Glypican-3 antibody

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

 

Background of invention

The scope of the invention

The present invention relates to an antibody against glypican 3, inhibitor of cell growth and anti-cancer agent, which contain the antibody as an active ingredient.

Description related field

Glypican 3 (GPC3) is a member of pipikaula family heparansulfate that are present on cell surfaces. This suggests that GPC3 is involved in cell division during the development or growth of cancer cells, but its function is not yet clear.

It was found that a certain type of antibody binding to the GPC3, inhibits cell growth by antibody-dependent cretaceouspaleogene cytotoxicity (ADCC) and complementability cytotoxicity (CDC) (international patent application WO 2003/000883). In addition, I believe that GPC3 is cleaved in vivo and enters the blood in the form of Sekretareva form of GPC3, therefore, cancer can be diagnosed using an antibody capable of contact Sekretareva form of GPC3 (international patent application WO 2004/022739, WO 03/100429 and WO 2004/018667).

For the development of anticancer funds based on the cytotoxic activity of the antibody is preferably used antibody had a high ADCC activity or CDC activity. The meet is but you want antibody against GPC3 with high cytotoxicity, as the antibody that recognizes GPC3.

The aim of the present invention is to provide an antibody against GPC3, which has higher ADCC activity and CDC activity than traditional antibody.

Brief description of the invention

The authors of the present invention could obtain an antibody with a higher cytotoxicity than traditional antibodies against glypican 3. In addition, the authors analyzed the epitopes of such antibodies, and they were able to identify areas of GPC3 recognized by the antibody with high cytotoxic activity.

In one aspect the present invention provides an antibody comprising the variable plot heavy chain that contains lots of CDR 1, 2 and 3 on any of the items(1)-(12):

(1) CDR1 having the amino acid sequence described in SEQ ID NO: 123, CDR2, having the amino acid sequence described in SEQ ID NO: 124, and CDR3 having the amino acid sequence described in SEQ ID NO: 125;

(2) CDR1 having the amino acid sequence described in SEQ ID NO: 109, CDR2, having the amino acid sequence described in SEQ ID NO: 110, and CDR3 having the amino acid sequence described in SEQ ID NO: 111;

(3) CDR1 having the amino acid sequence described in SEQ ID NO: 106, CDR2 having aminoxy the pilot sequence, described in SEQ ID NO: 107, and CDR3 having the amino acid sequence described in SEQ ID NO: 108;

(4) CDR1 having the amino acid sequence described in SEQ ID NO: 132, CDR2, having the amino acid sequence described in SEQ ID NO: 133, and CDR3 having the amino acid sequence described in SEQ ID NO: 134;

(5) CDR1 having the amino acid sequence described in SEQ ID NO: 106, CDR2, having the amino acid sequence described in SEQ ID NO: 135, and CDR3 having the amino acid sequence described in SEQ ID NO: 136;

(6) CDR1 having the amino acid sequence described in SEQ ID NO: 126, CDR2, having the amino acid sequence described in SEQ ID NO: 127, and CDR3 having the amino acid sequence described in SEQ ID NO: 128;

(7) CDR1 having the amino acid sequence described in SEQ ID NO: 129, CDR2, having the amino acid sequence described in SEQ ID NO: 130, and CDR3 having the amino acid sequence described in SEQ ID NO: 131;

(8) CDR1 having the amino acid sequence described in SEQ ID NO: 103, CDR2, having the amino acid sequence described in SEQ ID NO: 104, and CDR3 having the amino acid sequence described in SEQ ID NO: 105;

(9) CDR1 having the amino acid sequence described in SEQ ID NO: 118, CDR2, having the amino acid sequence described in SEQ ID NO: 121, and CR3, having the amino acid sequence described in SEQ ID NO: 122;

(10) CDR1 having the amino acid sequence described in SEQ ID NO: 115, CDR2, having the amino acid sequence described in SEQ ID NO: 116, and CDR3 having the amino acid sequence described in SEQ ID NO: 117;

(11) CDR1 having the amino acid sequence described in SEQ ID NO: 112, CDR2, having the amino acid sequence described in SEQ ID NO: 113, and CDR3 having the amino acid sequence described in SEQ ID NO: 114; or

(12) CDR1 having the amino acid sequence described in SEQ ID NO: 118, CDR2, having the amino acid sequence described in SEQ ID NO: 119, and CDR3 having the amino acid sequence described in SEQ ID NO: 120.

In another aspect, the invention provides an antibody comprising the variable area light chain contains the CDR 1, 2 and 3 on any of the items(1)-(13):

(1) CDR1 having the amino acid sequence described in SEQ ID NO: 143, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(2) CDR1 having the amino acid sequence described in SEQ ID NO: 143, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 145;

(3) DR1, having the amino acid sequence described in SEQ ID NO: 140, a CDR2 having the amino acid sequence described in SEQ ID NO: 141, and a CDR3 having the amino acid sequence described in SEQ ID NO: 142;

(4) CDR1 having the amino acid sequence described in SEQ ID NO: 167, CDR2, having the amino acid sequence described in SEQ ID NO: 168, and CDR3 having the amino acid sequence described in SEQ ID NO: 169;

(5) CDR1 having the amino acid sequence described in SEQ ID NO: 170, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 171;

(6) CDR1 having the amino acid sequence described in SEQ ID NO: 159, CDR2, having the amino acid sequence described in SEQ ID NO: 160, and CDR3 having the amino acid sequence described in SEQ ID NO: 161;

(7) CDR1 having the amino acid sequence described in SEQ ID NO: 162, CDR2, having the amino acid sequence described in SEQ ID NO: 147, and CDR3 having the amino acid sequence described in SEQ ID NO: 163;

(8) CDR1 having the amino acid sequence described in SEQ ID NO: 164, the CDR2 having the amino acid sequence described in SEQ ID NO: 165, and CDR3 having the amino acid sequence described in SEQ ID NO: 166;

(9) CDR1 having aminokislotna the sequence, described in SEQ ID NO: 137, CDR2, having the amino acid sequence described in SEQ ID NO: 138, and CDR3 having the amino acid sequence described in SEQ ID NO: 139;

(10) CDR1 having the amino acid sequence described in SEQ ID NO: 155, CDR2, having the amino acid sequence described in SEQ ID NO: 156, and CDR3 having the amino acid sequence described in SEQ ID NO: 157;

(11) CDR1 having the amino acid sequence described in SEQ ID NO: 149, CDR2, having the amino acid sequence described in SEQ ID NO: 150, and CDR3 having the amino acid sequence described in SEQ ID NO: 151;

(12) CDR1 having the amino acid sequence described in SEQ ID NO: 146, CDR2, having the amino acid sequence described in SEQ ID NO: 147, and CDR3 having the amino acid sequence described in SEQ ID NO: 148; or

(13) CDR1 having the amino acid sequence described in SEQ ID NO: 152, CDR2, having the amino acid sequence described in SEQ ID NO: 153, and CDR3 having the amino acid sequence described in SEQ ID NO: 154.

Preferably the antibody of the present invention is selected from the group consisting of the antibody according to any one of paragraphs(1)-(13):

(1) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, according to the government, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 143, 144 and 158, respectively;

(2) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 109, 110 and 111, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 143, 144 and 145, respectively;

(3) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 106, 107 and 108, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 140, 141 and 142, respectively;

(4) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 132, 133 and 134, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 167, 168 and 169, respectively;

(5) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 106, 135 and 136, respectively, and variable ucast the light chain, containing CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 170, 144 and 171, respectively;

(6) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 126, 127 and 128, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 159, 160 and 161, respectively;

(7) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 129, 130 and 131, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 162, 147 and 163, respectively;

(8) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 129, 130 and 131, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 164, 165 and 166, respectively;

(9) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 103, 104 and 105, respectively, and variable area light chain contains the CDR 1, 2 and 3, it is matter of amino acid sequence, described in SEQ ID NO: 137, 138 and 139, respectively;

(10) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 118, 121 and 122, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 155, 156 and 157, respectively;

(11) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 115, 116 and 117, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 149, 150 and 151, respectively;

(12) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 112, 113 and 114, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 146, 147 and 148, respectively; and

(13) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 118, 119 and 120, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 52, 153 and 154, respectively.

In another aspect, the invention provides an antibody containing the variable section of the heavy chain according to any one of paragraphs(1)-(7):

(1) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 84;

(2) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 85;

(3) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 86;

(4) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 87;

(5) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 88;

(6) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 89; or

(7) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90.

In another aspect, the invention provides an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 92.

Preferably the antibody of the present invention is selected from the group consisting of the antibody according to any one of paragraphs(1)-(7):

(1) an antibody comprising the variable plot heavy chain having the amino acid consistently is th described in SEQ ID NO: 84, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92;

(2) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 85, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92;

(3) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 86, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92;

(4) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 87, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92;

(5) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 88, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92;

(6) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 89, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92; and

(7) and tetelo, includes variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90, and variable area light chain having the amino acid sequence described in SEQ ID NO: 92.

In another aspect, the invention provides an antibody comprising the variable area light chain contains the CDR 1, 2 and 3 on any of the items(1)-(15):

(1) CDR1 having the amino acid sequence described in SEQ ID NO: 174, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(2) CDR1 having the amino acid sequence described in SEQ ID NO: 175, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(3) CDR1 having the amino acid sequence described in SEQ ID NO: 176, a CDR2 having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(4) CDR1 having the amino acid sequence described in SEQ ID NO: 177, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(5) CDR1 having the amino acid sequence described in SEQ ID NO: 78, CDR2 having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(6) CDR1 having the amino acid sequence described in SEQ ID NO: 179, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(7) CDR1 having the amino acid sequence described in SEQ ID NO: 180, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(8) CDR1 having the amino acid sequence described in SEQ ID NO: 181, A CDR2 having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(9) CDR1 having the amino acid sequence described in SEQ ID NO: 182, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(10) CDR1 having the amino acid sequence described in SEQ ID NO: 183, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(11) CDR1 having the amino acid sequence described in SEQ ID NO: 184, CDR2, having the amino acid is tnou sequence, described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(12) CDR1 having the amino acid sequence described in SEQ ID NO: 185, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(13) CDR1 having the amino acid sequence described in SEQ ID NO: 186, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158;

(14) CDR1 having the amino acid sequence described in SEQ ID NO: 187, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158; or

(15) CDR1 having the amino acid sequence described in SEQ ID NO: 188, CDR2, having the amino acid sequence described in SEQ ID NO: 144, and CDR3 having the amino acid sequence described in SEQ ID NO: 158.

In another aspect, the invention provides an antibody selected from the group consisting of antibodies items(1)-(15):

(1) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3, there is the following amino acid sequence, described in SEQ ID NO: 174, 144 and 158, respectively;

(2) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 175, 144 and 158, respectively;

(3) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 176, 144 and 158, respectively;

(4) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 177, 144 and 158, respectively;

(5) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 178, 14, 158, respectively;

(6) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 179, 144 and 158, respectively;

(7) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 180, 144 and 158, respectively;

(8) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 181, 144 and 158, respectively;

(9) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 182, 144 and 158, respectively;

(1) antibody includes variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 183, 144 and 158, respectively;

(11) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 184, 144 and 158, respectively;

(12) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 185, 144 and 158, respectively;

(13) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 186, 144 and 158, respectively;

(14) an antibody comprising roebelinii plot heavy chain, containing CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 187, 144 and 158, respectively; and

(15) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 188, 144 and 158, respectively.

In the following aspect, the invention provides an antibody containing the variable area light chain variable regions selected from(1)-(15):

(1) variable area light chain having the amino acid sequence described in SEQ ID NO: 191;

(2) variable area light chain having the amino acid sequence described in SEQ ID NO: 192;

(3) variable area light chain having the amino acid sequence described in SEQ ID NO: 193;

(4) variable area light chain having the amino acid sequence described in SEQ ID NO: 194;

(5) variable area light chain having the amino acid sequence described in SEQ ID NO: 195;

(6) variable area light chain, imediaseeking sequence, described in SEQ ID NO: 196;

(7) variable area light chain having the amino acid sequence described in SEQ ID NO: 197;

(8) variable area light chain having the amino acid sequence described in SEQ ID NO: 198;

(9) variable area light chain having the amino acid sequence described in SEQ ID NO: 199;

(10) variable area light chain having the amino acid sequence described in SEQ ID NO: 200;

(11) the variable area light chain having the amino acid sequence described in SEQ ID NO: 201;

(12) variable area light chain having the amino acid sequence described in SEQ ID NO: 202;

(13) the variable area light chain having the amino acid sequence described in SEQ ID NO: 203;

(14) variable area light chain having the amino acid sequence described in SEQ ID NO: 204; and

(15) variable area light chain having the amino acid sequence described in SEQ ID NO: 205.

In another aspect, the invention provides an antibody containing the variable area light chain selected from the group consisting of variable regions(1)-(15):

(1) variable area light chain having the amino acid sequence described in SEQ ID NO: 191;

(2) variable area light chain having aminokislotnoi sequence, described in SEQ ID NO: 192;

(3) variable area light chain having the amino acid sequence described in SEQ ID NO: 193;

(4) variable area light chain having the amino acid sequence described in SEQ ID NO: 194;

(5) variable area light chain having the amino acid sequence described in SEQ ID NO: 195;

(6) variable area light chain having the amino acid sequence described in SEQ ID NO: 196;

(7) variable area light chain having the amino acid sequence described in SEQ ID NO: 197;

(8) variable area light chain having the amino acid sequence described in SEQ ID NO: 198;

(9) variable area light chain having the amino acid sequence described in SEQ ID NO: 199;

(10) variable area light chain having the amino acid sequence described in SEQ ID NO: 200;

(11) the variable area light chain having the amino acid sequence described in SEQ ID NO: 201;

(12) variable area light chain having the amino acid sequence described in SEQ ID NO: 202;

(13) the variable area light chain having the amino acid sequence described in SEQ ID NO: 203;

(14) variable area light chain having the amino acid sequence described in SEQ ID NO: 204; and

(15) areally area light chain, having the amino acid sequence described in SEQ ID NO: 205;

and variable plot heavy chain selected from the group consisting of variable regions(1)-(7):

(1) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 84;

(2) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 85;

(3) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 86;

(4) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 87;

(5) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 88;

(6) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 89; and

(7) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90.

Variable plot heavy chains (H), variable area light chain (L) and amino acid sequence of CDR 1, 2 and 3, as well as non SEQ ID nos given in table 1.

