy chain expression universal vector (pEF1FCCU) was cleaved with the restriction enzyme BlpI, M30-H1, M30-H2, M30-H3, and M30-H4 type heavy chain expression vectors were constructed.

The resulting expression vectors were named "pEF1FCCU/M30-H1", "pEF1FCCU/M30-H2", "pEF1FCCU/M30-H3", and "pEF1FCCU/M30-H4", respectively.

10)-3 Production of humanized antibodies

Freestyle 293F cells (Invitrogen) of 1.2 × 10 ⁹ logarithmic growth phases were seeded in fresh 1.2 L of FreeStyle293 expression medium (Invitrogen), and 1 hour at 90 rpm in a 37°C, 8% CO ₂ incubator. The culture was shaken. 3.6 mg of Polyethyleneimine (Polyscience #24765) was dissolved in 20 ml of Opti-Pro SFM medium (Invitrogen), and then a heavy chain expression vector (0.4 mg) prepared using PureLink HiPure Plasmid kit (Invitrogen). And light chain expression vector (0.8 mg) was suspended in 20 ml of Opti-Pro SFM medium. To 20 ml of the polyethyleneethylene/Opti-Pro SFM mixture, 20 ml of the expression vector/Opti-Pro SFM mixture was added and stirred carefully, and after standing for an additional 5 minutes, it was added to FreeStyle 293F cells. The culture supernatant obtained by shaking culture at 37 rpm and 90 rpm in an 8% CO ₂ incubator for 7 days was filtered with a Disposable Capsule Filter (Advantec #CCS-045-E1H).

Humanized antibody of M30 obtained by combination of pEF1FCCU/M30-H1 and pEF6KCL/M30-L1 Humanized antibody of M30 obtained by combination of "M30-H1-L1", pEF1FCCU/M30-H1 and pEF6KCL/M30-L2 "M30-H1-L2", pEF1FCCU/M30-H3, pEF1FCCU/M30-H1 and pEF6KCL/M30- humanized antibodies obtained from M30-H1-L2, pEF1FCCU/M30-H1 and pEF6KCL/M30-L3 The humanized antibody of M30 obtained by the combination of L4 is "M30-H1-L4", the humanized antibody of M30 obtained by the combination of pEF1FCCU/M30-H4 and pEF6KCL/M30-L1 is "M30-H4-L1", Humanized antibody of M30 obtained by combination of pEF1FCCU/M30-H4 and pEF6KCL/M30-L2 Humanized antibody of M30 obtained by combination of "M30-H4-L2", pEF1FCCU/M30-H4 and pEF6KCL/M30-L3 "M30-H4-L4", pEF1FCCU/M30-H1 and pEF6KCL/M30- for humanized antibodies of M30 obtained by combining the antibody "M30-H4-L3", pEF1FCCU/M30-H4 and pEF6KCL/M30-L4 The humanized antibody of M30 obtained by the combination of L5 is "M30-H1-L5", the humanized antibody of M30 obtained by the combination of pEF1FCCU/M30-H1 and pEF6KCL/M30-L6 is "M30-H1-L6", And humanized antibody of M30 obtained by the combination of pEF1FCCU/M30-H1 and pEF6KCL/M30-L7 was named "M30-H1-L7".

10)-4 Purification of humanized antibody

The culture supernatant obtained in Example 6-1) was purified by a two-step process of rProteinA affinity chromatography (under 4-6°C) and ceramic hydroxyl apatite (under room temperature). The buffer substitution process after rProteinA affinity chromatography purification and after ceramic hydroxylapatite purification was performed at room temperature.

First, 1100-1200 ml of the culture supernatant was applied to MabSelectSuRe (HiTrap column manufactured by GE Healthcare Bioscience: volume 1 ml x 2 connection) equilibrated with PBS. After all of the culture solution entered the column, the column was washed with 15-30 ml of PBS. Next, eluted with a 2 M arginine hydrochloride solution (pH 4.0), the fractions containing the antibody were collected. The fraction was replaced with a buffer of 5 mM sodium phosphate/50 mM MES/20 mM NaCl/pH 6.5 using a desalting column (GE Healthcare Bioscience, HiTrap Desalting column: volume 5 ml×2 connection).

Further, the substituted antibody solution was equilibrated with a buffer of 5 mM NaPi/50 mM MES/20 mM NaCl/pH 6.5, ceramic hydroxylapatite column (Nippon Biorad, Bio-Scale CHT2-1 Hydroxyapatite Column: 2 ml volume) It was applied. A linear concentration gradient elution with sodium chloride was performed to collect fractions containing the antibody.

This fraction was subjected to liquid substitution with CBS (10 mM citric acid buffer/140 mM sodium chloride, pH 6.0) with a desalting column (GE Healthcare Bioscience, HiTrap Desalting column: volume 5 mL×2 connection).

Finally, it was concentrated in Centrifugal UF Filter Device VIVASPIN20 (fraction molecular weight 30 K, Sartorius, 4° C.), and the IgG concentration was adjusted to 1.0 mg/mL or more to prepare a purified sample.

10)-5 Humanized Antibodies to B7-H3 Antigen

The binding of the human B7-H3 antigen, a humanized M30 antibody, was measured by a surface plasmon resonance (SPR) device (BIACORE). By a static method, an anti-human IgG antibody is immobilized on a sensor chip, and after binding a purified sample of the humanized M30 antibody obtained in 10)-4 above, the extracellular region polypeptide of the B7-H3 variant 2 antigen at each concentration ( R&D Systems Inc.: #2318-B3-050/CF) was added, and the amount of binding to the antibody in a running buffer (phosphate buffer, 0.05% SP20) was measured over time. The measured binding amount was analyzed by dedicated software (BIA evaluation) to calculate the dissociation constant (KD [M]). The cM30 antibody was measured as a positive control for each measurement session. The results are shown in Table 3, and all of the humanized M30 antibodies had binding activity against the B7-H3 antigen.

Example 11.Measurement of contention inhibitory activity of B30-H3 antigen binding of M30 antibody, cM30 antibody and humanized M30 antibody

The competition inhibition activity of cM30 antibody, humanized M30 antibody (M30-H1-L4 antibody) to binding of M30 antibody to B7-H3 variant 1 and variant 2 was measured by the following method.

Using the EZ-Link Sulfo-NHS-LC Biotinylation Kit (#21435 manufactured by Thermo scientific), the mouse monoclonal antibody M30 was biotinylated according to the attached protocol (hereinafter, the biotinylated M30 is referred to as “bM30”, respectively). Notation). In addition, BD OPTI EIA (BD Biosciences #550536) was used for all of the buffers used in the following ELISA method.

Extracellular domain polypeptide of B7-H3 variant 1 (manufactured by R&D Systems: #1949-B3-050/CF) and extracellular domain polypeptide of B7-H3 variant 2 (manufactured by R&D Systems: #2318 -B3-050/CF) was diluted to 0.5 µg/ml with a coating buffer, dispensed at 100 µl/well in an immunoplate (#442404 manufactured by Nunc), and the protein was adsorbed onto the plate by standing still at 4°C overnight. The next day, the wells were dispensed with 200 μl/well of assay diluent, and allowed to stand still at room temperature for 4 hours.

After removing the solution in the well, 5 µg/ml of biotinylated antibody and various concentrations (0 µg/ml, 1 µg/ml, 5 µg/ml, 25 µg/ml, 50 µg/ml, 125 µg/ml) The mixed solution of unlabeled antibody was dispensed at 100 µl/well each in the assay diluent, and allowed to stand still at room temperature for 1 hour.

After washing the wells twice with Wash buffer, Streptavidin-horseradish Peroxidase Conjugate (#RPN1231V manufactured by GE Healthcare Bio-Sciences) diluted 500-fold with an assay diluent was added at 100 µl/well and stopped at room temperature for 1 hour.

The solution in the well was removed, the well was washed twice with wash buffer, and Substrate Solution was added at 100 µl/well to develop a color reaction while stirring. After color development, blocking buffer was added at 100 µl/well to stop the color reaction, and absorbance at 450 nm was measured with a plate reader.

As a result, the absorbance of the wells to which only bM30 was added was 2.36 ± 0.05, 1.90 ± 0.20 (average ± ±), respectively, on the plate to which the extracellular domain polypeptide of B7-H3 variant 1 and the extracellular domain polypeptide of B7-H3 variant 2 were attached. Standard deviation (n = 12)).

The absorbance in the graph of FIG. 10 is represented by an average value±standard deviation (n=3), respectively. Control IgG did not inhibit binding of bM30 to B7-H3.

On the other hand, the binding of bM30 to B7-H3 is the M30 antibody itself or its chimeric antibody in any of the plates to which the extracellular domain polypeptide of B7-H3 variant 1 and the extracellular domain polypeptide of B7-H3 variant 2 are attached. It was shown to be inhibited by cM30 antibody and humanized antibody M30-H1-L4.

That is, it was shown that the cM30 antibody and the humanized antibody (M30-H1-L4 antibody) recognize the same B7-H3 antigen epitope as the M30 antibody.

Example 12. Activity of humanized M30 antibody

12)-1 ADCP activity of humanized M30 antibody

Normal PBMCs were isolated according to the normal method. It was suspended in RPMI-10% FCS and seeded in a flask. Incubation was carried out overnight in a CO ₂ incubator. The culture supernatant was discarded, RPMI-10% FCS with M-CSF and GM-CSF (PeproTech) added to the cells attached to the flask, and cultured for 2 weeks. Cells were recovered by detaching with TrypLE. 500 μl/well (1×10 ⁵ cell/well) was added to a 24 well plate and incubated overnight at 37°C. This cell was used as an effector cell.

Labeling of NCI-H322 cells to be target cells was performed using a PKH26 dye labeling kit (Sigma). Target cells were peeled off with TrypLE and washed twice with PBS. Cells were suspended with Diluent C to 1 x 10 ⁷ cells/ml. The PKH26 dye stock (1 mM) was diluted with Diluent C to 8 μM, and the cell suspension and the same amount of dye dilution were added. It was left at room temperature for 5 minutes. Serum 1 ml was added, additional serum-containing medium was added, and washing was performed twice.

The M30 antibody, cM30 antibody and humanized M30 antibody (M30-H1-L4 antibody) were diluted to 20 μg/ml with RPMI-10% FCS (Invitrogen). Target cells (NCI-H322 cells) were mixed by 2×10 ⁶ cells/100 μl/tube dispensing. It was allowed to stand still for 30 minutes on the ice. The supernatant was discarded and washed twice with the culture medium. It was suspended in 500 µl of the culture.

The supernatant was removed from the effector cells, treated with M30 antibody, cM30 antibody and humanized M30 antibody (M30-H1-L4 antibody), and suspended cells were added to the culture medium and mixed. Incubated for 3 hours in a CO ₂ incubator.

The cells were detached with Trypsin-EDTA, and the cells were recovered.

FITC-labeled anti-mouse CD11b antibody (Becton Dickinson) was added to the recovered cells, and allowed to stand still for 30 minutes on ice.

The supernatant was discarded and washed twice with the culture medium.

The recovered cells were suspended in a medium of 300 uL, and measured by FACS Calibur (Becton Dickinson). In the CD11b positive macrophage cells, the PKH26 positive fraction was evaluated as a phagocytic positive cell.

The results are shown in Fig. 11.

In the group to which the humanized M30 antibody (M30-H1-L4 antibody) was added, ADCP activity was observed against NCI-H322 cells in the same manner as the group to which the M30 antibody and cM30 antibody were added.

12)-2 ADCC activity of humanized M30 antibody

Normal PBMCs were isolated according to the normal method. It was suspended in RPMI-10% FCS and seeded in a flask. Incubation was carried out overnight in a CO ₂ incubator.

Suspended cells were collected and washed to make peripheral blood lymphocytes (PBL). The obtained PBL was suspended in a phenol red-free RPMI1640 (Invitrogen Co., Ltd.) containing 10% Fetal Bovine Serum (Invitrogen Co., Ltd.) (hereinafter abbreviated as "ADCC medium"), and a cell strainer (pore size 40). (Micrometer: BD Biosciences), and the number of live cells was measured by a trypan blue pigment exclusion test. After centrifugation of the spleen cell suspension, the medium was removed, and resuspended in ADCC medium to give a cell density of 2.5 x 10 ⁶ cells/ml to obtain effector cells.

Chromium-51 (5550 kBq) of NCI-H322 cells treated with trypsin, washed with 10% FBS-containing RPMI1640, resuspended in 10% FBS-containing RPMI1640, and sterilized 4 x 10 ⁶ cells with a 0.22 μm filter ), and labeled for 1 hour at 37°C under 5% CO ₂ conditions. The labeled cells were washed 3 times in ADCC medium and resuspended to 2 x 10 ⁵ cells/mL in ADCC medium to obtain target cells.

Target cells at 2 x 10 ⁵ cells/ml were dispensed into a 96 hole U bottom microplate at 50 μl/well. Thereafter, cM30 antibody diluted with ADCC medium and humanized M30 antibody (M30-H1-L4, M30-H4-L4, M30-H1- were diluted with ADCC medium to a final concentration of 1, 10, 100, 1000 ng/ml after adding effector cells. L5, M30-H1-L6, and each antibody of M30-H1-L7) was added in 50 µl. To this, 100 µl of effector cells of 2.5 x 10 ⁶ cells/ml were added, and cultured for 4 hours under conditions of 37°C and 5% CO ₂ . The supernatant was collected in LumaPlate (manufactured by PerkinElmer), and the gamma dose emitted from the gamma counter was measured. Cell lysis rate by ADCC activity was calculated by the following equation.

Cell lysis rate (%) = (A-B)/(C-B) × 100

A: Sample well count

B: Average value of natural release (antibody/effector cell-free well) count (n = 3). At the time of antibody addition and effector cell addition, 50 µl and 100 µl of ADCC medium were added, respectively. Other than that, the same operation as the sample well was performed.

C: Average of the maximum release (wells in which target cells were lysed with surfactant) count (n=3). When the antibody was added, 50 µl of ADCC medium and 100 µl of ADCC medium containing 2% (v/v) of Triton-X100 when effector cells were added were added. Other than that, the same operation as the sample well was performed.

The results are shown in Table 4 and FIG. 12.

The average value of three experiments, the error bar indicates the standard deviation, and the P value was calculated by Student's t-test.

ADCC activity was observed in the M30-H1-L4 antibody addition group, similarly to the cM30 antibody addition group. ADCC activity was similarly observed for other humanized M30 antibodies (M30-H4-L4, M30-H1-L5, M30-H1-L6, and M30-H1-L7).

12)-3 In vivo anti-tumor effect of humanized M30 antibody

After MDA-MB-231 cells were trypsinized and peeled from the culture flask, they were suspended in RPMI1640 medium (Life Technologies, Inc.) containing 10% FBS, centrifuged, and the supernatant was removed. After washing the cells twice in the same medium, they were suspended in a BD Matrigel basement membrane matrix (manufactured by BD Biosciences) and 5× in 6-week-old mice (FOX CHASE SCID CB 17/Icr-scid/scidJcl, Nippon Clea Co., Ltd.). 10 ⁶ cells/mouse were implanted subcutaneously. 10, 1, 0.1, and 0.01 mg/kg of humanized M30 antibody (M30-H1-L4 antibody) at days 14, 21, 28, 35, and 42 on the day of transplantation (about 200, 20, 2, 0.2 respectively) Μg/mouse). Tumor volumes were measured on days 14, 18, 21, 25, 28, 31, 35, 39, 42, 45 and 49, and the antitumor effect by antibody administration was examined.

As a result, in the 10, 1, 0.1 mg/kg administration group of the humanized M30 antibody (M30-H1-L4 antibody), tumor proliferation was significantly inhibited compared to the untreated group without administration of the antibody. Tumor growth inhibition rate (%) (= 100-(antibody) of the 10, 1 and 0.1 mg/kg administered groups of humanized M30 antibody (M30-H1-L4 antibody) in comparison with the untreated group relative to tumor weight at day 49 The average value of the tumor weight of the administration group)/(the average value of the tumor weight of the non-treatment group) × 100) was 67, 54, and 51%, respectively, and all P values were P<0.0001. The P value was calculated by Dunnett's multiple comparison.

In addition, the tumor growth inhibition rate (%) of the M30-H1-L4 antibody at day 49 (%) (= 100-(average of tumor volume in the antibody-administered group)/(average of tumor volume in the untreated group) × 100) was 10, 84, 68, 61, 30%, respectively, in the 1, 0.1, and 0.01 mg/kg administration groups, and the humanized M30 antibody (M30-H1-L4 antibody) is the same as the M30 antibody and cM30 antibody, and has a very strong antitumor effect in vivo. Was observed, and the dose reactivity was confirmed to the effect (Fig. 38).

Industrial availability

The anti-B7-H3 antibody of the present invention has anti-tumor activity, and the pharmaceutical composition containing the anti-B7-H3 antibody can be an anti-cancer agent.

Array table free text

SEQ ID NO: 1-PCR primer 1

SEQ ID NO: 2-PCR primer 2

SEQ ID NO: 3-CMV promoter primer: Primer 3

SEQ ID NO: 4-BGH reverse primer: primer 4

SEQ ID NO: 5-Nucleotide sequence of B7-H3 variant 1

SEQ ID NO: 6-Amino acid sequence of B7-H3 variant 1

SEQ ID NO: 7-PCR primer 5

SEQ ID NO: 8-PCR primer 6

SEQ ID NO: 9-Nucleotide sequence of B7-H3 variant 2

SEQ ID NO: 10-Amino acid sequence of B7-H3 variant 2

SEQ ID NO: 11-PCR primer 7

SEQ ID NO: 12-PCR primer 8

SEQ ID NO: 13-PCR primer 9

SEQ ID NO: 14-PCR primer 10

SEQ ID NO: 15-PCR primer 11

SEQ ID NO: 16-PCR primer 12

SEQ ID NO: 17-PCR primer 13

SEQ ID NO: 18-PCR primer 14

SEQ ID NO: 19-PCR primer 15

SEQ ID NO: 20-Nucleotide sequence of B7-H3 IgV1

SEQ ID NO: 21-Amino acid sequence of B7-H3 IgV1

SEQ ID NO: 22-Nucleotide sequence of B7-H3 IgC1

SEQ ID NO: 23-Amino acid sequence of B7-H3 IgC1

SEQ ID NO: 24-Nucleotide sequence of B7-H3 IgV2

SEQ ID NO: 25-Amino acid sequence of B7-H3 IgV2

SEQ ID NO: 26-Nucleotide sequence of B7-H3 IgC2

SEQ ID NO: 27-Amino acid sequence of B7-H3 IgC2

SEQ ID NO: 28-Nucleotide sequence of B7-H3 IgC1-V2-C2

SEQ ID NO: 29-Amino acid sequence of B7-H3 IgC1-V2-C2

SEQ ID NO: 30-Nucleotide sequence of B7-H3 IgV2-C2

SEQ ID NO: 31-Amino acid sequence of B7-H3 IgV2-C2

SEQ ID NO: 32-Nucleotide sequence of B7RP-1

SEQ ID NO: 33-Amino acid sequence of B7RP-1

SEQ ID NO: 34-Nucleotide sequence of B7-H1

Amino acid sequence of SEQ ID NO: 35-B7-H1

SEQ ID NO: 36-Nucleotide sequence of B7-DC

SEQ ID NO: 37-Amino acid sequence of B7-DC

SEQ ID NO: 38-Nucleotide sequence of CD80

SEQ ID NO: 39-Amino acid sequence of CD80

SEQ ID NO: 40-Nucleotide sequence of CD86

SEQ ID NO: 41-Amino acid sequence of CD86

SEQ ID NO: 42-Nucleotide sequence of B7-H4

Amino acid sequence of SEQ ID NO: 43-B7-H4

SEQ ID NO: 44-N-terminal amino acid sequence of the mouse antibody M30 heavy chain

SEQ ID NO: 45-N-terminal amino acid sequence of mouse antibody M30 light chain

SEQ ID NO: 46-PCR primer 16

SEQ ID NO: 47-PCR primer 17

SEQ ID NO: 48-PCR primer 18

SEQ ID NO: 49-PCR primer 19

SEQ ID NO: 50-Nucleotide sequence of cDNA encoding M30 antibody heavy chain

SEQ ID NO: 51-amino acid sequence of the M30 antibody heavy chain

SEQ ID NO: 52-Nucleotide sequence of cDNA encoding M30 antibody light chain

SEQ ID NO: 53-Amino acid sequence of the M30 antibody light chain

SEQ ID NO: 54-PCR primer 20

SEQ ID NO: 55-PCR primer 21

SEQ ID NO: 56-DNA sequence encoding human κ chain secretion signal, human κ chain normal region and human polyA addition signal

SEQ ID NO: 57-DNA fragment comprising a human IgG1 signal sequence and a DNA sequence encoding the amino acid of the normal region

SEQ ID NO: 58-Nucleotide sequence of cDNA encoding the M30 antibody chimeric type light chain

SEQ ID NO: 59-Amino acid sequence of the M30 antibody chimeric type light chain

SEQ ID NO: 60-PCR primer 22

SEQ ID NO: 61-PCR primer 23

SEQ ID NO: 62-Nucleotide sequence of cDNA encoding the M30 antibody chimeric type heavy chain