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Table 1
Antibody and variable plotsSEQ ID NO
M3C11H22
M13B3H23
M1E7H24
M3B8H25
M11F1H26
M19B11H27
M6B1H28
M18D4H29
M5B9H30
M10D2H31
L9G11H32
M3C11L44
M13B3L45
M1E7L46
M3B8 L47
M11F1L48
M19B11L49
M6B1L50
M18D4L51
M5B9L52
M10D2L53
L9G11L54
GC199H60
GC202H61
GC33H62
GC179H63
GC194H64
GC199L71
GC202LGC33L73
GC179L74
GC194(1)L75
GC194(2)L76
GC33.ver.aH84
GC33.ver.cH85
GC33.ver.fH86
GC33.ver.hH87
GC33.ver.iH88
GC33.ver.jH89
GC33.ver.kH90
GC33.ver.aL92
M13B3(H)CDR1103
CDR2 104
CDR3105
M3B8(H)CDR1106
CDR2107
CDR3108
M11F1(H)CDR1109
CDR2110
CDR3111
M5B9(H)CDR1112
CDR2113
CDR3114
M6B1(H)CDR1115
CDR2116
CDR3117
M10D2(H)CDR111
CDR2119
CDR3120
L9G11(H)CDR1118
CDR2121
CDR3122
GC33(H)CDR1123
CDR2124
CDR3125
GC179(H)CDR1126
CDR2127
CDR3128
GC194(H)CDR1129
CDR2130
CDR3131
GC199(H)CDR1132
CDR2133
CDR3134
GC202(H)CDR1106
CDR2135
CDR3136
M13B3(L)CDR1137
CDR2138
CDR3139
M3B8(L)CDR1140
CDR2141
CDR3142
M11F1(L)CDR1143
CDR2144
CDR3145
M5B9(L)CDR1146
CDR2147
CDR3148
M6B1(L)CDR1149
CDR2150
CDR3151
M10D2(L)CDR1152
CDR2153
CDR3154
L9G11(L)CDR1155
CDR2156
CDR3157
GC33(L)CDR1143
the CDR2144
CDR3158
GC179(L)CDR1159
CDR2160
CDR3161
GC194(L)1CDR1162
CDR2147
CDR3163
GC194(L)2CDR1164
CDR2165
CDR3166
GC199(L)CDR1167
CDR2168
CDR3169
GC202(L) CDR1170
CDR2144
CDR3171
GC33(L)G34A174
GC33(L)G34D175
GC33(L)G34E176
GC33(L)G34F177
GC33(L)G34H178
GC33(L)G34N179
GC33(L)G34P180
GC33(L)G34Q181
GC33(L)G34I182
GC33(L)G34K183
GC33(L)G34L184
GC33(L)G34V185
GC33(L)G34W186
GC33(L)G34Y187
GC33(L)G34R188

In addition, the present invention describes an antibody having activity equivalent to the activity described above, antibodies, and having the above-described amino acid sequence in which one or more amino acid residues substituted, deleted or added and/or inserted.

Preferably the antibody of the present invention is humanitariannet antibody.

Thus, in another aspect, the invention provides humanitariannet antibody capable of contacting glypican 3.

In the following aspect, the invention provides an antibody capable of contacting a peptide consisting of the sequence of amino acid residues 524-563 glypican 3.

Preferably the antibody of the present invention is able to contact the peptide consisting of the sequence of amino acid residues 537-563 glypican 3. More preferably the antibody of the present invention is not associated with the peptide consisting of the posledovatelnosti amino acid residues 550-563 glypican 3.

Preferably the antibody is able to bind with a peptide consisting of the sequence of amino acid residues 544-553 glypican 3, or a peptide consisting of the sequence of amino acid residues 546-551 glypican 3.

In the following aspect, the invention provides an antibody capable of contacting the epitope with which it can communicate secondary antibody where the above-mentioned secondary antibody includes a variable plot heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 143, 144 and 158, respectively. Namely, the antibody of the present invention can compete with the secondary antibody for binding to GPC3.

In the preferred embodiment the antibody of the present invention can communicate with glypican 3 and has a high CDC activity against cells expressing glypican 3, and/or high ADCC activity against cells expressing glypican 3.

In another aspect, the invention provides polynucleotide encoding variable plot heavy chain or variable area light chain of the antibody of the present invention.

Preferably polynucleotide this image is etenia has the sequence described in SEQ ID NO: 11-21, 33-43, 55-59, 65-70 and 77-83.

In the following aspect, the invention provides an inhibitor of cell growth and anti-cancer agent containing as an active ingredient the antibody of the present invention. Preferably an anti-cancer agent of the present invention used for the treatment of hepatoma.

In the following aspect, the invention provides a peptide containing a sequence of amino acid residues 524-563 glypican 3, the sequence of amino acid residues 537-563 glypican 3, the sequence of amino acid residues 544-553 glypican 3 or the sequence of amino acid residues 546-551 glypican 3.

Brief description of drawings

Figure 1 shows the binding activity of the antibody against GPC3 in the CHO cell, a CHO cell expressing full-GPC3, HepG2 and HuH-7, as measured by flow cytometry. M1E7 (solid line) and M11F1 (dashed line) used at a concentration of 5 µg/ml, respectively.

Figure 2 is a table showing the results of the classification epitopes obtained using a competitive ELISA. The degree of competitive inhibition of binding of biotinylated antibody against GPC3 is given in percentage. Epitopes are divided into five groups, from a to e, according to the nature of competitive inhibition.

On figue the results of Western blotting, which shows how a fragment of a soluble form of the crustal GPC3 protein binds an antibody against GPC3: N-terminal fragment has a size of 40 kDa or C-terminal fragment with a size of 30 kDa. It was found that L9G11 associated with the N-terminal fragment, and M3C11 associated with C-end fragment.

Figure 4 shows the results of detection Sekretareva form of GPC3 in the culture supernatant HepG2 using the sandwich ELISA method. Secretiruema form well detected using combinations of antibodies that bind to N-terminal fragment, such as M6B1, M18D4 or M19B11, and poorly detected using combinations of antibodies that bind to the C-terminal fragment, such as M3C11, M13B3 or M3B8.

Figure 5 shows the results thus culture supernatant HepG2 using antibody against GPC3 and detection Sekretareva form of GPC3. Medium as a control (lines 1 and 3) and the culture supernatant HepG2 (lines 2 and 4) were thus using M1E7 (lines 1 and 2) and M10D2 (lines 3 and 4). Secretory GPC3 detects using M10D2, which binds to N-terminal fragment.

Figure 6 shows the results of the analysis of the epitope of an antibody that binds to C-terminal fragment of GPC3, by the method of Western blotting using a hybrid protein C-terminal peptide GPC3 and GST. The core protein p is storemay form of GPC3 (line 1), GST (line 2), GC-1 (line 3), GC-2 (line 4), GC-3 (line 5), GC-4 (line 6) and GC-5 (line 7) is subjected to SDS-electrophoresis in reducing conditions and detection method Western blotting using M3C11 and M11F1.

7 shows the results of measurement of CDC activity of mouse-human chimeric antibody against GPC3 in CHO cells, which Express GPC3.

On Fig shows the results of measurement of ADCC activity of murine-human chimeric antibody against GPC3 in CHO cells, which Express GPC3 and HepG2.

Figure 9 shows the results of measurement of ADCC activity GC33 on cell line human hepatoma, HuH-7, using effector cells mouse bone marrow.

Figure 10 shows the results of measurement of antitumor activity of GC33 antibody on mouse models transplanted with human hepatoma.

Figure 11 shows the results of measurement of CDC activity of mouse-human chimeric antibody GC33 on the CHO cells, which Express GPC3.

On Fig shows the results of measurement of ADCC activity of murine-human chimeric antibody GC33 on HepG2.

On Fig shows the sequence GPC3 contained in proteins, hybridized with GST (GC-4, 5, 6, 7, 8, 9, 11, 12, 13 and 14)obtained for the analysis of epitope GC33.

On Fig shows the results of Western blotting using GC33 after the separation of the GST, GC-7, 8, 9, 11, 12, 13 14 the method of SDS-PAGE in reducing conditions.

On Fig shows the results of measurement of the binding activity gumanitarnogo GC33 against GPC3 using ELISA method.

On Fig table antibodies, which includes the isotypes and the results of ELISA, BIAcore, FACS, epitope analysis and thus for clones derived from mice immunized with the soluble form of GPC3.

On Fig table antibodies, which includes the isotypes and the results of ELISA, FACS and epitope analysis of clones derived from mice immunized with GC-3.

On Fig shows the results of measurement of the binding activity of the modified antibody against korovou protein soluble form of GPC3 using ELISA method. Gly34, which is located in the CDR1 of the variable segment of the L-chain gumanitarnogo GC33 replace any of the 17 amino acids other than Cys and Met.

On Fig shows the results of measurement of CDC activity of mouse-human chimeric antibody GC33, M3C11 and M1E7 on CHO cells expressing full-GPC3.

On Fig shows the results of measurement of ADCC activity of murine-human chimeric antibody GC33, M3C11 and M1E7 on cell line human hepatoma SK-03 expressing full-GPC3.

Detailed description of the invention

Antibody

The present invention provides antibodies that are described in the following paragraphs (I)to(XI).

(I) an Antibody comprising variable is castke heavy chain, containing CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID nos given in any of the following items(1)-(12):

(1) SEQ ID NO: 123, 124 and 125 (GC33),

(2) SEQ ID NO: 109, 110 and 111 (M11F1),

(3) SEQ ID NO: 106, 107 and 108 (M3B8),

(4) SEQ ID NO: 132, 133 and 134 (GC199),

(5) SEQ ID NO: 106, 135 and 136 (GC202),

(6) SEQ ID NO: 126, 127 and 128 (GC179),

(7) SEQ ID NO: 129, 130 and 131 (GC194),

(8) SEQ ID nos: 103, 104 and 105 (M13B3),

(9) SEQ ID NO: 118, 121 and 122 (L9G11),

(10) SEQ ID NO: 115, 116 and 117 (M6B1),

(11) SEQ ID NO: 112, 113 and 114 (M5B9) and

(12) SEQ ID NO: 118, 119 and 120 (M10D2).

Among the antibodies described in paragraphs (1)to(12), the preferred antibodies described in paragraphs (1)to(8), the preferred antibodies described in paragraphs (1)to(5), and particularly preferably the antibody described in paragraph (1). Antibodies, as described in paragraphs (1)to(8), recognize C-terminal peptide glypican 3 (peptide containing 374-580 amino acids glypican 3) and used as therapeutic antibodies. In addition, the antibodies described in paragraphs (9)to(12), recognize the N-terminal peptide glypican 3 (peptide containing amino acids 1-373 glypican 3) and are used as diagnostic antibodies.

(II) the Antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID nos given in any of the following items(1)-(13):

(1) SEQ ID NO: 143, 144 and 158 (GC33),

(2) SEQ ID NO: 143, 144 and 145 (M11F1),

(3) SEQ ID NO: 140, 141 and 142 (M3B8),

(4) SEQ ID NO: 167 168 and 169 (GC199),

(5) SEQ ID NO: 170, 144 and 171 (GC202),

(6) SEQ ID NO: 159, 160 and 161 (GC179),

(7) SEQ ID NO: 162, 147 and 163 (GC194 (1)),

(8) SEQ ID NO: 164, 165 and 166 (GC194 (2)),

(9) SEQ ID NO: 137, 138 and 139 (M13B3),

(10) SEQ ID nos: 155, 156 and 157 (L9G11),

(11) SEQ ID NO: 149, 150 and 151 (M6B1),

(12) SEQ ID NO: 146, 147 and 148 (M5B9) and

(13) SEQ ID NO: 152, 153 and 154 (M10D2).

Among the antibodies described in paragraphs (1)to(13), the preferred antibodies described in paragraphs (1)to(8), the preferred antibodies described in paragraphs (1)to(5), and particularly preferably the antibody described in paragraph (1). Antibodies, as described in paragraphs (1)to(8), recognize C-terminal peptide glypican 3 (peptide containing 374-580 amino acids glypican 3) and used as therapeutic antibodies. In addition, the antibodies described in paragraphs (9)to(13), recognize the N-terminal peptide glypican 3 (peptide containing amino acids 1-373 glypican 3) and are used as diagnostic antibodies.

(III) an Antibody selected from the group consisting of antibodies, are described in the following paragraphs(1)-(13):

(1) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 143, 144 and 158 (GC33),

(2) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid is basic sequence, described in SEQ ID NO: 109, 110 and 111, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 143, 144 and 145 (M11F1),

(3) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 106, 107 and 108, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 140, 141 and 142 (M3B8),

(4) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 132, 133 and 134, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 167, 168 and 169 (GC199),

(5) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 106, 135 and 136, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 170, 144 and 171 (GC202),

(6) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 126, 127 and 128, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence opican the e in SEQ ID NO: 159, 160 and 161 (GC179),

(7) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 129, 130 and 131, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 162, 147 and 163 (GC194 (1)),

(8) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 129, 130 and 131, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 164, 165 and 166 (GC194 (2)),

(9) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 103, 104 and 105, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 137, 138 and 139 (M13B3),

(10) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 118, 121 and 122, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 155, 156 and 157 (L9G11),

(11) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid after which outermost, described in SEQ ID NO: 115, 116 and 117, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 149, 150 and 151 (M6B1),

(12) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 112, 113 and 114, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 146, 147 and 148 (M5B9),

(13) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 118, 119 and 120, and the flexible sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 152, 153 and 154 (M10D2).

Among the antibodies described in paragraphs (1)to(13), the preferred antibodies described in paragraphs (1)to(8), the preferred antibodies described in paragraphs (1)to(5), and particularly preferably the antibody described in paragraph (1). Antibodies, as described in paragraphs (1)to(8), recognize C-terminal peptide glypican 3 (peptide containing 374-580 amino acids glypican 3) and used as therapeutic antibodies. In addition, the antibodies described in paragraphs (9)to(13), recognize the N-terminal peptide glypican 3 (peptide containing amino acids 1-373 glypican 3) and are used as diagnostic antibodies is A.

(IV) an Antibody containing the variable plot heavy chain described in any of the following items(1)-(7):

(1) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 84 (GC33 VH ver.a),

(2) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 85 (GC33 VH ver.c),

(3) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 86 (GC33 VH ver.f),

(4) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 87 (GC33 VH ver.h),

(5) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 88 (GC33 VH ver.i),

(6) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 89 (GC33 VH ver.j), and

(7) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90 (GC33 VH ver.k).

Among the antibodies described in paragraphs (1)to(7), particularly preferred antibodies described in paragraphs(2)-(7).

(V) an Antibody comprising the variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a).

(VI) an Antibody selected from the group consisting of antibodies, are described in the following paragraphs(1)-(7):

(1) an antibody that includes variations is a recreational area heavy chain, having the amino acid sequence described in SEQ ID NO: 84 (GC33 VH ver.a), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a),

(2) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 85 (GC33 VH ver.c), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a),

(3) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 86 (GC33 VH ver.f), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a),

(4) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 87 (GC33 VH ver.h), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a),

(5) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 88 (GC33 VH ver.i), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a),

(6) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 89 (GC33 VH ver.j), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a), and

(7) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90 (GC33 VH ver.k), and variable area light chain having the amino acid sequence described in SEQ ID NO: 92 (GC33 VL ver.a).

Among the antibodies described in paragraphs (1)to(7), particularly preferred antibodies described in paragraphs(2)-(7).

(VII) the Antibody described in any of the following items(1)-(15):

(1) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 174, 144 and 158,

(2) the antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 175, 144 and 158,

(3) the antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 176, 144 and 158,

(4) the antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 177, 144 and 158,

(5) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence is, described in SEQ ID NO: 178, 144 and 158,

(6) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 179, 144 and 158,

(7) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 180, 144 and 158,

(8) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 181, 144 and 158,

(9) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 182, 144 and 158,

(10) the antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 183, 144 and 158,

(11) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 184, 144 and 158,

(12) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 185, 144 and 158,

(13) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 186, 144 I,

(14) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 187, 144 and 158, and

(15) an antibody comprising the variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 188, 144 and 158.

Among the antibodies described in paragraphs (1)to(15), preferably the antibody described in the item (15).

(VIII) the Antibody described in any of the following items(1)-(15):

(1) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 174, 144 and 158,

(2) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 175, 144 and 158,

(3) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid placenta is successive, described in SEQ ID NO: 176, 144 and 158,

(4) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 177, 144 and 158,

(5) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 178, 144 and 158,

(6) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 179, 144 and 158,

(7) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 180, 144 and 158,

(8) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid serial is a major, described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 181, 144 and 158,

(9) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 182, 144 and 158,

(10) the antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 183, 144 and 158,

(11) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 184, 144 and 158,

(12) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 185, 144 is 158,

(13) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 186, 144 and 158,

(14) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 187, 144 and 158, and

(15) an antibody comprising the variable sites of the heavy chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 123, 124 and 125, and variable sections of the light chain contains the CDR 1, 2 and 3 having the amino acid sequence described in SEQ ID NO: 188, 144 and 158.

Among the antibodies described in paragraphs (1)to(15), preferably the antibody described in the item (15).

(IX) the Antibody described in any of the following items(1)-(15):

(1) an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 191,

(2) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 192,

(3) the antibody containing the e variable area light chain, having the amino acid sequence described in SEQ ID NO: 193,

(4) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 194,

(5) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 195,

(6) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 196,

(7) an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 197,

(8) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 198,

(9) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 199,

(10) the antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 200,

(11) an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 201,

(12) an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 202,

(13) an antibody containing the variable area light chain having the amino acid placenta is the sequence, described in SEQ ID NO: 203,

(14) an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 204, and

(15) an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 205.

Among the antibodies described in paragraphs (1)to(15), preferably the antibody described in the item (15).

(X) an Antibody containing the variable area light chain selected from the group consisting of the variable regions of light chains described in the following paragraphs(1)-(15):

(1) variable area light chain having the amino acid sequence described in SEQ ID NO: 191,

(2) variable area light chain having the amino acid sequence described in SEQ ID NO: 192,

(3) variable area light chain having the amino acid sequence described in SEQ ID NO: 193,

(4) variable area light chain having the amino acid sequence described in SEQ ID NO: 194,

(5) variable area light chain having the amino acid sequence described in SEQ ID NO: 195,

(6) variable area light chain having the amino acid sequence described in SEQ ID NO: 196,

(7) variable area light chain having the amino acid sequence described in SEQ ID NO: 197,

(8) variable is castac light chain, having the amino acid sequence described in SEQ ID NO: 198,

(9) variable area light chain having the amino acid sequence described in SEQ ID NO: 199,

(10) variable area light chain having the amino acid sequence described in SEQ ID NO: 200,

(11) the variable area light chain having the amino acid sequence described in SEQ ID NO: 201,

(12) variable area light chain having the amino acid sequence described in SEQ ID NO: 202,

(13) the variable area light chain having the amino acid sequence described in SEQ ID NO: 203,

(14) variable area light chain having the amino acid sequence described in SEQ ID NO: 204, and

(15) variable area light chain having the amino acid sequence described in SEQ ID NO: 205 and variable plot heavy chain selected from the group consisting of the variable regions of the heavy chain described in the following paragraphs(1)-(7):

(1) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 84,

(2) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 85,

(3) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 86,

(4) variable participants of the heavy chain, having the amino acid sequence described in SEQ ID NO: 87,

(5) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 88,

(6) variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 89, and

Among the above-described antibody is preferably an antibody containing the variable area light chain having the amino acid sequence described in SEQ ID NO: 205 and variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90.

(XI) an Antibody having described in any of the above paragraphs (I)to(X) the amino acid sequence in which one or more amino acids substituted, deleted, added and/or inserted, and which has activity equivalent to the activity of the antibody described in any of paragraphs (I)to(X).

In the present invention, the phrase "activity equivalent to the activity of the antibody described in any of paragraphs (I)to(X)" means the equivalence of the binding activity of antibodies against human glypican 3, or cytotoxic activity against a cell expressing human glypican 3 (for example, HepG2 or recombinant CHO cells expressing human glypican 3, and others).

Humanitariannet antibody

One is repectfully embodiment of antibodies in accordance with the present invention is humanitariannet antibody which binds to glypican 3. Humanitariannet antibody can be obtained in a known manner.

Humanitariannet antibody is also referred to as modified human antibody that is produced by perezhivanija plot, complementarity determining (CDR) of an antibody of a mammal other than human, such as mouse antibodies, the CDR of a human antibody. To obtain such antibodies are also common methods of recombinant DNA (see European patent application EP 125023 and international patent application WO 96/02576).

For example, if a CDR derived from a murine antibody, a DNA sequence, which is designed to connect the CDR of a mouse antibody with the skeleton plot (FR) of a human antibody is synthesized by PCR using several oligonucleotides as primers, which receive so that the slices overlap each other at both ends of the CDR and FR (see the method described in international patent application WO 98/13388).

Choose wireframe plot of human antibodies, which in conjunction with the CDR allows you to get the hypervariable area with desirable antigennegative center. If necessary, the amino acid in wireframe plot of the variable segment antibodies can be replaced so that the hypervariable area modified the human is th antibodies could form a suitable antigennegative center (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

C-part of the human antibodies can be used as a C-section chimeric antibodies or gumanitarnogo antibodies, for example, Cγ1, Cγ2, Cγ3, and Cγ4 can be used as constituent parts of the H-chain, and Cκ and Cλ can be used as constituent parts of the L-chain. C-part of the human antibodies can also be modified to improve the stability of the antibody or to facilitate its reception. For humanization, you can use any isotype human antibodies, such as IgG, IgM, IgA, IgE and IgD, preferably IgG, more preferably IgG1 or IgG3, and particularly preferably IgG1. In the present invention IgG1 now, if the antibody is used as an anticancer tool, i.e. has a high cytotoxic activity (Chemical immunology, 65: 88 (1997)).

In addition, after receiving gumanitarnogo antibody amino acid in the variable (for example, in FR) or constant area can be replaced by a different amino acid.

The origin of the CDR in humanitariannet the antibody is not particularly limited, and CDR can be obtained from any animal. For example, you can use a sequence derived from mouse antibodies, rat antibodies, rabbit antibodies, camel antibodies, or the like, it is Preferable to use the sequence of the CDRs of murine antibody.

Typically,when the humanization of antibodies, it is difficult to keep the agonistic activity of the original antibody. However, in the present invention successfully get humanitariannet antibody having agonistic activity equivalent to the activity of the parent murine antibody. Since the antigenicity gumanitarnogo antibodies in the body decreases, it can be used for administration to man for therapeutic purposes.

Preferred examples gumanitarnogo antibodies against glypican 3 of the present invention include, for example, the antibody containing the variable plot heavy chain described in SEQ ID NO: 84 (GC33 VH ver.a), SEQ ID NO: 85 (GC33 VH ver.c), SEQ ID NO: 86 (GC33 VH ver.f), SEQ ID NO: 87 (GC33 VH ver.h), SEQ ID NO: 88 (GC33 VH ver.i), SEQ ID NO: 89 (GC33 VH ver.j) or SEQ ID NO: 90 (GC33 VH ver.k), or antibody containing the variable area light chain described in SEQ ID NO: 92 (GC33 VL ver.a). Particularly preferred examples of antibodies include antibody containing the variable plot heavy chain described in SEQ ID NO: 84 (GC33 VH ver.a), SEQ ID NO: 85 (GC33 VH ver.c), SEQ ID NO: 86 (GC33 VH ver.f), SEQ ID NO: 87 (GC33 VH ver.h), SEQ ID NO: 88 (GC33 VH ver.i), SEQ ID NO: 89 (GC33 VH ver.j) or SEQ ID NO: 90 (GC33 VH ver.k), and variable area light chain, described in SEQ ID NO: 92 (GC33 VL ver.a).

In addition, the preferred example gumanitarnogo antibodies against glypican 3 includes the antibody containing the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 90, and variable area light C is PI, having the amino acid sequence described in SEQ ID NO: 205.

The preferred embodiment of the antibodies of the present invention is an antibody which binds to the epitope which binds an antibody described in any of the following items(1)-(8):

(1) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 62, and variable area light chain having the amino acid sequence described in SEQ ID NO: 73 (GC33),

(2) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 26, and variable area light chain having the amino acid sequence described in SEQ ID NO: 48 (M11F1),

(3) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 25, and variable area light chain having the amino acid sequence described in SEQ ID NO: 47 (M3B8),

(4) the antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 60, and variable area light chain having the amino acid sequence described in SEQ ID NO: 71 (GC199),

(5) an antibody comprising the variable plot heavy chain, it is Mering amino acid sequence, described in SEQ ID NO: 61, and variable area light chain having the amino acid sequence described in SEQ ID NO: 72 (GC202),

(6) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 63, and variable area light chain having the amino acid sequence described in SEQ ID NO: 74 (GC179),

(7) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 64, and variable area light chain having the amino acid sequence described in SEQ ID NO: 75 (GC194 (1)), and

(8) an antibody comprising the variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 64, and variable area light chain having the amino acid sequence described in SEQ ID NO: 76 (GC194 (2)). More preferably the antibody binding to the epitope, which binds the antibody described in any of paragraphs (1)to(5), and particularly preferably the antibody binding to the epitope, which binds the antibody described in paragraph (1).

Use the antibody that binds to an epitope associated with any of the foregoing antibodies, because it has a particularly high cytotoxicity.

The antibody described in any of paragraphs (1) (), associated with the section from 524-th amino acid of up to 580-th amino acid of the human glypican 3. In particular, it is associated with the section from 524-th amino acid of up to 563-th amino acid. The antibody described in any of paragraphs (1)to(5), associated with the section from 537-th amino acid of up to 563-th amino acid of the human glypican 3. The antibody described in paragraph (1), associated with the section from 544-th amino acids to 553-th amino acid of the human glypican 3. In particular, it is associated with the section from 546-th amino acid of up to 551-th amino acid.

Antibodies that recognize the above epitopes, have high cytotoxicity, therefore, they can be used for the treatment of diseases such as cancer. In particular, it is possible to use an antibody which binds to the site from 546-th amino acid of up to 551-th amino acid, because it has a high cytotoxicity.

Accordingly, the present invention includes antibodies that bind to an epitope in the area from 524-th amino acid of up to 580-th amino acid of the human glypican 3, preferably in the area from 524-th amino acid of up to 563-th amino acids, more preferably in the area from 537-th amino acid of up to 563-th amino acids, even more preferably in the area from 544-th amino acids to 553-th amino acid, particularly preferably in the area from 546-th amino acid of up to 551 th AMI is ocelote.

Another preferred embodiment of the present invention is an antibody that recognizes a site from 524-th amino acid of up to 563-th amino acid of the human glypican 3 and does not recognize the plot from 537-th amino acid of up to 563-th amino acid.

Another preferred embodiment of the present invention is an antibody that recognizes a site from 537-th amino acid of up to 563-th amino acid of the human glypican 3 and does not recognize the plot from the 550th amino acids to 563-th amino acid.

Analysis of the epitope recognized by the antibody can be known in this field by the way, for example by the method of Western blotting, which is described below in the examples.

The antibody that recognizes the above areas as epitopes, you can get known in this field by the way. For example, it can be obtained by obtaining a peptide having the amino acid sequence of the target area based on the amino acid sequence of human glypican 3, followed by obtaining antibodies with the use of this peptide as an immunogen, or by obtaining antibodies in the usual way and determine the epitope, which recognizes the obtained antibody, followed by selection of antibodies that recognize the target epitope.

A preferred example antibodies p is otiv glypican 3 is an antibody, having high ADCC activity, or an antibody having a high CDC activity against a cell expressing glypican 3.

The phrase "high ADCC activity" or "high CDC activity" in this description means that the antibody of the present invention has higher ADCC activity or higher CDC activity than the known antibody against glypican 3. Known antibodies against glypican 3 include, for example, M3C11 and M1E07 described in international patent application WO 2004/22739.

ADCC activity or CDC activity can be measured using known in this area of the method. For example, it can be measured by chromium release. Specific conditions analysis of the release of chromium for the measurement of ADCC activity is not particularly limited, but, for example, it can be measured using the conditions described below in the examples.

Examples of cells expressing glypican 3 include, for example, the cell line hepatoma, such as HepG2 cell line CHO containing the gene encoding glypican 3, etc. For measuring the ADCC activity is preferable to use the cell line HepG2, and for measuring CDC activity is preferable to use a recombinant cell line CHO expressing GPC3. Recombinant cell line CHO expressing GPC3, can be obtained by any method, including its possible to obtain, for example, according to the method described below in the examples.

If the antibody against glypican 3 are used as anticancer means, it is preferable that it had the same level of ADCC activity as the antibody comprising variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 62, and variable area light chain having the amino acid sequence described in SEQ ID NO: 73 (GC33). If the antibody against glypican 3 are used as anticancer means, it is preferable that it had the same level of CDC activity as the antibody comprising variable plot heavy chain having the amino acid sequence described in SEQ ID NO: 62, and variable area light chain having the amino acid sequence described in SEQ ID NO: 73 (GC33).

In addition, the present invention includes an antibody having high binding activity with respect to glypican 3.

In the present invention, the binding activity of antibodies against glypican 3 can be measured is known in this field by the way. For example, it can be measured by means of surface plasmon resonance using BIAcore. Namely protein glypican 3 immobilized on the sensor chip for interaction with the antibody, and the interaction of antibodies is glypican 3 count as the reaction rate constant based on the measurement results. In addition, to measure the binding activity can be used enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay analysis (RIA) or immunofluorescent analysis. For example, in the case of enzyme immunoassay, the sample containing the analyzed antibody, such as culture supernatant of cells producing analyzed the antibody or the purified antibody is added to the tablet, pre-coated with antigen, associated with the analyzed antibody. Then add the secondary antibody labeled with an enzyme such as alkaline phosphatase, after which the tablets are incubated and washed. Then add the enzyme's substrate, such as p-nitrophenylphosphate, and measure the absorption, by which I hope antigennegative activity. The upper bound binding activity is not particularly limited. However, for example, the upper boundary may be in the range, which is determined by the technical capabilities from the point of view of a specialist in this field. It should be understood that as technology improved technically possible interval will be expanded.

Further, in the present invention the amino acid to be dezaminirovanie or neighboring amino acid may be replaced by another amino acid, for example, to suppress desametasone with C is poured increase the stability of the antibody. Be dezaminirovanie amino acids include asparagine and glutamine, preferably asparagine. The amino acid located adjacent to asparagine, not particularly limited and may be any amino acid. It is known that the sequence asparagine-glycine is particularly sensitive to dezaminirovanie therefore, preferably, adjacent to asparagine was glycine. The amino acid used for substitution are not particularly limited and may be any amino acid other than asparagine, and glutamine. Preferably, this differs from the amino acid valine and Proline. Therefore, in the present invention, if there is desametasone antibodies, it is preferable to substitute amino acid at amino acid other than asparagine, glutamine, valine and Proline. Suppression of desametasone by replacing amino acids can be, for example, by the method described in international patent application WO 03/057881. Preferably, after the replacement of amino acids to suppress desametasone remained antigennegative activity that was present before the replacement.

Another embodiment of the stabilization of antibodies includes replacement of glutamic acid with another amino acid. The present invention also found that, if 6-I Amin the acid heavy chain antibodies represents a glutamic acid, the antibody can largely be stabilized by substitution of glutamic acid for glutamine. Accordingly, the present invention also relates to a method of stabilizing antibody by replacing the glutamic acid in the 6th position of the heavy chain of the antibody on glutamine. The numbering of the amino acids in the antibody known to specialists in this field (e.g., Kabat, E. A. et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).

The antibody of the present invention may be a conjugated antibody, i.e. an antibody can be connected with different molecules, such as molecules of polyethylene glycol (PEG), radioactive substances and toxins. Such conjugated antibody can be obtained by chemical modification of previously obtained antibodies. Methods of modification of the antibodies known in the field. The antibody of the present invention includes such conjugated antibody.