SEQ ID NO: 63-M30 antibody chimeric type heavy chain amino acid sequence

SEQ ID NO: 64-PCR primer 24

SEQ ID NO: 65-PCR primer 25

SEQ ID NO: 66-PCR primer 26

SEQ ID NO: 67-PCR primer 27

SEQ ID NO: 68-PCR primer 28

SEQ ID NO: 69-PCR primer 29

SEQ ID NO: 70-Nucleotide sequence of light chain of type M30-L1

SEQ ID NO: 71-Amino acid sequence of the M30-L1 type light chain

SEQ ID NO: 72-Nucleotide sequence of M30-L2 type light chain

SEQ ID NO: 73-Amino acid sequence of the M30-L2 type light chain

SEQ ID NO: 74-M30-L3 type light chain nucleotide sequence

SEQ ID NO: 75-Amino acid sequence of the M30-L3 type light chain

SEQ ID NO: 76-M30-L4 type light chain nucleotide sequence

SEQ ID NO: 77-Amino acid sequence of the M30-L4 type light chain

SEQ ID NO: 78-M30-L5 type light chain nucleotide sequence

SEQ ID NO: 79-Amino acid sequence of the M30-L5 type light chain

SEQ ID NO: 80-M30-L6 light chain nucleotide sequence

SEQ ID NO: 81-Amino acid sequence of the M30-L6 type light chain

SEQ ID NO: 82-M30-L7 type light chain nucleotide sequence

SEQ ID NO: 83-Amino acid sequence of the M30-L7 type light chain

SEQ ID NO: 84-M30-H1 heavy chain nucleotide sequence

SEQ ID NO: 85-amino acid sequence of the M30-H1 type heavy chain

SEQ ID NO: 86-M30-H2 type heavy chain nucleotide sequence

SEQ ID NO: 87-M30-H2 type heavy chain amino acid sequence

SEQ ID NO: 88-M30-H3 heavy chain nucleotide sequence

SEQ ID NO: 89-Amino acid sequence of the M30-H3 type heavy chain

SEQ ID NO: 90-M30-H4 heavy chain nucleotide sequence

SEQ ID NO: 91-M30-H4 heavy chain amino acid sequence

SEQ ID NO: 92-Amino acid sequence of CDRH1 of the M30 antibody

SEQ ID NO: 93-Amino acid sequence of CDRH2 of the M30 antibody

SEQ ID NO: 94-Amino acid sequence of CDRH3 of the M30 antibody

SEQ ID NO: 95-Amino acid sequence of CDRL1 of M30 antibody

SEQ ID NO: 96-Amino acid sequence of CDRL2 of M30 antibody

SEQ ID NO: 97-Amino acid sequence of CDRL3 of the M30 antibody

SEQUENCE LISTING <110> Daiichi Sankyo Company, Limited <120> Anti B7-H3 antibody <130> DSPCT-FP1214 <150>?JP 2011-097645 <151>?2011-04-25 <150>?US 61/478878 <151>?2011-04-25 <160> 97 <170> PatentIn version 3.4 <210> 1 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 1 ctatagggag acccaagctg gctagcatgc tgcgtcggcg gggcag 46 <210> 2 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 2 aacgggccct ctagactcga gcggccgctc aggctatttc ttgtccatca tcttctttgc 60 tgtcag 66 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 cgcaaatggg cggtaggcgt g 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 tagaaggcac agtcgagg 18 <210> 5 <211> 1815 <212> DNA <213> Homo sapiens <400> 5 atgctgcgtc ggcggggcag ccctggcatg ggtgtgcatg tgggtgcagc cctgggagca 60 ctgtggttct gcctcacagg agccctggag gtccaggtcc ctgaagaccc agtggtggca 120 ctggtgggca ccgatgccac cctgtgctgc tccttctccc ctgagcctgg cttcagcctg 180 gcacagctca acctcatctg gcagctgaca gataccaaac agctggtgca cagctttgct 240 gagggccagg accagggcag cgcctatgcc aaccgcacgg ccctcttccc ggacctgctg 300 gcacagggca acgcatccct gaggctgcag cgcgtgcgtg tggcggacga gggcagcttc 360 acctgcttcg tgagcatccg ggatttcggc agcgctgccg tcagcctgca ggtggccgct 420 ccctactcga agcccagcat gaccctggag cccaacaagg acctgcggcc aggggacacg 480 gtgaccatca cgtgctccag ctaccagggc taccctgagg ctgaggtgtt ctggcaggat 540 gggcagggtg tgcccctgac tggcaacgtg accacgtcgc agatggccaa cgagcagggc 600 ttgtttgatg tgcacagcat cctgcgggtg gtgctgggtg caaatggcac ctacagctgc 660 ctggtgcgca accccgtgct gcagcaggat gcgcacagct ctgtcaccat cacaccccag 720 agaagcccca caggagccgt ggaggtccag gtccctgagg acccggtggt ggccctagtg 780 ggcaccgatg ccaccctgcg ctgctccttc tcccccgagc ctggcttcag cctggcacag 840 ctcaacctca tctggcagct gacagacacc aaacagctgg tgcacagttt caccgaaggc 900 cgggaccagg gcagcgccta tgccaaccgc acggccctct tcccggacct gctggcacaa 960 ggcaatgcat ccctgaggct gcagcgcgtg cgtgtggcgg acgagggcag cttcacctgc 1020 ttcgtgagca tccgggattt cggcagcgct gccgtcagcc tgcaggtggc cgctccctac 1080 tcgaagccca gcatgaccct ggagcccaac aaggacctgc ggccagggga cacggtgacc 1140 atcacgtgct ccagctaccg gggctaccct gaggctgagg tgttctggca ggatgggcag 1200 ggtgtgcccc tgactggcaa cgtgaccacg tcgcagatgg ccaacgagca gggcttgttt 1260 gatgtgcaca gcgtcctgcg ggtggtgctg ggtgcgaatg gcacctacag ctgcctggtg 1320 cgcaaccccg tgctgcagca ggatgcgcac ggctctgtca ccatcacagg gcagcctatg 1380 acattccccc cagaggccct gtgggtgacc gtggggctgt ctgtctgtct cattgcactg 1440 ctggtggccc tggctttcgt gtgctggaga aagatcaaac agagctgtga ggaggagaat 1500 gcaggagctg aggaccagga tggggaggga gaaggctcca agacagccct gcagcctctg 1560 aaacactctg acagcaaaga agatgatgga caagaaatag cctgagcggc cgccactgtg 1620 ctggatatct gcagaattcc accacactgg actagtggat ccgagctcgg taccaagctt 1680 aagtttaaac cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 1740 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 1800 aaatgaggaa attgc 1815 <210> 6 <211> 534 <212> PRT <213> Homo sapiens <400> 6 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln 225 230 235 240 Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val 245 250 255 Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro 260 265 270 Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285 Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300 Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln 305 310 315 320 Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly 325 330 335 Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val 340 345 350 Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu 355 360 365 Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser 370 375 380 Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln 385 390 395 400 Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu 405 410 415 Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala 420 425 430 Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp 435 440 445 Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro 450 455 460 Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu 465 470 475 480 Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys 485 490 495 Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly 500 505 510 Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp 515 520 525 Asp Gly Gln Glu Ile Ala 530 <210> 7 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 7 ggggacaagt ttgtacaaaa aagcaggctt caccatgctg cgtcggcggg gcagccctg 59 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 8 ggggaccact ttgtacaaga aagctgggtc ggctatttct tgt 43 <210> 9 <211> 948 <212> DNA <213> Homo sapiens <400> 9 atgctgcgtc ggcggggcag ccctggcatg ggtgtgcatg tgggtgcagc cctgggagca 60 ctgtggttct gcctcacagg agccctggag gtccaggtcc ctgaagaccc agtggtggca 120 ctggtgggca ccgatgccac cctgtgctgc tccttctccc ctgagcctgg cttcagcctg 180 gcacagctca acctcatctg gcagctgaca gataccaaac agctggtgca cagctttgct 240 gagggccagg accagggcag cgcctatgcc aaccgcacgg ccctcttccc ggacctgctg 300 gcacagggca acgcatccct gaggctgcag cgcgtgcgtg tggcggacga gggcagcttc 360 acctgcttcg tgagcatccg ggatttcggc agcgctgccg tcagcctgca ggtggccgct 420 ccctactcga agcccagcat gaccctggag cccaacaagg acctgcggcc aggggacacg 480 gtgaccatca cgtgctccag ctaccggggc taccctgagg ctgaggtgtt ctggcaggat 540 gggcagggtg tgcccctgac tggcaacgtg accacgtcgc agatggccaa cgagcagggc 600 ttgtttgatg tgcacagcgt cctgcgggtg gtgctgggtg cgaatggcac ctacagctgc 660 ctggtgcgca accccgtgct gcagcaggat gcgcacggct ctgtcaccat cacagggcag 720 cctatgacat tccccccaga ggccctgtgg gtgaccgtgg ggctgtctgt ctgtctcatt 780 gcactgctgg tggccctggc tttcgtgtgc tggagaaaga tcaaacagag ctgtgaggag 840 gagaatgcag gagctgagga ccaggatggg gagggagaag gctccaagac agccctgcag 900 cctctgaaac actctgacag caaagaagat gatggacaag aaatagcc 948 <210> 10 <211> 316 <212> PRT <213> Homo sapiens <400> 10 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln 225 230 235 240 Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250 255 Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265 270 Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285 Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His 290 295 300 Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315 <210> 11 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 11 ggggacaagt ttgtacaaaa aagcaggctt cggagccctg gaggtccagg tc 52 <210> 12 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 12 ggggacaagt ttgtacaaaa aagcaggctt cgctccctac tcgaagccca gcatg 55 <210> 13 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 13 ggggacaagt ttgtacaaaa aagcaggctt cggagccgtg gaggtccagg tc 52 <210> 14 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 14 ggggacaagt ttgtacaaaa aagcaggctt cgctccctac tcgaagccca gcatg 55 <210> 15 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 15 ggggaccact ttgtacaaga aagctgggtc tcaggctatt tcttgtccat catc 54 <210> 16 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 16 gggaatgtca taggctgccc ggccacctgc aggctgacgg cag 43 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 17 gggaatgtca taggctgccc tgtggggctt ctctggggtg tg 42 <210> 18 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 18 gggaatgtca taggctgccc ggccacctgc aggctgacgg cag 43 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 19 gggcagccta tgacattccc cccagag 27 <210> 20 <211> 576 <212> DNA <213> Homo sapiens <400> 20 ggagccctgg aggtccaggt ccctgaagac ccagtggtgg cactggtggg caccgatgcc 60 accctgtgct gctccttctc ccctgagcct ggcttcagcc tggcacagct caacctcatc 120 tggcagctga cagataccaa acagctggtg cacagctttg ctgagggcca ggaccagggc 180 agcgcctatg ccaaccgcac ggccctcttc ccggacctgc tggcacaggg caacgcatcc 240 ctgaggctgc agcgcgtgcg tgtggcggac gagggcagct tcacctgctt cgtgagcatc 300 cgggatttcg gcagcgctgc cgtcagcctg caggtggccg ggcagcctat gacattcccc 360 ccagaggccc tgtgggtgac cgtggggctg tctgtctgtc tcattgcact gctggtggcc 420 ctggctttcg tgtgctggag aaagatcaaa cagagctgtg aggaggagaa tgcaggagct 480 gaggaccagg atggggaggg agaaggctcc aagacagccc tgcagcctct gaaacactct 540 gacagcaaag aagatgatgg acaagaaata gcctga 576 <210> 21 <211> 191 <212> PRT <213> Homo sapiens <400> 21 Gly Ala Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val 1 5 10 15 Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe 20 25 30 Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln 35 40 45 Leu Val His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala 50 55 60 Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser 65 70 75 80 Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys 85 90 95 Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val 100 105 110 Ala Gly Gln Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val 115 120 125 Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val 130 135 140 Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala 145 150 155 160 Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro 165 170 175 Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 180 185 190 <210> 22 <211> 552 <212> DNA <213> Homo sapiens <400> 22 gctccctact cgaagcccag catgaccctg gagcccaaca aggacctgcg gccaggggac 60 acggtgacca tcacgtgctc cagctaccag ggctaccctg aggctgaggt gttctggcag 120 gatgggcagg gtgtgcccct gactggcaac gtgaccacgt cgcagatggc caacgagcag 180 ggcttgtttg atgtgcacag catcctgcgg gtggtgctgg gtgcaaatgg cacctacagc 240 tgcctggtgc gcaaccccgt gctgcagcag gatgcgcaca gctctgtcac catcacaccc 300 cagagaagcc ccacagggca gcctatgaca ttccccccag aggccctgtg ggtgaccgtg 360 gggctgtctg tctgtctcat tgcactgctg gtggccctgg ctttcgtgtg ctggagaaag 420 atcaaacaga gctgtgagga ggagaatgca ggagctgagg accaggatgg ggagggagaa 480 ggctccaaga cagccctgca gcctctgaaa cactctgaca gcaaagaaga tgatggacaa 540 gaaatagcct ga 552 <210> 23 <211> 183 <212> PRT <213> Homo sapiens <400> 23 Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu 1 5 10 15 Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr 20 25 30 Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr 35 40 45 Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp 50 55 60 Val His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser 65 70 75 80 Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val 85 90 95 Thr Ile Thr Pro Gln Arg Ser Pro Thr Gly Gln Pro Met Thr Phe Pro 100 105 110 Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala 115 120 125 Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser 130 135 140 Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu 145 150 155 160 Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu 165 170 175 Asp Asp Gly Gln Glu Ile Ala 180 <210> 24 <211> 576 <212> DNA <213> Homo sapiens <400> 24 ggagccgtgg aggtccaggt ccctgaggac ccggtggtgg ccctagtggg caccgatgcc 60 accctgcgct gctccttctc ccccgagcct ggcttcagcc tggcacagct caacctcatc 120 tggcagctga cagacaccaa acagctggtg cacagtttca ccgaaggccg ggaccagggc 180 agcgcctatg ccaaccgcac ggccctcttc ccggacctgc tggcacaagg caatgcatcc 240 ctgaggctgc agcgcgtgcg tgtggcggac gagggcagct tcacctgctt cgtgagcatc 300 cgggatttcg gcagcgctgc cgtcagcctg caggtggccg ggcagcctat gacattcccc 360 ccagaggccc tgtgggtgac cgtggggctg tctgtctgtc tcattgcact gctggtggcc 420 ctggctttcg tgtgctggag aaagatcaaa cagagctgtg aggaggagaa tgcaggagct 480 gaggaccagg atggggaggg agaaggctcc aagacagccc tgcagcctct gaaacactct 540 gacagcaaag aagatgatgg acaagaaata gcctga 576 <210> 25 <211> 191 <212> PRT <213> Homo sapiens <400> 25 Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val 1 5 10 15 Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe 20 25 30 Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln 35 40 45 Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ala 50 55 60 Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser 65 70 75 80 Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys 85 90 95 Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val 100 105 110 Ala Gly Gln Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val 115 120 125 Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val 130 135 140 Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala 145 150 155 160 Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro 165 170 175 Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 180 185 190 <210> 26 <211> 534 <212> DNA <213> Homo sapiens <400> 26 gctccctact cgaagcccag catgaccctg gagcccaaca aggacctgcg gccaggggac 60 acggtgacca tcacgtgctc cagctaccgg ggctaccctg aggctgaggt gttctggcag 120 gatgggcagg gtgtgcccct gactggcaac gtgaccacgt cgcagatggc caacgagcag 180 ggcttgtttg atgtgcacag cgtcctgcgg gtggtgctgg gtgcgaatgg cacctacagc 240 tgcctggtgc gcaaccccgt gctgcagcag gatgcgcacg gctctgtcac catcacaggg 300 cagcctatga cattcccccc agaggccctg tgggtgaccg tggggctgtc tgtctgtctc 360 attgcactgc tggtggccct ggctttcgtg tgctggagaa agatcaaaca gagctgtgag 420 gaggagaatg caggagctga ggaccaggat ggggagggag aaggctccaa gacagccctg 480 cagcctctga aacactctga cagcaaagaa gatgatggac aagaaatagc ctga 534 <210> 27 <211> 177 <212> PRT <213> Homo sapiens <400> 27 Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu 1 5 10 15 Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr 20 25 30 Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr 35 40 45 Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp 50 55 60 Val His Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser 65 70 75 80 Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser Val 85 90 95 Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val 100 105 110 Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala 115 120 125 Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala 130 135 140 Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu 145 150 155 160 Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile 165 170 175 Ala <210> 28 <211> 1188 <212> DNA <213> Homo sapiens <400> 28 gctccctact cgaagcccag catgaccctg gagcccaaca aggacctgcg gccaggggac 60 acggtgacca tcacgtgctc cagctaccag ggctaccctg aggctgaggt gttctggcag 120 gatgggcagg gtgtgcccct gactggcaac gtgaccacgt cgcagatggc caacgagcag 180 ggcttgtttg atgtgcacag catcctgcgg gtggtgctgg gtgcaaatgg cacctacagc 240 tgcctggtgc gcaaccccgt gctgcagcag gatgcgcaca gctctgtcac catcacaccc 300 cagagaagcc ccacaggagc cgtggaggtc caggtccctg aggacccggt ggtggcccta 360 gtgggcaccg atgccaccct gcgctgctcc ttctcccccg agcctggctt cagcctggca 420 cagctcaacc tcatctggca gctgacagac accaaacagc tggtgcacag tttcaccgaa 480 ggccgggacc agggcagcgc ctatgccaac cgcacggccc tcttcccgga cctgctggca 540 caaggcaatg catccctgag gctgcagcgc gtgcgtgtgg cggacgaggg cagcttcacc 600 tgcttcgtga gcatccggga tttcggcagc gctgccgtca gcctgcaggt ggccgctccc 660 tactcgaagc ccagcatgac cctggagccc aacaaggacc tgcggccagg ggacacggtg 720 accatcacgt gctccagcta ccggggctac cctgaggctg aggtgttctg gcaggatggg 780 cagggtgtgc ccctgactgg caacgtgacc acgtcgcaga tggccaacga gcagggcttg 840 tttgatgtgc acagcgtcct gcgggtggtg ctgggtgcga atggcaccta cagctgcctg 900 gtgcgcaacc ccgtgctgca gcaggatgcg cacggctctg tcaccatcac agggcagcct 960 atgacattcc ccccagaggc cctgtgggtg accgtggggc tgtctgtctg tctcattgca 1020 ctgctggtgg ccctggcttt cgtgtgctgg agaaagatca aacagagctg tgaggaggag 1080 aatgcaggag ctgaggacca ggatggggag ggagaaggct ccaagacagc cctgcagcct 1140 ctgaaacact ctgacagcaa agaagatgat ggacaagaaa tagcctga 1188 <210> 29 <211> 395 <212> PRT <213> Homo sapiens <400> 29 Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu 1 5 10 15 Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr 20 25 30 Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr 35 40 45 Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp 50 55 60 Val His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser 65 70 75 80 Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val 85 90 95 Thr Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val 100 105 110 Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg 115 120 125 Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu 130 135 140 Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu 145 150 155 160 Gly Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro 165 170 175 Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg 180 185 190 Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe 195 200 205 Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro 210 215 220 Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val 225 230 235 240 Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe 245 250 255 Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser 260 265 270 Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg 275 280 285 Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro 290 295 300 Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro 305 310 315 320 Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val 325 330 335 Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys 340 345 350 Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp 355 360 365 Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser 370 375 380 Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 385 390 395 <210> 30 <211> 873 <212> DNA <213> Homo sapiens <400> 30 ggagccgtgg aggtccaggt ccctgaggac ccggtggtgg ccctagtggg caccgatgcc 60 accctgcgct gctccttctc ccccgagcct ggcttcagcc tggcacagct caacctcatc 120 tggcagctga cagacaccaa acagctggtg cacagtttca ccgaaggccg ggaccagggc 180 agcgcctatg ccaaccgcac ggccctcttc ccggacctgc tggcacaagg caatgcatcc 240 ctgaggctgc agcgcgtgcg tgtggcggac gagggcagct tcacctgctt cgtgagcatc 300 cgggatttcg gcagcgctgc cgtcagcctg caggtggccg ctccctactc gaagcccagc 360 atgaccctgg agcccaacaa ggacctgcgg ccaggggaca cggtgaccat cacgtgctcc 420 agctaccggg gctaccctga ggctgaggtg ttctggcagg atgggcaggg tgtgcccctg 480 actggcaacg tgaccacgtc gcagatggcc aacgagcagg gcttgtttga tgtgcacagc 540 gtcctgcggg tggtgctggg tgcgaatggc acctacagct gcctggtgcg caaccccgtg 600 ctgcagcagg atgcgcacgg ctctgtcacc atcacagggc agcctatgac attcccccca 660 gaggccctgt gggtgaccgt ggggctgtct gtctgtctca ttgcactgct ggtggccctg 720 gctttcgtgt gctggagaaa gatcaaacag agctgtgagg aggagaatgc aggagctgag 780 gaccaggatg gggagggaga aggctccaag acagccctgc agcctctgaa acactctgac 840 agcaaagaag atgatggaca agaaatagcc tga 873 <210> 31 <211> 290 <212> PRT <213> Homo sapiens <400> 31 Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val 1 5 10 15 Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe 20 25 30 Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln 35 40 45 Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ala 50 55 60 Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser 65 70 75 80 Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys 85 90 95 Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val 100 105 110 Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp 115 120 125 Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly 130 135 140 Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu 145 150 155 160 Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe 165 170 175 Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr 180 185 190 Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser 195 200 205 Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro Glu Ala Leu Trp 210 215 220 Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val Ala Leu 225 230 235 240 Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn 245 250 255 Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala 260 265 270 Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu 275 280 285 Ile Ala 290 <210> 32 <211> 909 <212> DNA <213> Homo sapiens <400> 32 atgcggctgg gcagtcctgg actgctcttc ctgctcttca gcagccttcg agctgatact 60 caggagaagg aagtcagagc gatggtaggc agcgacgtgg agctcagctg cgcttgccct 120 gaaggaagcc gttttgattt aaatgatgtt tacgtatatt ggcaaaccag tgagtcgaaa 180 accgtggtga cctaccacat cccacagaac agctccttgg aaaacgtgga cagccgctac 240 cggaaccgag ccctgatgtc accggccggc atgctgcggg gcgacttctc cctgcgcttg 300 ttcaacgtca ccccccagga cgagcagaag tttcactgcc tggtgttgag ccaatccctg 360 ggattccagg aggttttgag cgttgaggtt acactgcatg tggcagcaaa cttcagcgtg 420 cccgtcgtca gcgcccccca cagcccctcc caggatgagc tcaccttcac gtgtacatcc 480 ataaacggct accccaggcc caacgtgtac tggatcaata agacggacaa cagcctgctg 540 gaccaggctc tgcagaatga caccgtcttc ttgaacatgc ggggcttgta tgacgtggtc 600 agcgtgctga ggatcgcacg gacccccagc gtgaacattg gctgctgcat agagaacgtg 660 cttctgcagc agaacctgac tgtcggcagc cagacaggaa atgacatcgg agagagagac 720 aagatcacag agaatccagt cagtaccggc gagaaaaacg cggccacgtg gagcatcctg 780 gctgtcctgt gcctgcttgt ggtcgtggcg gtggccatag gctgggtgtg cagggaccga 840 tgcctccaac acagctatgc aggtgcctgg gctgtgagtc cggagacaga gctcactggc 900 cacgtttga 909 <210> 33 <211> 302 <212> PRT <213> Homo sapiens <400> 33 Met Arg Leu Gly Ser Pro Gly Leu Leu Phe Leu Leu Phe Ser Ser Leu 1 5 10 15 Arg Ala Asp Thr Gln Glu Lys Glu Val Arg Ala Met Val Gly Ser Asp 20 25 30 Val Glu Leu Ser Cys Ala Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn 35 40 45 Asp Val Tyr Val Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr 50 55 60 Tyr His Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr 65 70 75 80 Arg Asn Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe 85 90 95 Ser Leu Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe His 100 105 110 Cys Leu Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val Leu Ser Val 115 120 125 Glu Val Thr Leu His Val Ala Ala Asn Phe Ser Val Pro Val Val Ser 130 135 140 Ala