The antibody of the present invention may also provide bespecifically antibody (see, e.g., Journal of Immunology, 1994, 152, 5368-5374). Bespecifically antibody can recognize glypican 3 and another antigen, or it may recognize different epitopes on the molecule GPC3.

In addition, the antibody of the present invention can bear a certain protein, heriditary with N - or C-end of antibodies (Clinical Cancer Research, 2004, 10, 1274-1281). Protein for gibr is ditali with the antibody can be suitably selected by a person skilled in the field.

The antibody of the present invention also includes an antibody with increased cytotoxicity. Examples of antibodies with increased cytotoxicity include antibody, lost fucose, antibody containing separating N-acetylglucosamine (GlcNAc)attached to its carbohydrate chain, and the antibody with altered binding activity against the Fcγ receptor, obtained by substitution of one or several amino acids in the plot Fc. Such antibodies with increased cytotoxicity can get well-known in this field by the way.

A method of obtaining antibodies

Antibody that binds to glypican 3 can be known in this field by the way. For example, hybridoma producing monoclonal antibody can be obtained as follows, using mostly known method. That is, hybridoma can be obtained by immunization of a mammal in the traditional way, using as a sensitizing antigen protein glypican 3 or cells expressing glypican 3. Thus obtained immunocyt hybridized with a known parent cell using the conventional method of hybridization of cells, and then using traditional screening method for selecting cells producing monoclonal antibodies.

Specifically, a monoclonal antibody can get the ü in the following way. First get protein glypican 3 based on gene/amino acid sequence glypican 3 described in SEQ ID NO: 3 and 4, which is used as a sensitizing antigen for obtaining antibodies. More specifically, a gene sequence encoding glypican 3, is inserted in a known vector expression system, the received vector, transforming a suitable cell host and then target human protein glypican 3 separated from the host cell or from the culture supernatant by a known method.

Then this purified protein glypican 3 is used as a sensitizing antigen. Alternative as a sensitizing antigen can be used peptide fragment glypican 3. In this case, the peptide fragment can also be obtained by chemical synthesis in accordance with the amino acid sequence of human glypican 3.

Epitope molecules glypican 3, which is recognized by the antibody against glypican 3 of the present invention is not limited to a specific epitope. Antibody against glypican 3 can recognize any epitope, if the epitope is present on the molecule glypican 3. Accordingly, to obtain antibodies against glypican 3 of the present invention as an antigen can be any fragment that contains the epitope PR is sustuli on the molecule glypican 3.

A mammal that are subjected to immunization sensitizing antigen is not particularly limited, but preferably is chosen from the viewpoint of compatibility with the parent cell used for hybridization of cells. For example, typically use rodents, such as mice, rats and hamsters, rabbits or monkeys.

Immunization of animals sensitizing antigen is carried out in a known manner. For example, immunization is carried out on the General way in which the sensitizing antigen is administered to the mammal intraperitoneally or subcutaneously. Namely, the sensitizing antigen is diluted with a suitable quantity or suspended in an appropriate amount of PBS (phosphate buffered saline), physiological saline and the like, if necessary, the product is mixed with an appropriate amount of standard adjuvant, such as complete beta-blockers, and then the solution emuleret and administered to the mammal multiple times over every 4-21 days. For immunization sensitizing antigen is also possible to use suitable media.

Mammal subjected to immunization by the above method and then confirm that the serum is elevated levels of the target antibodies. Then the mammal take immunocytes and expose their cell hybridization. Especially predpochtitelnei is splenocyte.

As the parent cell partner for hybridization with the above immunocytes use cell myeloma mammal. Examples of cell lines, myeloma, preferably used in the present invention include various known cell lines such as P3 (P3x63 Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63 Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D. H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323) and R210 (Galfre, G. et al., Nature (1979) 277, 131-133).

Hybridization of the above-mentioned immune cells with myeloma cells mainly can be performed using a known method such as a method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

More specifically, the above-mentioned cell hybridization is carried out in conventional nutrient solution for culturing in the presence of, for example, substances that accelerate the hybridization of cells. As a substance that accelerates the hybridization of cells, using, for example, polyethylene glycol (PEG), hemagglutinine Japanese virus (HVJ). If desired to further increase the efficiency of hybridization is possible to add an adjuvant such as dimethyl sulfoxide.

You can choose a suitable value of immunocytes and myeloma cells. For example, PR is doctitle, to the number of immunocytes was in 1-10 times higher than the number of myeloma cells. The culture solution used for the above-mentioned hybridization of cells includes, for example, a culture solution RPMI1640 or cultural MEM solution, suitable for the cultivation of the above myeloma cell lines, or other conventional culture solution used for this type of cell culture. In addition, in combination with this solution you can use a serum Supplement such as fetal calf serum (FCS).

Hybridization of cells is carried out as follows. A predetermined number of the above immunocytes and myeloma cells are thoroughly mixed in the above-mentioned culture solution, add heated to approximately 37°C a solution of PEG (e.g., with an average molecular weight from about 1000 to 6000) (total concentration from 30% to 60% (wt./vol.), then the solution is stirred and get the target hybrid cell (hybridoma). After that add suitable culture solution and repeatedly carry out the stage of removal of the supernatant by centrifugation to remove the reagent for hybridization of cells or other reagent that adversely affect the growth of hybridoma.

Thus obtained hybridoma selected by culturing hybridoma standard CE is Acciona culture solution, such as a culture solution HAT culture solution containing gipoksantin, aminopterin and thymidine). Cultivation in the above-mentioned culture solution HAT continue for a time period sufficient for the cells other than the cells of the target hybridoma (dehybridization cells), died (usually from several days to several weeks). Then using the standard method of limiting dilutions select a monoclone of hybridoma, which produces the target antibody.

In addition to the method of immunizing non-human animal with an antigen with the purpose of obtaining hybridoma target human antibody having binding activity against glypican 3, can also be obtained by sensitizing human lymphocytes with glypican 3 in vitro and hybridization of sensitized lymphocytes with a cell myeloma man capable of continuous division (see JP-B-1-59878). Another way to get the cells producing antibody against glypican 3, the antigen glypican 3 enter a transgenic animal containing the entire set of genes of a human antibody, then the received cells immortalized, and human antibody against glypican 3 is obtained from immortalized cells producing antibody against glypican 3 (see international patent application WO 94/25585, WO 93/12227, WO 92/03918 the WO 94/02602).

Thus obtained hybridoma, which produces monoclonal antibody, it is possible to cultivate passages in standard culture solution or it can be stored for long time in liquid nitrogen.

One example of the method used to obtain monoclonal antibodies from hybridoma, is the cultivation of hybridoma and obtaining the monoclonal antibody from the culture supernatant using the standard method. Another method involves the introduction of hybridoma a mammal compatible with hybridomas to ensure its proliferation, and obtaining monoclonal antibodies from ascites. The first method is suitable for obtaining antibodies of high purity, while the latter method is suitable for producing large amounts of antibodies.

You can also obtain recombinant antibody using the method of genetic engineering by cloning the gene of the antibody from hybridoma, introduction of the gene into a suitable vector, introducing the vector into the host with the subsequent production of the master of recombinant antibodies (e.g., see Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).

Specifically, mRNA encoding the variable (V) plot antibodies against glypican 3, separated from hybridoma producing antibody against glypican 3. mRNA allocate a known method, such as method ultrace is trippynova with guanidine (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) or the AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), and get the total RNA, and then the target mRNA obtained using the dial to highlight mRNA (Pharmacia) or the like, in Addition, mRNA can be directly obtained by using the dial to highlight the QuickPrep mRNA (Pharmacia).

cDNA V-phase antibodies are synthesized using reverse transcriptase and obtained as described above mRNA. cDNA can be synthesized using a kit for synthesis of single-stranded cDNA with AMV reverse transcriptase (SEIKAGAKU CORPORATION) or the like For synthesis and amplification of cDNA can also be used, for example, a set of 5'-Ampli FINDER RACE (Clontech), the method of 5'-RACE using PCR (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932).

The target DNA fragment is recovered from the obtained PCR product and then are ligated with a DNA vector. The recombinant vector is obtained from the product, then the vector is introduced into E. coli or the like and carry out selection of colonies, resulting in the target recombinant vector. Then determine the nucleotide sequence of the target DNA using a known method such as dideoxynucleotide method of chain termination.

After obtaining the DNA encoding the V-phase target antibodies against glypican 3, it is inserted into the expression vector containing DNA encoding the constant segment (C-section) of the target antibody.

Gene antibodies can be Express by separately introducing the expression vector of polynucleotide encoding the H-chain, or polynucleotide encoding the L-chain, followed by the simultaneous transformation of the host cell vectors, or by introducing into one expression vector of polynucleotides encoding the H-chain and L-chain, followed by transformation of the host cell vector (see international patent application WO 94/11523).

Polynucleotide

In another aspect the present invention provides polynucleotide encoding variable plot heavy chain or variable area light chain of the antibody of the present invention. Preferably polynucleotide the present invention has a nucleotide sequence described in any of SEQ ID nos: 11-21, 33-43, 55-59, 65-70 and 77-83. The present invention also encompasses polynucleotide that hybridizes with the above polynucleotides in stringent conditions and encodes an antibody having an activity equivalent to the activity of ant the body of the present invention.

Polynucleotide of the present invention is not particularly limited provided that it encodes the antibody of the present invention. It is a polymer composed of nucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It may contain a basis different from occurring. Polynucleotide of the present invention can be used to obtain antibodies using the method of genetic engineering. In addition, polynucleotide of the present invention can be used as a probe for screening antibodies that are functionally equivalent to the antibody of the present invention. That is, polynucleotide encoding the antibody of the present invention, or fragment, can be used as a probe to retrieve DNA, which hybridizes with this polynucleotides in stringent conditions and encodes an antibody having an activity equivalent to the activity of the antibodies of the present invention, using methods such as a method of hybridization, a gene amplification method (e.g., PCR). This DNA is also included in polynucleotide of the present invention.

Methods of hybridization (Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) are well known to specialists in this field. Examples of hybridization conditions include, for example, conditions of low jesd the STI. To conditions of low stringency include, for example, the following conditions: 42°C in 0.1×SSC and 0.1% SDS, preferably, 50°C, in 0.1×SSC and 0.1% SDS, if the washing is carried out after hybridization. More preferred examples of hybridization conditions include, for example, conditions of high stringency. To conditions of high stringency include, for example, the following conditions: 65°C, 5×SSC and 0.1% SDS. Under these conditions it can be expected that higher temperatures will be effective formation of polynucleotide with a higher degree of homology. Besides, there are several factors that affect the accuracy of hybridization, such as temperature and salt concentration, and by the proper selection of these factors specialists in this area can achieve similar accuracy.

The antibody is functionally equivalent to the antibody of the present invention encoded by polynucleotides obtained in this hybridization method and the method of gene amplification, has a high degree of homology with the antibody terms of amino acid sequence. The antibody of the present invention also includes an antibody functionally equivalent to the antibody of the present invention, and has a high degree of homology in relation to the amino acid sequence of the antibody. A high degree of homology means, ka is the rule, at least 50% or higher identity, preferably 75% or greater identity, more preferably 85% or higher identity, and even more preferably 95% or higher identity on amino acid level. To determine the homology of polypeptides, you can use the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad. Sci. USA (1983) 80, 726-730).

The present invention also provides a vector containing polynucleotide of the present invention. This vector can be used to obtain antibodies of the present invention. If the owner uses E. coli, the vector of the present invention, for example, are not particularly limited as long as it has "ori" for amplification in E. coli with the aim of obtaining and amplification of the vector in a large amount in E. coli (e.g., JM109, DH5α, HB101 or XLlBlue), and contains a marker gene for selecting transformed E. coli (e.g., the gene of resistance to the drug, which can be identified using drugs, such as ampicillin, tetracycline, kanamycin, or chloramphenicol). Examples of vectors include vectors series M13 vectors series pUC, pBR322, pBluescript, pCR-Script, etc. in Addition to sublimirovanny and extract the cDNA along with the above-described vectors can be used pGEM-T, pDIRECT, and pT7.

As a vector infusion is his invention, it is preferable to use the expression vector. For example, the expression vector designed for use in E. coli, should have the characteristics specified for amplification in E. coli. In addition, if the host cell using E. coli, such as JM109, DH5α, HB101, or XL1-Blue, the vector should have a promoter, for example, lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), T7 promoter or the like, which provides an effective expression of the target product in E. coli. Examples of such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen), pEGFP, and pET (in this case, the host is preferably BL21, which expresses RNA polymerase T7), etc. as well as the vectors described above.

The vector may also contain a signal sequence for secretion of the polypeptide. If the polypeptide is produced in periplasmic E. coli, as a signal sequence for secretion of the polypeptide can be used signal pelB sequence (Lei, S. P. et al., J. Bacteriol (1987) 169, 4379). Introduction of the vector into the cell host can be performed, for example, using calcium chloride and the electroporation method.

As the vector of the present invention, in addition to E. coli, can be used, for example, expression vectors derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res. (1990) 18(17), p5322), pEF, and pCDM8), expression vectors, is received from insect cells (for example, "the baculovirus expression system Bac-to-BAC" (GIBCO BRL) and pBacPAK8), expression vectors derived from plants (for example, pMH1 and pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, and pAdexLcw), expression vectors derived from retroviruses (e.g., pZIPneo), expression vectors derived from yeast (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and expression vectors derived from Bacillus subtilis (for example, pPL608 and pKTH50).

For expression in animal cell such as a CHO cell, a COS cell, a cell NIH3T3, etc., the vector should have a promoter necessary for expression in the cell, for example, SV40 promoter (Mulligan et al., Nature (1979) 277, 108), the promoter MMTV-LTR, the promoter EFlα (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter or the like, and, more preferably, it should contain a marker gene (such as the gene of resistance to the drug, which can be identified using drugs, such as neomycin or G418), helping to select transformed cells. Examples of vectors having such characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Further, to provide a stable gene expression and simultaneously increase the number of copies of the gene in the cell, a CHO cell with a deficit in the way of synthesis of nucleic acids enter the vector (for example, pCHOI and others), containing the gene for DHFR, to fill the deficit, and Ampl liziruut with methotrexate (MTX). To ensure temporal gene expression, transformation, make use of a vector (such as pcD)containing the site of initiation of replication of SV40, and using COS cells containing a gene on chromosome ensuring the expression of the antigen of SV40 t as the site of initiation of replication, you can use the site of initiation of virus polyoma, adenovirus, virus, bovine papillomavirus (BPV), etc. in Order to increase the number of copies of the gene in the system of the host cell, the expression vector may include, as a marker for selection, gene aminoglycosides (APH), gene timedancing (TK), gene contingenciesliabilities E. coli (Ecogpt)gene digidrofolatreduktazy (dhfr), etc.

To obtain the antibody of the present invention, the vector is introduced into a cell of the host. A host cell, into which is injected the vector is not particularly limited, but it includes, for example, E. coli or any animal cell. For example, the cell-master can be used as a production system for the production or expression of the antibodies of the present invention. As for the system of production used to obtain the polypeptide, there is a system of production in vitro and the system of production in vivo. In the system of production used in vitro eukaryotic and prokaryotic cells.

As eukariotic the ski cells can be used, for example, animal cells, plant cells, or fungal cells. Known animal cells include mammalian cells such as CHO cells (J. Exp. Med. (1995) 108, 945), COS cells, 3T3 cells, myeloma cells, cells, baby hamster kidney (BHK), HeLa cells and Vero cells, cells of amphibians such as Xenopus oocytes (Valle, et al., Nature (1981) 291, 358-340), or insect cells such as Sf9, Sf21, and Tn5. In the present invention preferably uses cells CHO-DG44, CHO-DXB11, COS7, BHK. To enable large-scale expression of animal cells, preferably CHO cells. Introduction of the vector into the cell host can be performed, for example, using calcium phosphate, DEAE-dextran, cationic ribosomes DOTAP (Boehringer Mannheim), electroporation method, a method of lipofectin or similar

From plant cells as a system for producing proteins using, for example, cells of Nicotiana tabacum, which can be subjected calluses cultivation. Examples of fungal cells include yeast cells, for example, belonging to the genus Saccharomyces, more specifically Saccharomyces cerevistae and Saccharomyces pombe, and hyphomycetes, for example, belonging to the genus Aspergillus, more specifically Aspergillus niger.

In the case of prokaryotic cells, you can use the system of production using bacterial cells. Examples of bacterial cells include Escherichiacoli (E. coli, such as JM109, DH5α and HB101, and Bacillus subtilis.

The preparation of recombinant antibodies

The antibody of the present invention can be obtained by culturing the above-mentioned host cells. The antibody can be obtained by in vitro culturing cells transformed target polynucleotide. The cultivation can be performed using a known method. The culture medium for animal cells include, for example, DMEM, MEM, RPMI 1640 and IMDM. The medium may be supplemented with serum, such as FBS or fetal calf serum (FCS), or you can use the medium does not contain serum. the pH of the cultivation is preferably in the range from 6 to 8. The cultivation is usually carried out at about 30-40°C for approximately 15-200 hours, if necessary, with the change of environment, with aeration and shaking.

On the other hand, the system for obtaining a polypeptide in vivo include, for example, the production system, which uses the animal, and the system of production, which uses the plant. Target polynucleotide injected into the animal or plant, and in the body of an animal or a plant polypeptide is produced, which is then removed. The term "a host cell" in this description covers such an animal and a plant.

In the case of animal available missile defense system is ucirvine using mammalian or insect. As mammals you can use goats, pigs, sheep, mice and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). You can also use transgenic mammal.

For example, the target polynucleotide receive in the form of a hybrid gene with the gene coding for the polypeptide, which in nature is produced in milk, such as goat β casein. Then the DNA fragment containing this hybrid gene is introduced into the embryo goats and embryo transplanted goat. The target antibody can be obtained from the milk of transgenic goats born goat who received the embryo or its descendant. To increase the quantity of produced milk containing antibody transgenic goat if necessary, you can give hormone (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

As an insect, for example, you can use the silkworm. In the case of silkworm, its infecting baculovirus, which is embedded in polynucleotide encoding the target antibody. The target antibody can be obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-594).

As plants can be used, for example, tobacco. When using tobacco polynucleotide encoding the target antibody is inserted into an expression vector for plants, for example, pMON 530, and then the vector is introduced into a bacterium such as Agrobacterium tumefaciens. Then tobacco, this is AK Nicotiana tabacum, infect bacteria and as a result of the tobacco leaf and get targeted antibody (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

Thus obtained antibody can be distinguished from internal or external (cultural medium and the like) environment of the host cell and then it can be clear to almost pure state and homogeneous antibodies. Isolation and purification of antibodies can be performed using the method of separation and purification, which is usually used for the purification of polypeptides. For example, the polypeptides can be extracted and cleaned using any techniques, including column chromatography, filter, ultrafiltration, salting out, precipitation with a solvent, solvent extraction, distillation, immunoprecipitation, electrophoresis on SDS-polyacrylamide gel, gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and their combination.

Examples of chromatographic methods include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, chromatography on reversed phase adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographic methods can be performed using liquid chromatography, such as HPLC and FPLC (fast protein liquid chromatography - liquid ek is press-chromatography of proteins). Examples of columns used for affinity chromatography include a column of protein A or column with protein G. the Example column with protein A is Hyper D, POROS, Sepharose F. F. (Pharmacia).

In addition, before or after cleaning, as necessary, the antibody can be modified, or can be removed partially peptides by use of a suitable enzyme that modifies proteins. Used for this purpose, the enzyme that modifies proteins include, for example, trypsin, chymotrypsin, lisianthius, a protein kinase, a glucosidase.

Diagnostic method

In another aspect the present invention provides a method of diagnosing diseases, such as cancer, by detecting GPC3 protein in the test sample using the antibody of the present invention.

Used in this description, the detection includes quantitative detection and qualitative detection. Non-quantitative detection includes, for example, simply determining the presence of GPC3 protein, the presence of a specified number or more GPC3 protein, comparing the number of GPC3 protein with its number in another sample (e.g., control sample). Quantitative detection includes determining the concentration of protein GPC3, the determination of the number of GPC3 protein.

The analyzed sample is not particularly limited, while it performance, is made an example of, which may contain GPC3 protein, but preferably it is a sample taken from a living organism such as a mammal, and more preferably it represents a sample taken from the man. Specific examples of the analyzed sample can include, for example, blood, interstitial fluid, plasma, extravascular fluid, cerebral fluid, synovial fluid, pleural fluid, serum, lymphatic fluid, saliva, preferably blood, serum and plasma. In addition, the term "analyzed the sample of the present invention also includes a sample obtained from the analyzed sample, such as a culture solution of cells collected from a living organism.

View the diagnosed cancer is not particularly limited, and specific examples may include liver cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, leukemia and lymphoma, preferably liver cancer. Detected GPC3 not particularly limited and may be either full-GPC3 or its fragment. If it detects a fragment of GPC3, it can be either N-terminal fragment or the C-terminal fragment, however, the N-terminal fragment is preferred. In addition, GPC3 protein can be a GPC3-added Gepar the sulfate or the core GPC3 protein.

Method of detecting GPC3 protein contained in the analyzed sample is not particularly limited, but the detection is preferably carried out immunological method using an antibody against GPC3. Examples of immunological methods include, for example, radioimmunoassay analysis, enzyme-linked immunosorbent assay, immunofluorescence assay, immunofluorescently analysis, immunoprecipitation, turbidimetric immunological analysis. It is preferable to use enzyme-linked immunosorbent assay, particularly preferably, the enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA method). The above immunological assay, such as ELISA, can be known in this field method.

A General method of detection using antibody against GPC3 includes immobilization of the antibody against GPC3 on the substrate, adding the analyzed sample, incubation of the substrate in order to ensure the binding of an antibody against GPC3 protein GPC3, rinsing the substrate and the detection of GPC3 binding protein with the substrate by antibody against GPC3 in the analyzed sample.

The binding of an antibody against GPC3 protein GPC3 usually occurs in the buffer. The buffers used in this invention include, for example, phosphate buffer, Tris-buffer. Incubation is carried out in normal conditions, for example, at temperatures from 4°C to the room, within 1-24 hours. Washing after incubation can be performed by any method that is not accompanied by inhibition of binding protein with GPC3 antibody against GPC3, using, for example, a buffer containing a surfactant, such as Tween 20.

In the method of detecting GPC3 protein of the present invention in addition to the sample analyzed in GPC3 protein may control sample. Control samples include samples of the negative control, which contains no GPC3 protein, and samples of positive control, which contains GPC3 protein. In this case, the GPC3 protein in the analyzed sample can be detected by comparing the results obtained using a sample negative control that contains no GPC3 protein, with the results obtained using a sample of the positive control, which contains GPC3 protein. GPC3 protein contained in the analyzed sample can be quantitatively determined by the results of the detection, control samples, and analyzed sample in the form of numeric values and comparing the obtained numerical values.

The preferred embodiment of the detection of GPC3 binding protein with the substrate by antibody against GPC3 is a method that uses an antibody against GPC3 labeled detectable label. For example, the trees GPC3 can be detected by bringing into contact of the analyzed sample with the antibody against GPC3, immobilized on the substrate, rinsing the substrate and then detecting GPC3 using a labeled antibody, which specifically binds with the protein GPC3.

Antibody against GPC3 can be marked by using a known method. Examples are known in this field detectable labels include fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance or a radioactive substance. Specific examples may include radioactive isotopes (32P,14C,125I3H,131I and the like), fluorescein, rhodamine, ancillary, umbelliferon, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, secrete lysozyme, SharedAccess, microbiocides, Biotin, etc. In the case of the use of Biotin as a detectable label, it is preferable to add labeled with Biotin antibody and then avidin conjugated with an enzyme such as alkaline phosphatase.

Specifically, a solution containing the antibody against GPC3 add to the substrate, such as a tablet, to mobilitat her antibody against GPC3. After washing the tablet block, for example, using BSA to prevent nonspecific binding of the protein. The tablet again washed and then analyzed sample is added to the tablet. After incubation the unbound washed and then add the labeled antibody against GPC3. After incubation under appropriate conditions, the tablet is washed and then detects labeled antibody against GPC3, remaining on the tablet. Protein can be used to detect known in the field method. For example, if the antibody have been labelled with a radioactive substance, the protein can be detected using a liquid scintillation analysis or method of RIA. If the antibody target of the enzyme protein can be detected by adding substrate and detection of enzymatic change of the substrate, such as the development of painting, using the reader's absorption. If the antibody have been labelled with a fluorescent substance, a protein can be detected using fluorimetry.

Especially preferred embodiment of the method of detecting GPC3 protein of the present invention is a method that uses an antibody against GPC3 labeled with Biotin and Avidya. Specifically, a solution containing the antibody against GPC3 add to the substrate, such as a tablet, to mobilitat her antibody against GPC3. After washing the tablet block, for example, using BSA to prevent nonspecific binding of the protein. The tablet again washed and then analyzed sample is added to the tablet. After incubation the unbound washed and then add the antibody against GPC3 labeled with Biotin. After incubation under appropriate conditions, the tablet p is washed and then add avidin, conjugated with an enzyme such as alkaline phosphatase or peroxidase. After incubation the unbound washed and then add the substrate of the enzyme conjugated to Avidya. Then GPC3 protein detected by enzymatic change of the substrate as indicator.

Another embodiment of the method of detecting GPC3 protein of the present invention is a method that uses a primary antibody that specifically binds to the GPC3 protein and a secondary antibody that specifically binds to the primary antibody. For example, the analyzed sample is brought into contact with an antibody against GPC3, immobilized on a substrate, the substrate is incubated, washed and bound protein GPC3 after washing detected using a primary antibody against GPC3 and secondary antibody, which specifically binds to the primary antibody. In this case, the secondary antibody is preferably mark detectable label.

Specifically, a solution containing the antibody against GPC3 add to the substrate, such as a tablet, to mobilitat her antibody against GPC3. After washing the tablet block, for example, using BSA to prevent nonspecific binding of the protein. The tablet again washed and then analyzed sample is added to the tablet. After incubation the unbound washed and then add the primary an is Italo against GPC3. After incubation under appropriate conditions, the tablet is washed and then add the secondary antibody, which specifically binds to the primary antibody. After incubation under appropriate conditions, the tablet is washed and then detects the secondary antibody remaining on the tablet. The secondary antibody can be used to detect the above-mentioned method.

The pharmaceutical composition

In another aspect the present invention provides a pharmaceutical composition comprising the antibody of the present invention. Pharmaceutical composition comprising the antibody of the present invention, used for the treatment and/or prevention of diseases associated with cell proliferation, such as cancer, and especially its use for the treatment and/or prevention of liver cancer. If the antibody of the present invention are used as pharmaceutical compositions, the antibody may be included in the dosage forms known in this field by the way. For example, the pharmaceutical composition can be entered by parenteral injection of a sterile solution or suspension in water or another pharmaceutically acceptable solution. For example, the antibody can be entered into the composition of the dosage form by mixing it in a suitable manner with a pharmaceutically acceptable carrier or solvent, such as sterile the haunted water, saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavorant, excipient, carrier, preservative, binder, to obtain a standard dosage forms, corresponding to the generally accepted standards use of drugs (Drug Implementation). The number of active ingredients in these preparations is chosen so as to ensure the introduction of a suitable dose at the specified interval.

A sterile composition for injection can be obtained using such media as distilled water for injection, in accordance with the General provisions on the use of drugs.

Examples of an aqueous solution for injection include, for example, saline solution, glucose solution and other isotonic fluid, which includes supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. They can be used in combination with a suitable solubilizer such as alcohol, especially ethanol, polyhydric alcohol such as propylene glycol and polyethylene glycol, and nonionic surfactant, such as Polysorbate 80 ™ and HCO-50.

Sesame oil or soybean oil can be used as the oily liquid in combination with benzyl benzoate or benzyl alcohol as a solubilizer. It can come in the ü in the composition together with a buffer, such as phosphate buffer or sodium acetate buffer, a painkiller, such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, or an antioxidant. The resulting solution for injection usually fill in an appropriate ampoule.

Preferably use injecting, specific examples of which include injection, transnasal insertion, transpulmonary introduction, percutaneous introduction, etc. Composition for injection can be entered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like

Depending on the patient's age and symptoms of the disease choose a suitable route of administration. For example, a single dose of a pharmaceutical composition comprising the antibody or polynucleotide encoding the antibody may be selected from a range from 0.0001 to 1000 mg per kg of body weight. Alternatively, for example, the dose can be selected from the range from 0.001 to 100000 mg/body of the patient, although it is not always limited to these numeric values. The dose and method of administration chosen by the person skilled in the art depending on the body weight and the patient's age and symptoms.

All patents and references cited herein, incorporated in it by reference. All content of revelation is passed in the description and drawings of the patent application of Japan No. 2004-203637, to which this application claims priority, are included in this description by reference.

EXAMPLES

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to these examples.

Example 1

cDNA cloning of human glypican 3 (GPC3)

Full-size cDNA encoding human GPC3, amplified by PCR using a set of Advantage 2 (CLONTECH) and single-stranded cDNA obtained in the usual method of cell lines of colon cancer, Caco2, as a matrix. More specifically, 50 μl of reaction mixture containing 2 µl cDNA, obtained from Caco2, 1 μl sense primer (GATATC-ATGGCCGGGACCGTGCGCACCGCGT: SEQ ID NO: 1), 1 μl antisense primer (GCTAGC-TCAGTGCACCAGGAAGAAGAAGCAC: SEQ ID NO: 2), 5 μl of 10× buffer for PCR Advantage 2, 8 μl of dNTP mixture (1.25 mm) and 1.0 ál of polymerase mix Advantage subjected to 35 cycles comprising 94°C for 1 min, 63°C for 30 seconds, and 68°C for 3 minutes. Amplificatory the reaction product of PCR is inserted into the TA vector, pGEM-T Easy using the pGEM-T Easy Vector System I (Promega). The sequence confirmed by DNA sequencing machine (ABI 3100. Thus, isolated cDNA encoding a full-sized human GPC3. The sequence described in SEQ ID NO: 3 corresponds to the nucleotide sequence of the gene of human GPC3, and p is the sequence, described in SEQ ID NO: 4 corresponds to the amino acid sequence of human GPC3 protein.

Example 2

Obtaining a soluble form of GPC3

Get immune protein for the formation of antibody against GPC3, a soluble form of the protein GPC, which removed hydrophobic region at the C-terminal side (amino acids 564-580).