Pro His Ser Pro Ser Gln Asp Glu Leu Thr Phe Thr Cys Thr Ser 145 150 155 160 Ile Asn Gly Tyr Pro Arg Pro Asn Val Tyr Trp Ile Asn Lys Thr Asp 165 170 175 Asn Ser Leu Leu Asp Gln Ala Leu Gln Asn Asp Thr Val Phe Leu Asn 180 185 190 Met Arg Gly Leu Tyr Asp Val Val Ser Val Leu Arg Ile Ala Arg Thr 195 200 205 Pro Ser Val Asn Ile Gly Cys Cys Ile Glu Asn Val Leu Leu Gln Gln 210 215 220 Asn Leu Thr Val Gly Ser Gln Thr Gly Asn Asp Ile Gly Glu Arg Asp 225 230 235 240 Lys Ile Thr Glu Asn Pro Val Ser Thr Gly Glu Lys Asn Ala Ala Thr 245 250 255 Trp Ser Ile Leu Ala Val Leu Cys Leu Leu Val Val Val Ala Val Ala 260 265 270 Ile Gly Trp Val Cys Arg Asp Arg Cys Leu Gln His Ser Tyr Ala Gly 275 280 285 Ala Trp Ala Val Ser Pro Glu Thr Glu Leu Thr Gly His Val 290 295 300 <210> 34 <211> 873 <212> DNA <213> Homo sapiens <400> 34 atgaggatat ttgctgtctt tatattcatg acctactggc atttgctgaa cgcatttact 60 gtcacggttc ccaaggacct atatgtggta gagtatggta gcaatatgac aattgaatgc 120 aaattcccag tagaaaaaca attagacctg gctgcactaa ttgtctattg ggaaatggag 180 gataagaaca ttattcaatt tgtgcatgga gaggaagacc tgaaggttca gcatagtagc 240 tacagacaga gggcccggct gttgaaggac cagctctccc tgggaaatgc tgcacttcag 300 atcacagatg tgaaattgca ggatgcaggg gtgtaccgct gcatgatcag ctatggtggt 360 gccgactaca agcgaattac tgtgaaagtc aatgccccat acaacaaaat caaccaaaga 420 attttggttg tggatccagt cacctctgaa catgaactga catgtcaggc tgagggctac 480 cccaaggccg aagtcatctg gacaagcagt gaccatcaag tcctgagtgg taagaccacc 540 accaccaatt ccaagagaga ggagaagctt ttcaatgtga ccagcacact gagaatcaac 600 acaacaacta atgagatttt ctactgcact tttaggagat tagatcctga ggaaaaccat 660 acagctgaat tggtcatccc agaactacct ctggcacatc ctccaaatga aaggactcac 720 ttggtaattc tgggagccat cttattatgc cttggtgtag cactgacatt catcttccgt 780 ttaagaaaag ggagaatgat ggatgtgaaa aaatgtggca tccaagatac aaactcaaag 840 aagcaaagtg atacacattt ggaggagacg taa 873 <210> 35 <211> 290 <212> PRT <213> Homo sapiens <400> 35 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 36 <211> 909 <212> DNA <213> Homo sapiens <400> 36 atgcggctgg gcagtcctgg actgctcttc ctgctcttca gcagccttcg agctgatact 60 caggagaagg aagtcagagc gatggtaggc agcgacgtgg agctcagctg cgcttgccct 120 gaaggaagcc gttttgattt aaatgatgtt tacgtatatt ggcaaaccag tgagtcgaaa 180 accgtggtga cctaccacat cccacagaac agctccttgg aaaacgtgga cagccgctac 240 cggaaccgag ccctgatgtc accggccggc atgctgcggg gcgacttctc cctgcgcttg 300 ttcaacgtca ccccccagga cgagcagaag tttcactgcc tggtgttgag ccaatccctg 360 ggattccagg aggttttgag cgttgaggtt acactgcatg tggcagcaaa cttcagcgtg 420 cccgtcgtca gcgcccccca cagcccctcc caggatgagc tcaccttcac gtgtacatcc 480 ataaacggct accccaggcc caacgtgtac tggatcaata agacggacaa cagcctgctg 540 gaccaggctc tgcagaatga caccgtcttc ttgaacatgc ggggcttgta tgacgtggtc 600 agcgtgctga ggatcgcacg gacccccagc gtgaacattg gctgctgcat agagaacgtg 660 cttctgcagc agaacctgac tgtcggcagc cagacaggaa atgacatcgg agagagagac 720 aagatcacag agaatccagt cagtaccggc gagaaaaacg cggccacgtg gagcatcctg 780 gctgtcctgt gcctgcttgt ggtcgtggcg gtggccatag gctgggtgtg cagggaccga 840 tgcctccaac acagctatgc aggtgcctgg gctgtgagtc cggagacaga gctcactggc 900 cacgtttga 909 <210> 37 <211> 302 <212> PRT <213> Homo sapiens <400> 37 Met Arg Leu Gly Ser Pro Gly Leu Leu Phe Leu Leu Phe Ser Ser Leu 1 5 10 15 Arg Ala Asp Thr Gln Glu Lys Glu Val Arg Ala Met Val Gly Ser Asp 20 25 30 Val Glu Leu Ser Cys Ala Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn 35 40 45 Asp Val Tyr Val Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr 50 55 60 Tyr His Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr 65 70 75 80 Arg Asn Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe 85 90 95 Ser Leu Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe His 100 105 110 Cys Leu Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val Leu Ser Val 115 120 125 Glu Val Thr Leu His Val Ala Ala Asn Phe Ser Val Pro Val Val Ser 130 135 140 Ala Pro His Ser Pro Ser Gln Asp Glu Leu Thr Phe Thr Cys Thr Ser 145 150 155 160 Ile Asn Gly Tyr Pro Arg Pro Asn Val Tyr Trp Ile Asn Lys Thr Asp 165 170 175 Asn Ser Leu Leu Asp Gln Ala Leu Gln Asn Asp Thr Val Phe Leu Asn 180 185 190 Met Arg Gly Leu Tyr Asp Val Val Ser Val Leu Arg Ile Ala Arg Thr 195 200 205 Pro Ser Val Asn Ile Gly Cys Cys Ile Glu Asn Val Leu Leu Gln Gln 210 215 220 Asn Leu Thr Val Gly Ser Gln Thr Gly Asn Asp Ile Gly Glu Arg Asp 225 230 235 240 Lys Ile Thr Glu Asn Pro Val Ser Thr Gly Glu Lys Asn Ala Ala Thr 245 250 255 Trp Ser Ile Leu Ala Val Leu Cys Leu Leu Val Val Val Ala Val Ala 260 265 270 Ile Gly Trp Val Cys Arg Asp Arg Cys Leu Gln His Ser Tyr Ala Gly 275 280 285 Ala Trp Ala Val Ser Pro Glu Thr Glu Leu Thr Gly His Val 290 295 300 <210> 38 <211> 867 <212> DNA <213> Homo sapiens <400> 38 atgggccaca cacggaggca gggaacatca ccatccaagt gtccatacct caatttcttt 60 cagctcttgg tgctggctgg tctttctcac ttctgttcag gtgttatcca cgtgaccaag 120 gaagtgaaag aagtggcaac gctgtcctgt ggtcacaatg tttctgttga agagctggca 180 caaactcgca tctactggca aaaggagaag aaaatggtgc tgactatgat gtctggggac 240 atgaatatat ggcccgagta caagaaccgg accatctttg atatcactaa taacctctcc 300 attgtgatcc tggctctgcg cccatctgac gagggcacat acgagtgtgt tgttctgaag 360 tatgaaaaag acgctttcaa gcgggaacac ctggctgaag tgacgttatc agtcaaagct 420 gacttcccta cacctagtat atctgacttt gaaattccaa cttctaatat tagaaggata 480 atttgctcaa cctctggagg ttttccagag cctcacctct cctggttgga aaatggagaa 540 gaattaaatg ccatcaacac aacagtttcc caagatcctg aaactgagct ctatgctgtt 600 agcagcaaac tggatttcaa tatgacaacc aaccacagct tcatgtgtct catcaagtat 660 ggacatttaa gagtgaatca gaccttcaac tggaatacaa ccaagcaaga gcattttcct 720 gataacctgc tcccatcctg ggccattacc ttaatctcag taaatggaat ttttgtgata 780 tgctgcctga cctactgctt tgccccaaga tgcagagaga gaaggaggaa tgagagattg 840 agaagggaaa gtgtacgccc tgtatag 867 <210> 39 <211> 288 <212> PRT <213> Homo sapiens <400> 39 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr 1 5 10 15 Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys 20 25 30 Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35 40 45 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 50 55 60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 65 70 75 80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 85 90 95 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100 105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 115 120 125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 130 135 140 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145 150 155 160 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165 170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp 180 185 190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met 195 200 205 Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210 215 220 Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230 235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly 245 250 255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg 260 265 270 Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 275 280 285 <210> 40 <211> 990 <212> DNA <213> Homo sapiens <400> 40 atggatcccc agtgcactat gggactgagt aacattctct ttgtgatggc cttcctgctc 60 tctggtgctg ctcctctgaa gattcaagct tatttcaatg agactgcaga cctgccatgc 120 caatttgcaa actctcaaaa ccaaagcctg agtgagctag tagtattttg gcaggaccag 180 gaaaacttgg ttctgaatga ggtatactta ggcaaagaga aatttgacag tgttcattcc 240 aagtatatgg gccgcacaag ttttgattcg gacagttgga ccctgagact tcacaatctt 300 cagatcaagg acaagggctt gtatcaatgt atcatccatc acaaaaagcc cacaggaatg 360 attcgcatcc accagatgaa ttctgaactg tcagtgcttg ctaacttcag tcaacctgaa 420 atagtaccaa tttctaatat aacagaaaat gtgtacataa atttgacctg ctcatctata 480 cacggttacc cagaacctaa gaagatgagt gttttgctaa gaaccaagaa ttcaactatc 540 gagtatgatg gtattatgca gaaatctcaa gataatgtca cagaactgta cgacgtttcc 600 atcagcttgt ctgtttcatt ccctgatgtt acgagcaata tgaccatctt ctgtattctg 660 gaaactgaca agacgcggct tttatcttca cctttctcta tagagcttga ggaccctcag 720 cctcccccag accacattcc ttggattaca gctgtacttc caacagttat tatatgtgtg 780 atggttttct gtctaattct atggaaatgg aagaagaaga agcggcctcg caactcttat 840 aaatgtggaa ccaacacaat ggagagggaa gagagtgaac agaccaagaa aagagaaaaa 900 atccatatac ctgaaagatc tgatgaagcc cagcgtgttt ttaaaagttc gaagacatct 960 tcatgcgaca aaagtgatac atgtttttag 990 <210> 41 <211> 329 <212> PRT <213> Homo sapiens <400> 41 Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met 1 5 10 15 Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe 20 25 30 Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln 35 40 45 Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val 50 55 60 Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser 65 70 75 80 Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg 85 90 95 Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile 100 105 110 His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser 115 120 125 Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile 130 135 140 Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile 145 150 155 160 His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys 165 170 175 Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn 180 185 190 Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro 195 200 205 Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys 210 215 220 Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln 225 230 235 240 Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val 245 250 255 Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys 260 265 270 Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu 275 280 285 Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro 290 295 300 Glu Arg Ser Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser 305 310 315 320 Ser Cys Asp Lys Ser Asp Thr Cys Phe 325 <210> 42 <211> 849 <212> DNA <213> Homo sapiens <400> 42 atggcttccc tggggcagat cctcttctgg agcataatta gcatcatcat tattctggct 60 ggagcaattg cactcatcat tggctttggt atttcaggga gacactccat cacagtcact 120 actgtcgcct cagctgggaa cattggggag gatggaatcc tgagctgcac ttttgaacct 180 gacatcaaac tttctgatat cgtgatacaa tggctgaagg aaggtgtttt aggcttggtc 240 catgagttca aagaaggcaa agatgagctg tcggagcagg atgaaatgtt cagaggccgg 300 acagcagtgt ttgctgatca agtgatagtt ggcaatgcct ctttgcggct gaaaaacgtg 360 caactcacag atgctggcac ctacaaatgt tatatcatca cttctaaagg caaggggaat 420 gctaaccttg agtataaaac tggagccttc agcatgccgg aagtgaatgt ggactataat 480 gccagctcag agaccttgcg gtgtgaggct ccccgatggt tcccccagcc cacagtggtc 540 tgggcatccc aagttgacca gggagccaac ttctcggaag tctccaatac cagctttgag 600 ctgaactctg agaatgtgac catgaaggtt gtgtctgtgc tctacaatgt tacgatcaac 660 aacacatact cctgtatgat tgaaaatgac attgccaaag caacagggga tatcaaagtg 720 acagaatcgg agatcaaaag gcggagtcac ctacagctgc taaactcaaa ggcttctctg 780 tgtgtctctt ctttctttgc catcagctgg gcacttctgc ctctcagccc ttacctgatg 840 ctaaaatag 849 <210> 43 <211> 282 <212> PRT <213> Homo sapiens <400> 43 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys 275 280 <210> 44 <211> 10 <212> PRT <213> Mus musculus <400> 44 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu 1 5 10 <210> 45 <211> 14 <212> PRT <213> Mus musculus <400> 45 Ile Val Leu Ser Gln Ser Pro Thr Ile Leu Ser Ala Ser Pro 1 5 10 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 46 aagaattcat ggaatggagt tggata 26 <210> 47 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 47 aagatatctc atttacccgg agtccgggag aa 32 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 48 aagaattcat ggattttctg gtgcag 26 <210> 49 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 49 aagatatctt aacactcatt cctgttgaag ct 32 <210> 50 <211> 1413 <212> DNA <213> Mus musculus <400> 50 atggaatgga gttggatatt tctctttctc ctgtcaggaa ctgcaggtgt ccactctgag 60 gtccagctgc agcagtctgg acctgagctg gtaaagcctg gggcttcagt gaagatgtcc 120 tgcaaggctt ctggatacac attcactaac tatgttatgc actgggtgaa gcagaagcct 180 gggcagggcc ttgagtggat tggatatatt aatccttaca atgatgatgt taagtacaat 240 gagaagttca aaggcaaggc cacacagact tcagacaaat cctccagcac agcctacatg 300 gagctcagca gcctgacctc tgaggactct gcggtctatt actgtgcaag atgggggtac 360 tacggtagtc ccttatacta ctttgactac tggggccaag gcaccactct cacagtctcc 420 tcagccaaaa caacagcccc atcggtctat ccactggccc ctgtgtgtgg agatacaact 480 ggctcctcgg tgactctagg atgcctggtc aagggttatt tccctgagcc agtgaccttg 540 acctggaact ctggatccct gtccagtggt gtgcacacct tcccagctgt cctgcagtct 600 gacctctaca ccctcagcag ctcagtgact gtaacctcga gcacctggcc cagccagtcc 660 atcacctgca atgtggccca cccggcaagc agcaccaagg tggacaagaa aattgagccc 720 agagggccca caatcaagcc ctgtcctcca tgcaaatgcc cagcacctaa cctcttgggt 780 ggaccatccg tcttcatctt ccctccaaag atcaaggatg tactcatgat ctccctgagc 840 cccatagtca catgtgtggt ggtggatgtg agcgaggatg acccagatgt ccagatcagc 900 tggtttgtga acaacgtgga agtacacaca gctcagacac aaacccatag agaggattac 960 aacagtactc tccgggtggt cagtgccctc cccatccagc accaggactg gatgagtggc 1020 aaggagttca aatgcaaggt caacaacaaa gacctcccag cgcccatcga gagaaccatc 1080 tcaaaaccca aagggtcagt aagagctcca caggtatatg tcttgcctcc accagaagaa 1140 gagatgacta agaaacaggt cactctgacc tgcatggtca cagacttcat gcctgaagac 1200 atttacgtgg agtggaccaa caacgggaaa acagagctaa actacaagaa cactgaacca 1260 gtcctggact ctgatggttc ttacttcatg tacagcaagc tgagagtgga aaagaagaac 1320 tgggtggaaa gaaatagcta ctcctgttca gtggtccacg agggtctgca caatcaccac 1380 acgactaaga gcttctcccg gactccgggt aaa 1413 <210> 51 <211> 471 <212> PRT <213> Mus musculus <400> 51 Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Gln Thr Ser Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr 130 135 140 Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr 145 150 155 160 Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu 165 170 175 Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser 195 200 205 Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn 210 215 220 Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro 225 230 235 240 Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro 245 250 255 Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys 260 265 270 Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val 275 280 285 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn 290 295 300 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr 305 310 315 320 Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp 325 330 335 Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu 340 345 350 Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg 355 360 365 Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys 370 375 380 Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp 385 390 395 400 Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys 405 410 415 Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser 420 425 430 Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser 435 440 445 Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser 450 455 460 Phe Ser Arg Thr Pro Gly Lys 465 470 <210> 52 <211> 705 <212> DNA <213> Mus musculus <400> 52 atggattttc tggtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60 agaggacaaa ttgttctctc ccagtctcca acaatcctgt ctgcatctcc aggggagaag 120 gtcacaatga cttgcagggc cagctcaaga ctaatttaca tgcattggta tcagcagaag 180 ccaggatcct cccccaaacc ctggatttat gccacatcca acctggcttc tggagtccct 240 gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagagtggag 300 gctgaagatg ctgccactta ttactgccag cagtggaata gtaacccacc cacgttcggt 360 actgggacca agctggagct gaaacgggct gatgctgcac caactgtatc catcttccca 420 ccatccagtg agcagttaac atctggaggt gcctcagtcg tgtgcttctt gaacaacttc 480 taccccaaag acatcaatgt caagtggaag attgatggca gtgaacgaca aaatggcgtc 540 ctgaacagtt ggactgatca ggacagcaaa gacagcacct acagcatgag cagcaccctc 600 acgttgacca aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag 660 acatcaactt cacccattgt caagagcttc aacaggaatg agtgt 705 <210> 53 <211> 235 <212> PRT <213> Mus musculus <400> 53 Met Asp Phe Leu Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Thr Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Arg Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Asn Ser Asn Pro Pro Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys 115 120 125 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 130 135 140 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 145 150 155 160 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 165 170 175 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 195 200 205 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 210 215 220 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 54 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 54 ccacgcgccc tgtagcggcg cattaagc 28 <210> 55 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 55 aaacccggga gctttttgca aaagcctagg 30 <210> 56 <211> 1704 <212> DNA <213> Artificial Sequence <220> <223> human kappa chain secretion signal, human kappa chain constant region and human poly A additional signal <400> 56 ggtaccaccc aagctggcta ggtaagcttg ctagcgccac catggtgctg cagacccagg 60 tgttcatctc cctgctgctg tggatctccg gcgcatatgg cgatatcgtg atgattaaac 120 gtacggtggc cgccccctcc gtgttcatct tccccccctc cgacgagcag ctgaagtccg 180 gcaccgcctc cgtggtgtgc ctgctgaata acttctaccc cagagaggcc aaggtgcagt 240 ggaaggtgga caacgccctg cagtccggga actcccagga gagcgtgacc gagcaggaca 300 gcaaggacag cacctacagc ctgagcagca ccctgaccct gagcaaagcc gactacgaga 360 agcacaaggt gtacgcctgc gaggtgaccc accagggcct gagctccccc gtcaccaaga 420 gcttcaacag gggggagtgt taggggcccg tttaaacggg tggcatccct gtgacccctc 480 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat 540 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag 600 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 660 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg 720 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac 780 caggctcacc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct 840 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt 900 acaggcgtga accactgctc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 960 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 1020 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 1080 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 1140 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 1200 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 1260 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 1320 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 1380 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 1440 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 1500 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 1560 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 1620 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 1680 aggcttttgc aaaaagctcc cggg 1704 <210> 57 <211> 1120 <212> DNA <213> Artificial Sequence <220> <223> human IgG1 signal sequence and constant region <400> 57 tgctagcgcc accatgaaac acctgtggtt cttcctcctg ctggtggcag ctcccagatg 60 ggtgctgagc caggtgcaat tgtgcaggcg gttagctcag cctccaccaa gggcccaagc 120 gtcttccccc tggcaccctc ctccaagagc acctctggcg gcacagccgc cctgggctgc 180 ctggtcaagg actacttccc cgaacccgtg accgtgagct ggaactcagg cgccctgacc 240 agcggcgtgc acaccttccc cgctgtcctg cagtcctcag gactctactc cctcagcagc 300 gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac 360 aagcccagca acaccaaggt ggacaagaga gttgagccca aatcttgtga caaaactcac 420 acatgcccac cctgcccagc acctgaactc ctggggggac cctcagtctt cctcttcccc 480 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 540 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 600 cataatgcca agacaaagcc ccgggaggag cagtacaaca gcacgtaccg ggtggtcagc 660 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 720 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg ccagccccgg 780 gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 840 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 900 ggccagcccg agaacaacta caagaccacc cctcccgtgc tggactccga cggctccttc 960 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcagggcaa cgtcttctca 1020 tgctccgtga tgcatgaggc tctgcacaac cactacaccc agaagagcct ctccctgtct 1080 cccggcaaat gagatatcgg gcccgtttaa acgggtggca 1120 <210> 58 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Light chain of chimeric M30 <400> 58 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 caaattgttc tctcccagtc tccaacaatc ctgtctgcat ctccagggga gaaggtcaca 120 atgacttgca gggccagctc aagactaatt tacatgcatt ggtatcagca gaagccagga 180 tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 240 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 300 gatgctgcca cttattactg ccagcagtgg aatagtaacc cacccacgtt cggtactggg 360 accaagctgg agctgaaacg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 59 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Light chain of chimeric M30 <400> 59 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Gln Ile Val Leu Ser Gln Ser Pro Thr Ile Leu Ser 20 25 30 Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys 50 55 60 Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg 85 90 95 Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 60 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 60 aaacatatgg ccaaattgtt ctctcccagt ctccaacaat cc 42 <210> 61 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 61 aaacgtacgt ttcagctcca gcttggtccc agtaccg 37 <210> 62 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain of chimeric M30 <400> 62 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagcgag 60 gtccagctgc agcagtctgg acctgagctg gtaaagcctg gggcttcagt gaagatgtcc 120 tgcaaggctt ctggatacac attcactaac tatgttatgc actgggtgaa gcagaagcct 180 gggcagggcc ttgagtggat tggatatatt aatccttaca atgatgatgt taagtacaat 240 gagaagttca aaggcaaggc cacacagact tcagacaaat cctccagcac agcctacatg 300 gaactcagca gcctgacctc tgaggactct gcggtctatt actgtgcaag atgggggtac 360 tacggtagtc ccttatacta ctttgactac tggggccaag gcaccactct cacagtcagc 420 tcagcctcca ccaagggccc aagcgtcttc cccctggcac cctcctccaa gagcacctct 480 ggcggcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc cgtgaccgtg 540 agctggaact caggcgccct gaccagcggc gtgcacacct tccccgctgt cctgcagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccctgcc cagcacctga actcctgggg 780 ggaccctcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccccggga ggagcagtac 960 aacagcacgt accgggtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aaggccagcc ccgggaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatggccag cccgagaaca actacaagac cacccctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg gcaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acccagaaga gcctctccct gtctcccggc aaa 1413 <210> 63 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of chimeric M30 <400> 63 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Gln Thr Ser Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 64 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 64 aaagctgagc gaggtccagc tgcagcagtc tggacctgag 40 <210> 65 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 65 gaggtcaggc tgctgagttc catgtaggct gtgctg 36 <210> 66 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 66 cagcacagcc tacatggaac tcagcagcct gacctc 36 <210> 67 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 67 aaagctgagc