Using as template cDNA full-sized human GPC3, carry out a PCR reaction using the antisense primer (ATA GAA TTC CAC CAT GGC CGG GAC CGT GCG C: SEQ ID NO: 5) and sense primer, to which is added, the sequence of the EcoRI recognition and a Kozak sequence (ATA GGA TCC CTT CAG CGG GGA ATG AAC GTT C: SEQ ID NO: 6). The fragment obtained by PCR reaction (1711 BP), clone in pCXND2-Flag. Design pCXND2-Flag for the expression of Flag-labeled protein by inserting a section providing for the expression of the DHFR gene pCHOI (Hirata et al., FEBS letter 1994; 356; 244-248)in the HindIII site pCXN2 (Niwa et al., Gene 1991; 108; 193-199) and add a marker sequence below section multicameraframe. The constructed expression plasmid DNA introduced into the cell line CHO DXB11 and cell line CHO, effectively expressing the soluble form of GPC3, get through selection using 500 μg/ml of geneticin. Conduct large-scale cultivation of the cell lines CHO, effectively expressing the soluble form of GPC3, uses the 1700 cm 2roller bottle, and remove the culture supernatant for the selection of antibodies. The culture supernatant is applied on a column of Fast Flow DEAE-separate (Amersham), after washing the antibody elute with buffer containing 500 mm NaCl, and subjected to his affinity purification using agarose affinity gel Anti-Flag M2 (SIGMA). Elution is carried out with the use of 200 μg/ml FLAG peptide. After concentration of the eluate using Centriprep-10 (Millipore) FLAG peptide is removed by gel-filtration using a Superdex 200 HR 10/30 (Amersham). Finally, the filtrate is concentrated and column using Fast Flow DEAE-separate, and elute PBS (containing 500 mm NaCl), not containing Tween 20, to secure a replacement buffer.

Example 3

Obtaining a soluble form of the crustal GPC3 protein

GPC3 modify heparansulfate getting macromolecule. To screening for antibody against GPC3 eliminate antibody against heparan sulfate receive a soluble form of the crustal GPC3 protein, which has a mutation in the binding site heparan sulfate, and use it at the screening.

Using the above-mentioned soluble form of GPC3 (1-563) as template, cDNA, in which the Ser residues at positions 509 495 and replaced by Ala, obtained by Assembly PCR using the primers designed to accommodate the addition of His marker to the C-end. The obtained cDNA CLO is irout in the vector pCXND3. pCXND3 design by inserting section pCHOI ensuring the expression of the DHFR gene, into the HindIII site pCXN2. The constructed expression plasmid DNA introduced into the cell line DXB11, and the cell line CHO, effectively expressing the soluble form of the crustal GPC3 protein produced in the result of selection using 500 μg/ml of geneticin.

Conduct large-scale cultivation using the 1700 cm2roller bottle, and remove the culture supernatant for the selection of antibodies. The culture supernatant is applied on a column of Fast Flow Q-separate (Amersham). After washing the antibody elute phosphate buffer containing 500 mm NaCl, and subjected to his affinity purification using column Fast Flow chelating separate (Amersham). Antibody elute with a gradient of 10-150 mm imidazole. Finally, the eluate is concentrated and column using Fast Flow Q-separate, and elute phosphate buffer containing 500 mm NaCl.

Electrophoresis on SDS-polyacrylamide gel under reducing conditions shows three bands, corresponding to 70 kDa, 40 kDa and 30 kDa. The results of amino acid sequencing using protein sequencing machine ABI492 (Applied Biosystems) shows that the band of 30 kDa corresponds to the amino acid sequence of GPC3, starting with 359, amino acids and below, or 375-th amino acids and below, suggesting that GPC3 is split between the have and Arg358 Ser359 or between Lys374 and Val375, therefore, it is divided into N-terminal fragment has a size of 40 kDa and C-terminal fragment of a size of 30 kDa.

Example 4

Obtaining cell line CHO expressing full-sized human GPC3

To assess the binding activity using flow cytometry get cell line CHO expressing full-GPC3.

Mix 10 micrograms of expression vector containing the gene of the full sized human GPC3, and 60 μl of SuperFect (QIAGEN). After the formation of the complex carry out the introduction of the gene by adding it to the cell line CHO DXB11. After culturing for 24 hours in CO2-incubator start breeding, using a medium αMEM (GIBCO BRL)containing geneticin with a final concentration of 0.5 mg/ml and 10% FBS. Collect the obtained resistant to geneticin colony and spend the cloning of cells, using the method of limiting dilutions. Each cell clone solubilizers and confirm the expression of a full-sized human GPC3 by the method of Western blotting using antibody against GPC3. Thus, the receive cell line with stable expression.

Example 5

Determination of binding activity by ELISA method

Soluble form of the crustal GPC3 protein was diluted to a concentration of 1 µg/ml buffer coating (0.1 mol/l NaHCO3(pH 9,6), 0,02% (wt./about.) NaN ), making immunoplate and leave at 4°C during the night that ended the coating of the tablet. After blocking the plate with dilution buffer (50 mmol/l Tris-HCl (pH 8,1), 1 mmol/l MgCl2, 150 mmol/l NaCl, 0,05% (vol./about.) Tween 20, 0.02 percent (wt./about.) NaN3, 1% (wt./about.) BSA) add antibody against GPC3 and leave to stand at room temperature for 1 hour. Washed with buffer for washing (0,05% (vol./about.) Tween 20, PBS) and then add the antibody against mouse IgG (ZYMED)labeled with alkaline phosphatase, and leave to stand at room temperature for 1 hour. Washed with buffer for washing, then add the SIGMA 104 (SIGMA), diluted to a concentration of 1 mg/ml substrate buffer (50 mmol/l NaHCO3(pH 9,8), 10 mmol/l MgCl2), and leave to stand at room temperature for 1 hour to develop color. Then measure the absorbance (at 405 nm, the wavelength comparison 655 nm) using a Benchmark Plus (BIO-RAD).

Example 6

Immunization with a soluble form of GPC3 and selection of hybridoma

Because the human GPC3 and mouse GPC3 have high homology, 94%, at the amino acid level, it is considered that it is difficult to obtain an antibody against GPC3, if subjected to immunization of normal mice. Therefore, as the immunized animal use mouse with autoimmune disease, mouse, MRL/MpJUmmCrj-lpr/lpr (hereinafter referred to as the mouse MRL/lpr supplied Chares River Japan, Inc.). Mice begin to immunize at the age of 7 or 8 weeks. The first immunization carried out as follows: receive the soluble form of GPC3 in the amount of 100 µg/specimen, emuleret using full beta-blockers (FCA, Becton Dickinson)and injected subcutaneously. Two weeks receive a soluble form of GPC3 in the amount of 50 µg/specimen, emuleret using incomplete adjuvant's adjuvant (FIA, Becton Dickinson)and injected subcutaneously. Then each week spend additional immunization, only 5 times. During the last two immunization immunized mice injected in the tail vein of the soluble form of GPC3, diluted with PBS to 50 ug/animal. On the fourth day after the last immunization, the spleen removed and receive cells of the spleen, which is mixed with cells of the mouse myeloma P3-X63Ag8U1 (P3U1, obtained from ATCC)in a 2:1 ratio. Hybridization of cells is carried out by gradual addition of PEG 1500 (Roche Diagnostic). Carefully add the medium RPMI 1640 (GIBCO BRL)to dilute PEG 1500, then PEG 1500 is removed by centrifugation, the cells are suspended in medium RPMI 1640 containing 10% FBS, and bring in 96-well culture plate in a quantity of 100 μl/well. The next day add the medium RPMI 1640 containing 10% FBS, Supplement to environment 1× HAT (SIGMA) and additive to clone hybridoma of 0.5× BM-Condimed H1 Hybridoma (Roche Diagnostic) (hereinafter referred to as the environment HAT), in the amount of 100 µl/well. 2, 3 and 5 days the second half of the culture solution replace on Wednesday HAT. After 7 days of conducting screening using culture supernatant. The screened using ELISA method, using immunoplates covered with a soluble form of the crustal GPC3 protein. A positive clone is subjected to monochlorophenol by the method of limiting dilutions. The result is 11 clones of antibodies (M3C11, M13B3, M1E7, M3B8, M11F1, L9G11, M19B11, M6B1, M18D4, M5B9 and M10D2), which have a strong binding activity against GPC3.

Example 7

Determination of the isotype and purification of antibody against GPC3

The isotype is determined by the method of the antigen-specific ELISA using the set I to determine the isotypes immunocyte monoclonal antibodies (PIERCE). Purification of the antibodies carried out as follows. The culture supernatant of hybridoma, cultivated with medium HAT, supplemented with FBS (Ultra low IgG) (GIBCO BRL), adsorb on Hi Trap ProteinG HP (Amersham) and washed with buffer for binding (20 mm sodium phosphate (pH 7.0)). Antibody elute buffer for elution (0,1M glycine-HCl (pH 2,7)). The eluate is immediately neutralized with buffer to neutralize (1M Tris-HCl (pH 9,0)) and cialiswhat against PBS during the day and night to replace the buffer.

Example 8

Determination of binding activity by ELISA method

To evaluate the binding activity obtained earlier antibody against GPC3, define dependent on the concentration of the binding of an antibody with monoplanet containing and is mobilized soluble form of the crustal GPC3 protein. Add the series 3-fold dilutions (12 dilutions) purified antibodies with a concentration of 10 μg/ml and as a secondary antibody add antibody against mouse IgG. The color development is performed with the use of SIGMA 104. Because the degree of development of the color depends on the time of color development, analyze the data received exactly 1 hour. Each antibody gives dependent on the concentration of the development of painting. Build the graph of the development of painting from the antibody concentration and using analyzing software, GraphPad Prism, get the approximated curve. Determine the EC50 value for antibodies and use it as an indicator of binding activity. The EC50 values for all clones is shown in Fig.

Example 9

Determination of binding activity using flow cytometry

Cells divide using 1mm EDTA pH 8.0 (GIBCO)/PBS and suspended in FACS buffer (1% FBS/PBS) at a concentration of 1×106cells/ml Suspension contribute to the filter tablet Multiscreen-HV (Millopre) in an amount of 100 μl/well and the supernatant is removed by centrifugation. Add antibody against GPC3, diluted to a suitable concentration, and allowed to interact on ice for 30 minutes. Cells are washed once with FACS buffer, add FITC-labeled antibody against mouse IgG and leave entries batch is VAT on ice for 30 minutes. After completion of the reaction, the cells are centrifuged at 500 rpm for 1 minute and the supernatant removed. Cells are suspended in 400 μl of FACS buffer and analyzed by flow cytometry. As the flow cytometer using EPICS ELITE ESP (Beckman Coulter). The area of living cells are selected on the histogram with the forward and side scattering. As shown in figure 1, an antibody against GPC3 (M3C11, M11F1) well associated with the CHO cell expressing GPC3, and is not associated with the parent CHO cell, indicating that the antibody specifically binds to GPC3, presented on the cell membrane. In addition, the antibody has a binding activity against cell lines hepatoma, HepG2 (obtained from ATCC) and HuH-7 (obtained from Health Science Research Resources Bank), suggesting that the antibody can specifically recognize the hepatoma. Determined by flow cytometry binding activity of clones derived from mice immunized with the soluble form of GPC3 shown in Fig where specified X-value histogram for the antibody concentration 5 ug/ml

Example 10

Classification epitopes using competitive ELISA

The resulting antibodies are classified according to the epitopes using a competitive ELISA. Antibodies biotinylated using the kit for labeling with Biotin (Roche). Soluble form of the crustal the tree GPC3 diluted with buffer to cover up to a concentration of 1 μg/ml, make the tablet in the amount of 100 μl/well and incubated at 4°C over night, so the tablet was formed coating. The next day, spend the blocking by adding 200 µl of substrate buffer. The tablet is left at 4°C overnight or longer, add antibody against GPC3 in the amount of 100 μl/well and allowed to interact at room temperature for 1 hour. Then, without rinsing, tablet, add 10 ál of labeled Biotin antibody against GPC3 in a concentration of 10 μg/ml and allowed to interact for 1 hour. The tablet is washed 3 times with buffer for washing in 300 µl/well. Conjugate AP-streptavidin (ZYMED) diluted 1000-fold with dilution buffer add 100 µl/well and allowed to interact at room temperature for 1 hour. The tablet is washed 5 times with buffer for washing in 300 µl/well. SIGMA 104 dilute to a concentration of 1 mg/ml substrate buffer and add 100 ál/well. After incubation at room temperature for 1 hour and measure the absorbance (at 405 nm, the wavelength comparison 655 nm).

The results of the competitive ELISA is shown in figure 2. It is believed that the epitopes of antibodies that competitively inhibits the binding of biotinylated antibodies in 50% or more in the three-dimensional conformations are close to each other. In the cut is ltate classification in accordance with the nature of the development of painting at competitive inhibition of binding of 8 types of biotinylated antibodies 11 clones, obtained from mice immunized with the soluble form of GPC3, divided into 5 groups (a, b, c, d and e) (Fig).

Example 11

Classification epitopes by the method of Western blotting

Soluble form of the crustal GPC3 protein is applied on the mini-plate in 10% SDS-PAGE (TEFCO) and subjected to electrophoresis in reducing conditions. Then transferred to Immobilon-P (Millipore), using a cell for semi-dry electrophoretic transfer Trans-Blot SD (BIO-RAD). After a brief washing of the membrane TBS-T (0.05% of Tween 20, TBS), her shaking in TBS-T containing 5% skim milk for 1 hour. The membrane shaking in TBS-T for about 10 minutes, then add each antibody against GPC3 diluted in TBS-T containing 1% skim milk, the membrane is shaken for 1 hour. The membrane was washed with TBS-T, shaken in a solution of HRP-antibodies against mouse IgG (Amersham)diluted in TBS-T containing 1% skim milk for 1 hour, and then washed with TBS-t For the development of painting, which will be detected using Hyperfilm ECL (Amersham)add ECL-Plus (Amersham).

As shown in figure 3, L9G11 defined as the antibody to bind to the N-end, as it is associated with a bandwidth corresponding to approximately 40 kDa. M3C11 determine how the antibody to bind to the C-end, as it is associated with a bandwidth corresponding to approximately 30 kDa. All antibodies, to which were referred to the group c, d or e according to the results of a competitive ELISA, contact N-end, and all antibodies specific to group a or b, contact C-end (Fig). According to the Western blot L9G11 has a higher detection sensitivity than other antibodies that bind to the N-end, suggesting that this antibody can be used for detection of N-terminal fragments by the method of Western blotting.

Example 12

Detection Sekretareva form of GPC3

After it was found that GPC3 is cleaved by 358-mu or 374-th amino-acid residue, the authors of this invention have hypothesized that secretiruema form of GPC3 enters the blood of a patient with liver cancer. In this regard, in order to detect secreterial the form of GPC3 was developed sandwich ELISA system for the detection of GPC3.

Immunoplate coated with antibody against GPC3 in a concentration of 10 μg/ml and block substrate buffer. Immunoplate kept for several hours at room temperature or over night at 4°C, then add the culture supernatant HepG2 and incubated 1 hour at room temperature. Immunoplate washed 3 times with buffer for washing in 300 µl/well, add labeled with Biotin antibody against GPC3, diluted to 10 μg/ml, and incubated for 1 hour at room temperature. Immunoplate washed 3 times buffet is ohms for washing in 300 µl/well, add AP-streptavidin and incubated for 1 hour at room temperature. Immunoplate washed 5 times with buffer for washing in 300 µl/well. Ensure the development of painting using AMPAK (DAKO) according to the attached manual and measure the absorption using a spectrophotometer to read the microplate. Antibodies that bind to the N-end (M6B1, M19B11 and M18D4), and antibodies that bind to the C-end (M3C11, M13B3 and M3B8), combine to create five sandwich ELISA systems. All these combinations have the same sensitivity standard curve obtained using Sekretareva form of GPC3. These systems are evaluated using the culture supernatant HepG2. When using combinations of antibodies that bind to the N-end detects a high concentration Sekretareva form of GPC3, approximately 1 µg/ml (figure 4). When using combinations of antibodies that bind to the C-end detects a low concentration, this suggests that Sekretareva the form of GPC3 is predominantly N-terminal fragment.

Then the culture supernatant HepG2 subjected thus, using an antibody against GPC3 for detection Sekretareva form of GPC3. When using M10D2, which binds to N-terminal fragment detects secreterial the form of GPC3 size of 40 kDa (figure 5). On the other hand, p and using M1E7, which is associated with C-end fragment, secreterial the form of GPC3 not find. Immunoprecipitation spend for all of the obtained antibody against GPC3. Secretiruema form of GPC3 well detected by all antibodies that bind to the N-terminal fragment, whereas the use of antibodies that bind to the C-end fragment, secretiruema form of GPC3 is not detected or detected bad (Fig). Presumably, the antibody that can detect secreterial the form of GPC3 method thus can be used for the diagnosis of hepatoma. In addition, assume that the antibody, which secretiruema form of GPC3 detected bad, can be used to develop therapeutic antibody having ADCC activity and CDC activity, because such an antibody may migrate to the area affected by hepatoma, not ullevaal Sekretareva form of GPC3 present in the blood.

Example 13

Cloning of the variable segment of the antibody against GPC3

Variable plot antibody against GPC3 amplified by PCR with reverse transcriptase using total RNA extracted from hybridoma producing antibody against GPC3. Total RNA extracted from 1×107cells hybridoma using kits RNeasy Plant Mini (QIAGEN). 5'-Terminal frag the UNT gene amplified using 1 µg total RNA kit for amplification of cDNA SMART RACE (CLONTECH) and any of the following synthetic oligonucleotides:

synthetic oligonucleotide MHC-IgG1, complementary sequences const plot murine IgG1:

GGG CCA GTG GAT AGACAG ATG (SEQ ID NO: 7);

synthetic oligonucleotide MHC-IgG2a, complementary sequences const plot murine IgG2a:

CAG GGG CCA GTG GAT AGA CCG ATG (SEQ ID NO: 8);

synthetic oligonucleotide MHC-IgG2b, complementary sequences const plot murine IgG2b:

CAG GGG CCA GTG GAT AGA CTG ATG (SEQ ID NO: 9); and

synthetic oligonucleotide Kappa, complementary sequences const plot of mouse Kappa-chain:

GCT CAC TGG ATG GTG GGA AGA TG (SEQ ID NO: 10).

The reverse transcription reaction was performed at 42°C for 1 hour and 30 minutes. Mixture for PCR (50 μl) containing 5 μl of buffer for PCR 10× Advantage 2, 5 µl of a mixture of universal primers And 10×, 0.2 mm dNTPs (dATP, dGTP, dCTP and dTTP), 1 μl polymerase mix Advantage 2 (all reagents from CLONTECH), and 2.5 μl of the reaction product of reverse transcription and 10 pmol of synthetic oligonucleotide MHC-IgG1, MHC-IgG2a, MHC-IgG2b or Kappa. PCR carried out using 5 cycles comprising 94°C for 30 seconds, 94°C for 5 seconds and 72°C for 3 minutes, 5 cycles comprising 94°C for 5 seconds, 70°C for 10 seconds and 72°C for 3 minutes, and 25 cycles, including all the I 94°C for 5 seconds, 68°C for 10 seconds and 72°C for 3 minutes. And, finally, the reaction product is heated at 72°C for 7 minutes. Each PCR product isolated from the agarose gel using the kit for gel extraction QIAquick (QIAGEN), clone in the vector pGEM-T Easy (Promega) and determine the nucleotide sequence.

Nucleotide sequence of the variable regions of H chain M3C11, M13B3, M1E7, M3B8, M11F1, M19B11, M6B1, M18D4, M5B9, M10D2 and L9G11 described in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21, respectively, and their amino acid sequences described in SEQ ID NO: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32, respectively. Nucleotide sequence of the variable regions of the L-chain of these antibodies is described in SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 43, respectively, and their amino acid sequences described in SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54, respectively.

Example 14

Classification epitopes using GST-hybrid protein

To conduct a detailed analysis of the epitopes of the antibodies to bind to the C-terminal fragment, a hybrid proteins sequentially shortened C-terminal peptides GPC3 with GST, namely GC-1 (Ser495 to Lys563), GC-2 (from Gly510 to Lys563), GC-3 (from Ala524 to Lys563), GC-4 (Gly537 to Lys563) and GC-5 (from Ser550 to Lys563). C-terminal site of GPC3 clone in pGEX-4T-3 (Amersham)to construct a plasmid DNA in which the C-terminal site of GPC3 Legerova with C-end of the GST. Plasmid DNA is introduced into DH5α and rez is ltate get the transformant. Then to the culture of transformant in the phase of logarithmic growth add IPTG to induce the expression of GST-hybrid protein. Bacterial cells are harvested after two hours of cultivation. Cells are homogenized by ultrasonic treatment and centrifuged at 35000 rpm for 30 minutes in an ultracentrifuge XL-80 (Beckman, rotor is 70.1 Ti). Then remove the culture supernatant and purified using modules purification of GST (Amersham). Treated thus GST-hybrid proteins share method SDS-PAGE in reducing conditions and carried out Western blotting using antibody against GPC3 (6). M3C11 and M1E7 allow to detect GC-1 and GC-2, but not GC-3, GC-4, GC-5, indicating that the epitopes recognized by these antibodies are located on a plot of GC-2, and that the plot GC-3 is not sufficient. M3B8 and M11F1 allow to detect GC-1, GC-2, GC-3 and GC-4, but not GC-5, indicating that the epitopes recognized by these antibodies are located on a plot of GC-4, and that the plot of GC-5 is not sufficient. The minimum areas of GST-hybrid protein, which may be contacted all antibodies are listed in the column under the heading "Western blotting" Fig.

Example 15

Receiving a mouse-human chimeric antibody against GPC3

Sequences of variable regions of the H-chain and L-chain antibody against GPC3 are ligated with the sequence is mi constant sections of a human IgG1 and Kappa-chain. PCR is performed using synthetic oligonucleotide, complementary to the 5'end of the nucleotide sequence of the variable segment H-chain of each antibody, which contains a Kozak sequence, and a synthetic oligonucleotide complementary to the 3'end of the nucleotide sequence that contains a NheI site. The resulting PCR product clone in the vector pB-CH, in which a constant area of the human IgG1 built into the vector pBluescript KS(+) (Toyobo). Variable plot of the mouse H-chain and the constant area of the human H-chain (chain γ1) are ligated via the NheI site. The obtained gene fragment H-chain clone in the expression vector, pCXND3. At the same time carry out PCR using synthetic oligonucleotide, complementary to the 5'end of the nucleotide sequence of the variable segment of the L-chain of each antibody, which contains a Kozak sequence, and a synthetic oligonucleotide complementary to the 3'end of the nucleotide sequence that contains a BsiWI site. The resulting PCR product clone in the vector pB-CL, in which a constant area of the human Kappa-chain built into the vector pBluescript KS(+) (Toyobo). Variable and constant parts of the human L-chain are ligated through the BsiWI site. The obtained gene fragment L-chain clone in the expression vector, pUCAG. Vector pUCA obtained by cloning the fragment size 2.6 thousand P.O. obtained by splitting pCXN (Niwa et al., Gene 1991; 108: 193-200) restriction enzyme BamHI BamHI site of the vector pUC19 (Toyobo).

To obtain the expression vector of murine-human chimeric antibody against GPC3, a gene fragment obtained by cleavage of the vector pUCAG containing the gene fragment of the L-chain, the restriction enzyme HindIII (Takara Shuzo), and clone into the HindIII site of the vector pCXND3, containing the gene for H-chain. This plasmid will Express the gene of resistance to neomycin, gene DHFR gene and the murine-human chimeric antibody against GPC3 in a cell of the animal.

Cell line CHO (cell line DG44), stably expressing the antibody obtained as follows. Gene is introduced into cells by means of electroporation using Gene Pulser II (Bio-Rad). A mixture obtained by mixing 25 mg of expression vector of each mouse-human chimeric antibody against GPC3 and 0.75 ml of suspension CHO cells in PBS (1×107cells/ml), cooled on ice for 10 minutes and transferred into a cuvette. Then use pulses at a voltage of 1.5 kV and a capacitance of 25 μf. After a 10-minute recovery period at room temperature, the cells subjected to electroporation, suspended in 40 ml of medium CHO-S-SFM II (Invitrogen)containing additive 1× HT (Invitrogen). The suspension was diluted 50 times with the same environment and contribute in 96-well culture plate in a quantity of 100 μl/well. Cultivate the CO 2-incubator (5% CO2within 24 hours, then add geneticin (Invitrogen) at a concentration of 0.5 mg/ml and the cells cultured for 2 weeks. Culture supernatant taken from wells containing a colony resistant to geneticin transformed cells, and the amount of IgG was measured using the following method to determine concentration. The cell line with a high level of products consistently grown, to obtain a cell line stably expressing the murine-human chimeric antibody against GPC3. The cell line is cultivated on a large scale and collect the culture supernatant. Cleaning of each mouse-human chimeric antibody against GPC3 is performed using a Hi Trap ProteinG HP (Amersham).

Example 16

Measurement of the activity associated with the complement-dependent cytotoxicity (CDC activity)

16.1. Receiving human albumin-weanling buffer (HAVB)

In water, treated at the facility milli-Q, dissolve was 12.75 g of NaCl (the highest category, Wako Pure Chemicals), 0,5625 g Na-barbitala (the highest category, Wako Pure Chemicals) and 0,8625 g barbitala (the highest category, Wako Pure Chemicals) to a final volume of 200 ml and autoclave at 121°C for 20 minutes. Then add 100 ml treated in the autoclave hot water milli-q pH is 7,43 (recommended value pH: 7.5). The solution is used in the form of 5× Veronello what about the buffer. In 50 ml of water, treated at the facility milli-Q, dissolve 0,2205 g CaCl2·2H2O (the highest category, Junsei Chemical) to a final concentration of 0.03 mol/l and the resulting solution is used as the solution of CaCl2. In 50 ml of water, treated at the facility milli-Q, dissolve 1,0165 g MgCl2·6H2O (the highest category, Junsei Chemical) to a final concentration of 0.1 mol/l and the resulting solution is used as the solution of MgCl2. In water, treated at the facility milli-Q, dissolve 100 ml of 5× weanling buffer, 4 ml of human serum albumin (25% Buminate (registered trademark), the concentration of human serum albumin: 250 mg/ml, Baxter Healthcare), 2.5 ml of a solution of CaCl2, 2.5 ml of a solution of MgCl2, 0.1 g KCl (the highest category, Junsei Chemical), 0.5 g of glucose, D(+)-glucose, anhydrous, the highest category, Wako Pure Chemicals) to obtain a final volume of 500 ml, and the resulting solution is used as HAVB. After sterilization by filtration HAVB stored at a predetermined temperature 5°C.

16.2. Obtaining target cells

The CHO cells expressing full-sized human GPC3 obtained in example 4 were cultured in the medium α-MEM containing nucleic acid (+) (GIBCO) and supplemented with 10% FBS and 0.5 mg/ml geneticin (GIBCO). Cells detach from a Cup, using a buffer for the separation of cells (Invitrogen Corp), make to each well of 96-well flat-bottomed tablet is (Falcon) in an amount of 1×10 4cells/well and cultured for 3 days. After culturing add to 5.55 MBq of chromium-51 and cells cultivated in an incubator in an atmosphere containing 5% carbon dioxide at 37°C for 1 hour. The obtained cells are washed twice HAVB add 50 μl of HAVB and used as target cells.

16.3. Analysis of the release of chromium (CDC activity)

Each chimeric antibody diluted HAVB, obtaining a solution with a concentration of 40 μg/ml To target cells are added 50 μl of a solution of each antibody and leave on ice for 15 minutes. Then to each well was added 100 μl of human serum from peripheral blood of healthy volunteers, diluted HAVB, obtaining a final concentration of 25% (the final concentration of antibody: 10 µg/ml) and left in the incubator in an atmosphere of 5% carbon dioxide at 37°C for 90 minutes. Tablet centrifuged, after which each of the selected wells 100 ál of supernatant and measure the radioactivity using a gamma counter. The specific rate of release of chromium calculated according to the following formula:

The specific rate of release of chromium (%) = (A-C)×100/(B-C)

where "A" denotes the amount of radioactivity (counts/min) in each well; "B" is the average amount of radioactivity (counts/min) in the holes in which the target cells are added 100 μl of 2% aqueous solution of NP-40 (Nonidet P-40, Code No. 252-23, Nacalai Tesque) and 50 μl of HVB; "C" indicates the average amount of radioactivity (counts/min) in the holes in which the target cells are added 150 μl of HAVB. The analysis is performed with the triple repetition and CDC activity calculated average value and standard deviation (%).

The results are shown in Fig.7. Among the 9 types of chimeric antibody against GPC3, M3B8 and M11F1, which are antibodies that recognize the C-end, high CDC activity against CHO cells expressing GPC3, but other antibodies CDC activity is not observed. M3B8 and M11F1 belong to the group "b" according to the results of the competitive ELISA method, and it is possible to detect the epitope plays an important role in the manifestation of a high CDC activity.

Example 17

Measurement of ADCC activity using PBMC obtained from human peripheral blood

17.1. Obtaining a solution containing human PBMC

Heparinized peripheral blood obtained from healthy volunteers was diluted 2 times with PBS(-) and layered on Ficoll-Paque PLUS TM (Amersham). After centrifugation at 500×g for 30 minutes at 20°C collect the middle layer, which represents the fraction of mononuclear leukocytes. Cells are washed 3 times, suspended in 10% FBS/RPMI and used as the solution containing human PBMC.

17.2. Obtaining target cells

The HepG2 cells, cultured in the medium of 10% FBS/RPMI 140, disconnect from the plate with Trypsin-EDTA (Invitrogen), make to each well of 96-well plate with a U-shaped bottom (Falcon) in an amount of 1×104cells/well and cultured for 2 days. Obtained in example 4 CHO cells expressing full-sized human GPC3, cultivated in the medium α-MEM containing nucleic acid (+) (GIBCO) and supplemented with 10% FBS and 0.5 mg/ml geneticin (GIBCO). Cells detach from a Cup, using a buffer for the separation of cells (Invitrogen Corp), make to each well of 96-well flat-bottomed tablet (Falcon) in an amount of 1×104cells/well and cultured for 3 days. To each well add chromium-51 (of 5.55 MBq) and cells cultured in the incubator in an atmosphere containing 5% carbon dioxide at 37°C for 1 hour. The obtained cells are washed once with medium, add 50 µl of medium with 10% FBS/RPMI 1640 and used as target cells.

17.3. Analysis of the release of chromium (ADCC activity)

The target cells are added 50 μl of a solution containing different concentrations of antibody, and allowed to interact on ice for 15 minutes. Then add 100 μl of the solution of human PBMC (5×105cells/well and cells were cultured in an incubator in an atmosphere of 5% carbon dioxide at 37°C for 4 hours. After culturing the tablet and centrifuged using a gamma counter to measure the radioactivity in 100 MK the culture supernatant. The specific rate of release of chromium calculated according to the following formula:

The specific rate of release of chromium (%) = (A-C)×100/(B-C)

where "A" denotes the average amount of radioactivity (counts/min) in each well; "B" is the average amount of radioactivity (counts/min) in the holes in which the target cells are added 100 μl of 2% aqueous solution of NP-40 (Nonidet P-40, Code No. 252-23, Nacalai Tesque) and 50 μl of medium with 10% FBS/RPMI; "C" denotes the average amount of radioactivity (counts/min) in the holes in which the target cells are added 150 μl of medium with 10% FBS/RPMI. The analysis is performed with the triple repetition and for ADCC activity calculated average value and standard deviation (%). The results are shown in Fig. Among the 9 types of chimeric antibody against GPC3 antibodies that recognize the C-end, have a tendency to exhibit a high ADCC activity.