tgactgtgag agtggtgcct tggccccag 39 <210> 68 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 68 aaagctgagc gaggtccagc tgcagcagtc tggacctgag 40 <210> 69 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 69 aaagctgagc tgactgtgag agtggtgcct tggccccag 39 <210> 70 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 70 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 gagatcgtgc tgacccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgagctgca gagccagcag ccgcctgatc tacatgcact ggtatcagca gaagcccggc 180 caggccccca gactgctgat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccgacttc accctgacca tctctcggct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 71 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 71 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Leu Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 72 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 72 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 gagatcgtgc tgacccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgagctgca gagccagcag caggctgatc tacatgcact ggtatcagca gaagcccggc 180 caggccccca gactgtggat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccgactac accctgacca tcagccgcct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 73 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 73 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Leu Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 74 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 74 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 cagatcgtgc tgtcccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgacctgca gagccagcag caggctgatc tacatgcact ggtatcagca gaagcccggc 180 agcgccccca agctgtggat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccagctac accctgacca tctcccgcct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 75 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 75 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Thr Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Lys 50 55 60 Leu Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 76 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 76 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 gagatcgtgc tgacccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgagctgca gagccagcag ccgcctgatc tacatgcact ggtatcagca gaagcccggc 180 caggccccca gacctctgat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccgacttc accctgacca tcagcagcct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 77 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 77 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 78 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 78 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 cagatcgtgc tgtcccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgacctgca gagccagcag caggctgatc tacatgcact ggtatcagca gaagcccggc 180 agcgccccca agccttggat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccagctac accctgacca tctcccgcct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 79 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 79 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Thr Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Lys 50 55 60 Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 80 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 80 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 gagatcgtgc tgacccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgagctgca gagccagcag ccgcctgatc tacatgcact ggtatcagca gaagcccggc 180 caggccccca gacctctgat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccgacttc accctgacca tcagccgcct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 81 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 81 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 82 <211> 699 <212> DNA <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 82 atggtgctgc agacccaggt gttcatctcc ctgctgctgt ggatctccgg cgcatatggc 60 gagatcgtgc tgacccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 120 ctgagctgca gagccagcag ccgcctgatc tacatgcact ggtatcagca gaagcccggc 180 caggccccca gacctctgat ctacgccacc agcaacctgg ccagcggcat ccccgccaga 240 ttttctggca gcggcagcgg caccgactac accctgacca tcagccgcct ggaacccgag 300 gacttcgccg tgtactactg ccagcagtgg aacagcaacc cccccacctt cggccagggc 360 accaaggtcg aaatcaagcg tacggtggcc gccccctccg tgttcatctt ccccccctcc 420 gacgagcagc tgaagtccgg caccgcctcc gtggtgtgcc tgctgaataa cttctacccc 480 agagaggcca aggtgcagtg gaaggtggac aacgccctgc agtccgggaa ctcccaggag 540 agcgtgaccg agcaggacag caaggacagc acctacagcc tgagcagcac cctgaccctg 600 agcaaagccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggcctg 660 agctcccccg tcaccaagag cttcaacagg ggggagtgt 699 <210> 83 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain of M30 <400> 83 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Arg 35 40 45 Leu Ile Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 50 55 60 Pro Leu Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Arg 85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser 100 105 110 Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 84 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 84 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 120 tgcaaggcca gcggctacac cttcaccaac tacgtgatgc actgggtgcg ccaggcccct 180 gggcagggac tggaatggat gggctacatc aacccctaca acgacgacgt gaagtacaac 240 gagaagttca agggcagagt gaccatcacc gccgacgaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag atggggctac 360 tacggcagcc ccctgtacta cttcgactac tggggccagg gcaccctggt gacagtcagc 420 tcagcctcca ccaagggccc aagcgtcttc cccctggcac cctcctccaa gagcacctct 480 ggcggcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc cgtgaccgtg 540 agctggaact caggcgccct gaccagcggc gtgcacacct tccccgctgt cctgcagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccctgcc cagcacctga actcctgggg 780 ggaccctcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccccggga ggagcagtac 960 aacagcacgt accgggtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aaggccagcc ccgggaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatggccag cccgagaaca actacaagac cacccctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg gcaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acccagaaga gcctctccct gtctcccggc aaa 1413 <210> 85 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 85 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 86 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 86 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagccag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 120 tgcaaggcca gcggctacac cttcaccaac tacgtgatgc actgggtgcg ccaggcccct 180 gggcagggac tggaatggat cggctacatc aacccctaca acgacgacgt gaagtacaac 240 gagaagttca agggcagagt gaccatcacc gccgacgaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag atggggctac 360 tacggcagcc ccctgtacta cttcgactac tggggccagg gcaccctggt gacagtcagc 420 tcagcctcca ccaagggccc aagcgtcttc cccctggcac cctcctccaa gagcacctct 480 ggcggcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc cgtgaccgtg 540 agctggaact caggcgccct gaccagcggc gtgcacacct tccccgctgt cctgcagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccctgcc cagcacctga actcctgggg 780 ggaccctcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccccggga ggagcagtac 960 aacagcacgt accgggtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aaggccagcc ccgggaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatggccag cccgagaaca actacaagac cacccctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg gcaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acccagaaga gcctctccct gtctcccggc aaa 1413 <210> 87 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 87 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 88 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 88 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagcgag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 120 tgcaaggcca gcggctacac cttcaccaac tacgtgatgc actgggtgaa acaggcccct 180 gggcagggcc tggaatggat cggctacatc aacccctaca acgacgacgt gaagtacaac 240 gagaagttca agggcaaggc caccatcacc gccgacgaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag atggggctac 360 tacggcagcc ccctgtacta cttcgactac tggggccagg gcaccctggt gacagtcagc 420 tcagcctcca ccaagggccc aagcgtcttc cccctggcac cctcctccaa gagcacctct 480 ggcggcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc cgtgaccgtg 540 agctggaact caggcgccct gaccagcggc gtgcacacct tccccgctgt cctgcagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccctgcc cagcacctga actcctgggg 780 ggaccctcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccccggga ggagcagtac 960 aacagcacgt accgggtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aaggccagcc ccgggaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatggccag cccgagaaca actacaagac cacccctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg gcaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acccagaaga gcctctccct gtctcccggc aaa 1413 <210> 89 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 89 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 90 <211> 1413 <212> DNA <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 90 atgaaacacc tgtggttctt cctcctgctg gtggcagctc ccagatgggt gctgagcgag 60 gtgcagctgg tgcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 120 tgcaaggcca gcggctacac cttcaccaac tacgtgatgc actgggtcaa gcaggcccct 180 gggcagggcc tggaatggat cggctacatc aacccctaca acgacgacgt gaagtacaac 240 gagaagttca agggcaaggc cacccagacc agcgacaaga gcaccagcac cgcctacatg 300 gaactgagca gcctgcggag cgaggacacc gccgtgtact actgcgccag atggggctac 360 tacggcagcc ccctgtacta cttcgactac tggggccagg gcaccctggt caccgtcagc 420 tcagcctcca ccaagggccc aagcgtcttc cccctggcac cctcctccaa gagcacctct 480 ggcggcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc cgtgaccgtg 540 agctggaact caggcgccct gaccagcggc gtgcacacct tccccgctgt cctgcagtcc 600 tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 660 acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gagagttgag 720 cccaaatctt gtgacaaaac tcacacatgc ccaccctgcc cagcacctga actcctgggg 780 ggaccctcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccccggga ggagcagtac 960 aacagcacgt accgggtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1080 tccaaagcca aaggccagcc ccgggaacca caggtgtaca ccctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatggccag cccgagaaca actacaagac cacccctccc 1260 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg gcaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acccagaaga gcctctccct gtctcccggc aaa 1413 <210> 91 <211> 471 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain of M30 <400> 91 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Val Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn 65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Gln Thr Ser Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 92 <211> 5 <212> PRT <213> Mus musculus <400> 92 Asn Tyr Val Met His 1 5 <210> 93 <211> 17 <212> PRT <213> Mus musculus <400> 93 Tyr Ile Asn Pro Tyr Asn Asp Asp Val Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 94 <211> 13 <212> PRT <213> Mus musculus <400> 94 Trp Gly Tyr Tyr Gly Ser Pro Leu Tyr Tyr Phe Asp Tyr 1 5 10 <210> 95 <211> 10 <212> PRT <213> Mus musculus <400> 95 Arg Ala Ser Ser Arg Leu Ile Tyr Met His 1 5 10 <210> 96 <211> 7 <212> PRT <213> Mus musculus <400> 96 Ala Thr Ser Asn Leu Ala Ser 1 5 <210> 97 <211> 9 <212> PRT <213> Mus musculus <400> 97 Gln Gln Trp Asn Ser Asn Pro Pro Thr 1 5

Claims (26)

An antibody or functional fragment of the antibody that is bound to a radioactive substance or a compound having a pharmacological action, wherein the antibody has the following characteristics or a functional fragment of the antibody:
(a) A molecule specifically binding to B7-H3, wherein B7-H3 is a molecule comprising the amino acid sequence set forth in SEQ ID NO: 6 or 10, and the antibody or functional fragment of the antibody is a domain of B7-H3, IgC1, IgC2, Or a domain that binds to IgC1 and IgC2, wherein IgC1 is the domain comprising the amino acid sequence of amino acids 140 to 244 in SEQ ID NO: 6, and IgC2 is the domain comprising the amino acid sequence of amino acids 358 to 456 of SEQ ID NO: 6 to be;
(b) has antibody dependent cell mediated phagocytosis (ADCP) activity;
(c) has anti-tumor activity in vivo; And
(d) CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 92, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 93 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 94 as the complementarity determining region in the heavy chain, and in the light chain As a complementarity determining region of, CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 95, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 96, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 97. According to claim 1,
An antibody having a competitive inhibitory activity with an M30 antibody for binding to B7-H3 or a functional fragment of the antibody. According to claim 1,
Antibodies with antibody dependent cytotoxicity (ADCC) activity, complement dependent cytotoxicity (CDC) activity, or both, or functional fragments of the antibodies. According to claim 1,
An antibody in which the tumor is cancer or a functional fragment of the antibody. The method of claim 4,
An antibody or functional fragment of the antibody wherein the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer or kidney cancer. According to claim 1,
An antibody comprising the heavy chain variable region consisting of the amino acid sequence of amino acid numbers 20 to 141 in SEQ ID NO: 51 and the light chain variable region consisting of the amino acid sequence of amino acid numbers 23 to 130 in SEQ ID NO: 53 or the antibody concerned Functional fragments. According to claim 1,
An antibody whose normal region is a human-derived normal region or a functional fragment of the antibody. The method of claim 7,
An antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 63 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 59, or a functional fragment of the antibody. According to claim 1,
A humanized antibody or functional fragment of the antibody. The method of claim 9,
(a) amino acid sequence of amino acid number 20-141 in sequence number 85, (b) amino acid sequence of amino acid number 20-141 in sequence number 87, (c) amino acid sequence of amino acid number 20-141 in sequence number 89, The variable region of the heavy chain consisting of the amino acid sequence described, and (d) the amino acid sequence selected from the group consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 91, and (g) amino acid numbers 21 to 21 of SEQ ID NO: The amino acid sequence shown in 128, (h) The amino acid sequence shown in amino acid number 21-128 in sequence number 73, (i) The amino acid sequence shown in amino acid number 21-128 in sequence number 75, (j) In sequence number 77 Amino acid sequence shown in amino acid number 21-128, (k) Amino acid sequence shown in amino acid number 21-128 in sequence number 79, (l) Amino acid sequence shown in amino acid number 21-128 in sequence number 81, and (m) An antibody comprising a light chain variable region consisting of an amino acid sequence selected from the group consisting of the amino acid sequences shown in amino acid numbers 21 to 128 in SEQ ID NO: 83 or a functional fragment of the antibody. The method of claim 10,
The variable region of the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 141 in SEQ ID NO: 85 and the variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 71, the amino acid in SEQ ID NO: 85 The variable region of the heavy chain consisting of the amino acid sequence of numbers 20 to 141 and the variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 of SEQ ID NO: 73 and the amino acid numbers 20 to 141 of SEQ ID NO: 85 The variable region of the heavy chain consisting of the amino acid sequence and the light chain variable region consisting of the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 75, the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 85 The variable region of the light chain consisting of the amino acid sequence of amino acid numbers 21 to 128 in SEQ ID NO: 77 and the variable region of SEQ ID NO: 77, the heavy chain variable region and the sequence number of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 85 The variable region of the light chain consisting of the amino acid sequence of amino acid numbers 21 to 128 in 79, the variable region of the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 141 of SEQ ID NO: 85, and the amino acid number 21 of SEQ ID NO: 81. The variable region of the light chain consisting of the amino acid sequence of 128 to 128, the variable region of the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 141 of SEQ ID NO: 85, and the amino acid sequence of amino acids 21 to 128 of SEQ ID NO: 83 The variable region of the light chain consisting of, the variable region and arrangement of the heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 141 in SEQ ID NO: 91 The variable region of the light chain consisting of the amino acid sequence of amino acids 21 to 128 in No. 71, the variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 91, and the amino acid number of SEQ ID NO: 73. The variable region of the light chain consisting of the amino acid sequence of 21 to 128, the variable region of the heavy chain consisting of the amino acid sequence of amino acid number 20 to 141 of SEQ ID NO: 91, and the amino acid of amino acid numbers 21 to 128 of SEQ ID NO: 75 The variable region of the light chain consisting of an array, and the light chain consisting of the variable region of the heavy chain consisting of the amino acid sequence of amino acids 20 to 141 in SEQ ID NO: 91 and the amino acid sequence of amino acids 21 to 128 in SEQ ID NO: 77 An antibody or functional fragment of an antibody comprising a heavy chain variable region and a light chain variable region selected from the group consisting of variable regions. The method of claim 10,
The heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 471 in SEQ ID NO: 85, the light chain consisting of the amino acid sequence of amino acid numbers 21 to 233 in SEQ ID NO: 71, the amino acid numbers 20 to 471 of SEQ ID NO: 85 The heavy chain consisting of the amino acid sequence, the light chain consisting of the amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 73, the heavy chain consisting of the amino acid sequence of amino acids 20 to 471 in SEQ ID NO: 85 and SEQ ID NO: 75 Light chain consisting of the amino acid sequence of amino acid numbers 21 to 233, heavy chain consisting of the amino acid sequence of amino acids 20 to 471 in SEQ ID NO: 85, and the light chain consisting of the amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 77 Chain, heavy chain consisting of the amino acid sequence of amino acid numbers 20 to 471 in SEQ ID NO: 85, light chain consisting of the amino acid sequence of amino acid numbers 21 to 233 in SEQ ID NO: 79, amino acid numbers 20 to 471 of SEQ ID NO: 85 The heavy chain consisting of the amino acid sequence of SEQ ID NO: 81, the light chain consisting of the amino acid sequence of amino acid number 21 to 233 in SEQ ID NO: 81, the heavy chain consisting of the amino acid sequence of amino acid number 20 to 471 in SEQ ID NO: 85 and SEQ ID NO: 83 In the light chain consisting of the amino acid sequence of amino acids 21 to 233, the heavy chain consisting of the amino acid sequences of amino acids 20 to 471 in SEQ ID NO: 91, and the amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 71 Consisting of light chains, amino acid sequence 20 to 471 in SEQ ID NO: 91 Heavy chain consisting of light chain consisting of amino acid sequence of amino acids 21 to 233 in SEQ ID NO: 73, heavy chain consisting of amino acid sequence of amino acids 20 to 471 in SEQ ID NO: 91 and amino acid number 21 of SEQ ID NO: 75 The light chain consisting of the amino acid sequence of 233 to 233, the heavy chain consisting of the amino acid sequence of amino acid number 20 to 471 in SEQ ID NO: 91, and the light chain consisting of the amino acid sequence of amino acid numbers 21 to 233 in SEQ ID NO: 77 An antibody comprising a heavy chain and a light chain selected from the group or a functional fragment of the antibody. The method of claim 10,
The heavy chain consisting of the amino acid sequence of SEQ ID NO: 85, the light chain consisting of the amino acid sequence of SEQ ID NO: 71, the heavy chain consisting of the amino acid sequence of SEQ ID NO: 85 and the light chain consisting of the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: The heavy chain consisting of the amino acid sequence of 85 and the light chain consisting of the amino acid sequence of SEQ ID NO: 75, the heavy chain consisting of the amino acid sequence of SEQ ID NO: 85, and the light chain of the amino acid sequence of SEQ ID NO: 77, of SEQ ID NO: 85 The heavy chain consisting of the amino acid sequence described, the light chain consisting of the amino acid sequence shown in SEQ ID NO: 79, the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 85, the light chain consisting of the amino acid sequence shown in SEQ ID NO: 81, the amino acid shown in SEQ ID NO: 85 To the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 91, the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 91, the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: Consisting of a heavy chain consisting of an amino acid sequence as set out in SEQ ID NO: 73, a light chain consisting of an amino acid sequence as shown in SEQ ID NO: 91, a light chain consisting of an amino acid sequence as shown in SEQ ID NO: 75, and an amino acid sequence as shown in SEQ ID NO: 91 An antibody comprising a heavy chain and a light chain selected from the group consisting of a heavy chain and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 77, or a functional fragment of the antibody. The method according to any one of claims 1 to 13,
Functional fragment of an antibody selected from the group consisting of Fab, F(ab)2, Fab' and Fv. A method for producing the antibody according to any one of claims 1 to 13 or a functional fragment of the antibody, comprising the steps of culturing the host cell and a target antibody or functional fragment of the antibody from the culture obtained in the culturing step A method for producing an antibody or a functional fragment of the antibody, characterized in that it comprises a step of harvesting. The method according to claim 1, comprising a step of culturing the host cell and a step of collecting a target antibody or a functional fragment of the antibody from a culture obtained in the cultivation step. As described antibody or functional fragment of the antibody,
The host cell is transformed with an expression vector,
The expression vector is an antibody comprising any one of the following 1 to 7 polynucleotides or functional fragments of the antibody:
1. The polynucleotide encoding the antibody according to any one of items 1 to 13 or a functional fragment of the antibody;
2. The polynucleotide of 1, wherein the polynucleotide comprises the nucleotide sequence of 58 to 423 in SEQ ID NO: 50, and the nucleotide sequence of 67 to 390 in SEQ ID NO: 52;
3. The polynucleotide of 1, wherein the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 62, and the nucleotide sequence of SEQ ID NO: 58;
4. In the polynucleotide of 1 above,
(a) The nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 84, (b) the nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 86, and (c) the nucleotide numbers of 58 to 423 in SEQ ID NO: 88. The nucleotide sequence described, and (d) a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth at nucleotide numbers 58 to 423 at SEQ ID NO: 90, and (f) the nucleotide sequences set forth at nucleotide numbers 61 to 384 at SEQ ID NO: 70, ( g) the nucleotide sequence of nucleotides 61 to 384 in SEQ ID NO: 72, (h) the nucleotide sequence of nucleotides 61 to 384 in SEQ ID NO: 74, (i) the nucleotides of 61 to 384 in SEQ ID NO: 76; Nucleotide sequence, (j) nucleotide sequence of nucleotide number 61 to 384 in sequence number 78, (k) nucleotide sequence of nucleotide number 61 to 384 in sequence number 80, and (l) nucleotide number of sequence number 82. A polynucleotide comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences described in 61 to 384;
5. In the polynucleotide of 4 above, the nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 84 and the nucleotide sequence of nucleotide numbers 61 to 384 in SEQ ID NO: 70, nucleotide numbers 58 to 423 in SEQ ID NO: 84 The nucleotide sequence as set forth in SEQ ID NO: 72 to nucleotide sequence 61 to 384, the nucleotide sequence as shown in nucleotide number 58 to 423 in SEQ ID NO: 84, and the nucleotide sequence as shown in nucleotide numbers 61 to 384 in SEQ ID NO: 74, The nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 84, the nucleotide sequence of nucleotide numbers 61 to 384 in SEQ ID NO: 76, the nucleotide sequence of nucleotide numbers 58 to 423 in SEQ ID NO: 84, and in SEQ ID NO: 78 Nucleotide sequence shown in nucleotide number 61-384, nucleotide sequence shown in nucleotide number 58-423 in sequence number 84, nucleotide sequence shown in nucleotide number 61-384 in sequence number 80, nucleotide number 58-423 in sequence number 84 The nucleotide sequence as set forth in SEQ ID NO: 82 and the nucleotide sequence as set forth in nucleotide number 61 to 384, the nucleotide sequence as shown in nucleotide number 58 to 423 in SEQ ID NO: 90 and the nucleotide sequence as shown in nucleotide numbers 61 to 384 in SEQ ID NO: 70, The nucleotide sequence as shown in nucleotide numbers 58 to 423 in SEQ ID NO: 90 and the nucleotide sequence as shown in nucleotide numbers 61 to 384 in SEQ ID NO: 72, nucleotide numbers 58 to in SEQ ID NO: 90 The nucleotide sequence set forth in 423 and the nucleotide sequence set forth in nucleotide numbers 61 to 384 in SEQ ID NO: 74, and the nucleotide sequence set forth in nucleotide numbers 58 to 423 in sequence number 90 and the nucleotide set forth in nucleotide numbers 61 to 384 in sequence number 76 A polynucleotide comprising a nucleotide sequence selected from the group consisting of sequences;
6. In the polynucleotide of 4 above, the nucleotide sequence of nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and the nucleotide sequence of nucleotide numbers 61 to 699 in SEQ ID NO: 70, nucleotide numbers 58 to 1413 in SEQ ID NO: 84 The nucleotide sequence as set forth in SEQ ID NO: 72 and the nucleotide sequence as shown in nucleotide number 61 to 699, the nucleotide sequence as shown in nucleotide number 58 to 1413 in SEQ ID NO: 84 and the nucleotide sequence as shown in nucleotide numbers 61 to 699 in SEQ ID NO: 74, The nucleotide sequence as set forth in nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and the nucleotide sequence as set forth in nucleotide numbers 61 to 699 in SEQ ID NO: 76, the nucleotide sequence as set forth in nucleotide numbers 58 to 1413 in SEQ ID NO: 84 and as shown in SEQ ID NO: 78 Nucleotide sequence shown in nucleotide number 61-699, nucleotide sequence shown in nucleotide number 58-1413 in sequence number 84, and nucleotide sequence shown in nucleotide number 61-699 in sequence number 80, nucleotide number 58-1414 in sequence number 84 The nucleotide sequence as set forth in SEQ ID NO: 82 and the nucleotide sequence as shown in nucleotide number 61 to 699, the nucleotide sequence as shown in nucleotide number 58 to 1413 in SEQ ID NO: 90 and the nucleotide sequence as shown in nucleotide numbers 61 to 699 in SEQ ID NO: 70, The nucleotide sequence as set forth in nucleotide numbers 58 to 1413 in SEQ ID NO: 90 and the nucleotide sequence as set forth in nucleotide numbers 61 to 699 in SEQ ID NO: 72, the nucleotides as shown in SEQ ID NO: 90 Nucleotide sequence shown in number 58-1413 and nucleotide sequence shown in nucleotide number 61-699 in sequence number 74, and nucleotide sequence shown in nucleotide number 58-1413 in sequence number 90, and nucleotide number 61-699 in sequence number 76 A polynucleotide comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences described in;
7. In the polynucleotide of 4 above, the nucleotide sequence set forth in SEQ ID NO: 84 and the nucleotide sequence set forth in SEQ ID NO: 70, the nucleotide sequence set forth in SEQ ID NO: 84 and the nucleotide sequence set forth in SEQ ID NO: 72, the nucleotide sequence set forth in SEQ ID NO: 84, and The nucleotide sequence shown in SEQ ID NO: 74, the nucleotide sequence shown in SEQ ID NO: 84 and the nucleotide sequence shown in SEQ ID NO: 76, the nucleotide sequence shown in SEQ ID NO: 84 and the nucleotide sequence shown in SEQ ID NO: 78, the nucleotide sequence shown in SEQ ID NO: 84 and the sequence number The nucleotide sequence of 80, the nucleotide sequence of SEQ ID NO: 84 and the nucleotide sequence of SEQ ID NO: 82, the nucleotide sequence of SEQ ID NO: 90 and the nucleotide sequence of SEQ ID NO: 70, the nucleotide sequence of SEQ ID NO: 90, and the SEQ ID NO: 72 A polynucleotide comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence described, the nucleotide sequence shown in SEQ ID NO: 90 and the nucleotide sequence shown in SEQ ID NO: 74, and the nucleotide sequence shown in SEQ ID NO: 90 and the nucleotide sequence shown in SEQ ID NO: 76. The method of claim 16,
Functional fragment of an antibody selected from the group consisting of Fab, F(ab)2, Fab' and Fv. The method according to any one of claims 1 to 13,
An antibody or functional fragment of an antibody whose sugar chain modification is regulated to enhance antibody-dependent cytotoxic activity. A pharmaceutical composition for the treatment or prevention of a tumor, comprising the antibody according to any one of claims 1 to 13 or a functional fragment of the antibody. The method of claim 19,
A pharmaceutical composition for the treatment of tumors. A pharmaceutical composition for treating a tumor, comprising the antibody according to any one of claims 1 to 13, or at least one of functional fragments of the antibody, and at least one therapeutic agent for cancer. The method of claim 20,
A pharmaceutical composition in which the tumor is cancer. The method of claim 22,
Pharmaceutical composition wherein the cancer is lung cancer, breast cancer, prostate cancer, pancreatic cancer, colon cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, stomach cancer, esophageal cancer or kidney cancer. A pharmaceutical composition for the treatment or prevention of a tumor, comprising at least one of the antibody according to claim 16 or a functional fragment of the antibody. A pharmaceutical composition for treating a tumor, comprising at least one of the antibody according to claim 16 or a functional fragment of the antibody, and at least one cancer therapeutic agent. The method of claim 21,
A pharmaceutical composition in which the tumor is cancer.

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