Example 18

Immunization using GC-3 and breeding hybridoma

Among the obtained antibody against GPC3 only M11F1 and M3B8 have high CDC activity, indicating that the CDC activity is epitope dependent. To obtain an antibody having as ADCC activity and CDC activity, for immunization using GST-hybrid protein containing the epitope recognized M11F1 and M3B8, which represent GC-3. Using the above method to purify a large number of GC-3. The buffer exchanged for PBS by gel-filtration is then on Superdex 75 (Amersham). The obtained product is used as an immune protein. Three Balb/c mice (obtained from Charles River Japan, Inc.) and three mice MRL/lpr subjected to immunization GC-3 in accordance with the above method. For the first immunization GC-3 get in the amount of 100 µg/specimen, emuleret using FCA and injected subcutaneously. Two weeks later, GC-3 get 50 µg/specimen, emuleret using FIA and injected subcutaneously. After the fifth immunization spend the last immunization (50 µg/individual) of all mice by injecting immune protein in the tail vein. After hybridization, cells carry out the screening of hybridomas by ELISA method using immunoplates covered with a soluble form of the crustal GPC3 protein. A positive clone is subjected to monochlorophenol by the method of limiting dilutions. The result is five clones of antibodies (GC199, GC202, GC33, GC179 and GC194)with high binding activity with respect to GPC3.

The antibody is recovered from the culture supernatant of hybridoma using Hi Trap proteinG HP and analyze the above method. The EC50 value determined by the ELISA method using immunoplates covered with a soluble form of the crustal GPC3 protein, and using flow cytometry get the X-value of the histogram at a concentration of 5 µg/ml (Fig). In accordance with the classification of the epitopes competitive method ELISA antibodies section of the ut group b (GC199, GC202 and GC33) and a new epitope group f (GC179 and GC194). Classification epitopes using GST-hybrid proteins shows that using GC199, GC202 and GC33 can be used to detect GC-1, GC-2, GC-3 and GC-4, but not GC-5, suggesting that the epitopes recognized by these antibodies are located on a plot of GC-4 as well as the epitopes recognized M11F1 and M3B8 and plot GC-5 is not sufficient. On the other hand, using GC179 and GC194 can be used to detect GC-1, GC-2 and GC-3, but not GC-4, GC-5, suggesting that the epitopes recognized by these antibodies remain on-site GC-3 and GC plot-4 is not sufficient. The minimum areas of GST-hybrid proteins, which may be contacted all antibodies are listed in the column under the heading "Western blotting" Fig.

Variable sites of H-chain and L-chain GC199, GC202, GC33, GC179 and GC194 clone in accordance with the above method and determine their sequence. In the case of L-chain GC194 clone two types of sequences. Nucleotide sequence of the variable regions of the H-chain GC199, GC202, GC33, GC179 and GC194 described in SEQ ID NO: 55, 56, 57, 58 and 59, respectively, and their amino acid sequences described in SEQ ID NO: 60, 61, 62, 63 and 64, respectively. Nucleotide sequence of the variable regions of the L-chain GC199, GC202, GC33, GC179, GC194(1) and GC194(2) described in SEQ ID NO: 65, 66, 67, 68, 69 and 70, respectively, and their amino acid members is Telenesti described in SEQ ID NO: 71, 72, 73, 74, 75 and 76, respectively.

In addition, determine the degree of homology amino acid sequence data by comparing them with a database of amino acid sequences of known antibodies, thus determining their hypervariable sites, which are shown in table 2.

143
Table 2
AntibodyCDRAmino acid sequenceSEQ ID NO
M13B3(H)CDR1NYAMS103
CDR2AINNNGDDTYYLDTVKD104
CDR3QGGAY105
M3B8(H)CDR1TYGMGVG106
CDR2NIWWYDAKYYNSDLKS107
CDR3MGLAWFAY18
M11F1(H)CDR1IYGMGVG109
CDR2NIWWNDDKYYNSALKS110
CDR3IGYFYFDY111
M5B9(H)CDR1GYWMH112
CDR2AIYPGNSDTNYNQKFKG113
CDR3SGDLTGGLAY114
M6B1(H)CDR1SYAMS115
CDR2AINSNGGTTYYPDTMKD116
CDR3HNGGYENYGWFAY117
M10D2(H)CDR1SYWMH118
CDR2EIDPSDSYTYYNQKFRG119
CDR3SNLGDGHYRFPAFPY120
L9G11(H)CDR1SYWMH118
CDR2TIDPSDSETHYNLQFKD121
CDR3GAFYSSYSYWAWFAY122
GC33(H)CDR1DYEMH123
CDR2ALDPKTGDTAYSQKFKG124
CDR3FYSYTY125
GC179(H)CDR1INAMN126
CDR2RIRSESNNYATYYGDSVKD127
CDR3EVTTFAY 128
GC194(H)CDR1ASAMN129
CDR2RIRSKSNNYAIYYADSVKD130
CDR3DPGYYGNPWFAY131
GC199(H)CDR1DYSMH132
CDR2WINTETGEPTYADDFKG133
CDR3LY134
GC202(H)CDR1TYGMGVG106
CDR2NIWWHDDKYYNSALKS135
CDR3IAPRYNKYEGFFAF136
M13B3(L)CDR1KSSQSLLDSDGKTYLN137
CDR2LVSKLDS138
CDR3WQGTHFPLT139
M3B8(L)CDR1KASQDINNYLS140
CDR2RANRLVD141
CDR3LQCDEFPPWT142
M11F1(L)CDR1RSSQSLVHSNGNTYLH143
CDR2KVSNRFS144
CDR3SQSTHVPWT145
M5B9(L)CDR1RSSKSLLHSNGITYLY146
CDR2QMSNLAS147
CDR3AQNLELPYT148
M6B1(L)CDR1KASQDINKNII149
CDR2YTSTLQP150
CDR3LQYDNLPRT151
M10D2(L)CDR1RASHSISNFLH152
CDR2YASQSIS153
CDR3QQSNIWSLT154
L9G11(L)CDR1RASESVEYYGTSLMQ155
CDR2GASNVES156
CDR3QQSRKVPYT157
GC33(L)CDR1RSSQSLVHSNGNTYLH
CDR2KVSNRFS144
CDR3SQNTHVPPT158
GC179(L)CDR1KSSKSLLHSNGNTYLN159
CDR2WMSNLAS160
CDR3MQHIEYPFT161
GC194(L)1CDR1RSSKSLLHSYDITYLY162
CDR2QMSNLAS147
CDR3AQNLELPPT163
GC194(L)2CDR1SASSSVSYMY164
CDR2DTSNLAS165
CDR3QQWSSYPLT166
GC199(L)CDR1KSSQSLLHSDGKTFLN167
CDR2LVSRLDS168
CDR3CQGTHFPRT169
GC202(L)CDR1RSSQSIVHSNGNTYLE170
CDR2KVSNRFS144
CDR3FQGSHVPWT171

Example 19

Determination of ADCC activity using effector cells from bone marrow mouse

19.1. Obtaining a solution containing effector cells in the bone marrow of the mouse

Bone marrow cells collected from the femur of SCID mice (CLEA Japan, Inc., male, age 10 weeks) and suspended in the medium of 10% FBS/RPMI 1640 at a concentration of 5×105cells/ml Murine GM-CSF (PeproTech) and human IL-2 (PeproTech) is added at a concentration of 10 ng/ml and 50 ng/ml, respectively, and glue the Ki cultivated in an incubator in the atmosphere, containing 5% carbon dioxide at 37°C for 5 days. After cultivation, the cells are scraped by the scraper and once washed with medium. The cells are then suspended in the medium of 10% FBS/RPMI 1640 at a concentration of 5×106cells/ml and used as a solution containing effector cells mouse bone marrow.

19.2. Obtaining target cells

Cell line human hepatoma, HuH-7, support and subcultured using DMEM medium (SIGMA)containing 10% FBS (ThermoTrace). Cells detach from the plate with the buffer for the separation of cells (Invitrogen), make to each well of 96-well plate with a U-shaped bottom (Falcon) in an amount of 1×104cells/well and cultured for 1 day. After culturing add to 5.55 MBq of chromium-51 and cells cultivated in an incubator in an atmosphere containing 5% carbon dioxide at 37°C for 1 hour. The obtained cells are washed once with medium, add 50 µl of medium with 10% FBS/RPMI 1640 and used as target cells.

19.3. Analysis of the release of chromium (ADCC activity)

The target cells are added 50 μl of a solution containing different concentrations of antibody, and allowed to interact on ice for 15 minutes. Then add 100 ál of a solution containing effector cells mouse bone marrow (5×105cells/well) and cells were cultured in an incubator in an atmosphere of 5% dio is sid carbon at 37°C for 4 hours. After culturing the tablet and centrifuged using a gamma counter to measure the radioactivity in 100 μl of culture supernatant. The specific rate of release of chromium calculated according to the following formula:

The specific rate of release of chromium (%) = (A-C)×100/(B-C)

where "A" denotes the average amount of radioactivity (counts/min) in each well; "B" is the average amount of radioactivity (counts/min) in the holes in which the target cells are added 100 μl of 2% aqueous solution of NP-40 (Nonidet P-40, Code No. 252-23, Nacalai Tesque) and 50 μl of medium with 10% FBS/RPMI; "C" denotes the average amount of radioactivity (counts/min) in the holes in which the target cells are added 150 μl of medium with 10% FBS/RPMI. The analysis is performed with the triple repetition and for ADCC activity calculated average value and standard deviation (%).

The results are shown in Fig.9. Found that the GC33 antibody exhibits ADCC activity, if the concentration of the antibody is 0.1 µg/ml or above, and that it possesses a higher activity than the antibody GC199.

Example 20

The antitumor activity of GC33 antibody on mouse models transplanted with human hepatoma

20.1. Getting a mouse model transplanted with human hepatoma

Receive a suspension cell line human hepatoma HuH-7, 5×107cells/ml in a solution containing DMEM and MATRIGEL(BD Bioscience) at a ratio of 1:1. The day before 100 μl of the solution of antibodies against asialo-GM1 (Wako Pure Chemicals, the contents of one vial was dissolved in 1 ml of distilled water for injection, then add 4 ml of saline) was injected intraperitoneally SCID mice (male, 5 weeks of age, CLEA Japan, Inc.). Mice subcutaneously transplanted in the abdominal region 100 μl of the above cell suspension (5×106cells/mouse).

20.2. The receipt and entry of antibodies

Starting from the 20th day after transplantation of the cells, the antibody solution obtained on the day of administration, with a concentration of 0.5 mg/ml (group, which is administered 5 mg/kg) and 0.1 mg/ml (group administered 1 mg/kg), PBS(-), injected mouse model transplanted with cells of human hepatoma in the amount of 10 ml/kg via the tail vein once a week for 3 weeks. As a negative control in the same way enter the environment, PBS(-), in the amount of 10 ml/kg via the tail vein once a week for 3 weeks. Both groups consist of 6 mice each.

20.3. Evaluation of the antitumor effect

Antitumor activity of GC33 antibody to a mouse model transplanted with cells hepatoma person is judged by the change in tumor volume over time and the tumor weight in 1 week after the last injection. Tumor volume calculated using the formula below:

the tumor volume = (major axis)×(minor axis)×(minor axis)/2.

As shown in figure 10, in the group receiving the GC33 antibody, there was a significant suppression of tumor growth compared with the group receiving environment.

Thus, it is shown that GC33 has antitumor activity in a mouse model transplanted with cells hepatoma person.

Example 21

Receiving a mouse-human chimeric antibody GC33

H-chain and L-chain GC33 amplified by PCR using synthetic oligonucleotide, complementary to the 5'-end a nucleotide sequence that contains a Kozak sequence and a HindIII site, and a synthetic oligonucleotide, complementary to the 3'-end nucleotide sequences, which contains a BamHI site. After cleavage with HindIII and BamHI resulting PCR product clone in the expression vector, HEFgγ1, in which is embedded a constant area of human IgG1, and in the expression vector, HEFgκ, in which is embedded a constant area of the human Kappa-chain (Sato et al., Mol Immunol. 1994; 371-381). The vector is introduced into CHO cells (cell line DG44) in accordance with the above method and receive cell line with stable expression. The antibody is recovered from the culture supernatant using Hi Trap ProteinG HP (Amersham). The concentration of IgG in the culture supernatant was measured by sandwich ELISA method for the human is a mini-IgG, using goat antibodies against human IgG (BIOSOURCE), and conjugate goat antibodies against human IgG with alkaline phosphatase (BIOSOURCE), and the concentration is determined by comparison with commercially available human IgG (Cappel).

Example 22

The definition of CDC activity and ADCC activity using mouse-human chimeric antibody GC33

CDC activity and ADCC activity of mouse-human chimeric antibody GC33, M3C11 and M1E7 measured using the methods described in examples 16 and 17. As target cells for the measurement of CDC activity using CHO cells expressing full-GPC3, and to measure ADCC activity using HepG2 cells. The results are shown at 11 and 12, respectively. Discovered that all analytical systems GC33, compared with the other two antibodies, shows high CDC activity and ADCC activity.

Example 23

Analysis of epitopes GC33

To further define the epitope recognized GC33, get a hybrid protein consisting of optionally shortened C-terminal peptide GPC3 and GST, and analyze them using Western blotting. Received GPC3 peptide sequence contained in the GST-hybrid protein is shown in Fig. Because GC33 can communicate with the GC-4 (amino acids 537-563), but cannot communicate with GC-5 (amino acids 550-563), it is assumed that e is iTop is in the area, includes at least a portion of the fragment from 537 to 550 amino acids. First get peptides GC-6 (G N S Q Q A T P K D N E I S (SEQ ID NO: 93)), GC-7 (G N S Q Q A T P (SEQ ID NO: 94)), GC-8 (Q Q A T P K D N (SEQ ID NO: 95)) and GC-9 (T P K D N E I S (SEQ ID NO: 96)). Receive direct oligo-DNA and reverse oligo-DNA, which are manufactured so that the cleavage site sequence of the EcoRI recognition was attached to the 5'-end, and the cleavage site sequence of the SalI recognition was attached to the 3'-end, respectively. Oligo-DNA synthesized using Espec Oligo Service. DNA allocate using cartridge (C-18, phosphorylate 5'-end and analyze. Mix 25 microlitres direct oligo-DNA (10 μm) and 25 μl reverse oligo-DNA (10 μm) and subjected to interaction at 94°C for 5 minutes at 37°C for 10 minutes and at room temperature for 15 minutes, then left at 4°C for 10 minutes to anneal direct oligo-DNA and reverse oligo-DNA. The concentration of oligonucleotides is determined by measuring absorption at a molar ratio of insert to vector was 3:1. Oligonucleotides clone into EcoRI - and SalI-cleaved vector pGEX4T-3 and confirm the nucleotide sequence. GST-get a hybrid protein as described above and purified using glutathione-sepharose 4B. Purified proteins share method SDS-PAGE in reducing conditions and analyzed by the method of Western blotting with what ispolzovaniem GC33. The results show that the antibody GC33 not reliably detect any GST-hybrid proteins, therefore, we can assume that for binding GC33 require a longer sequence at the C-end (Fig). Based on the above assumption, receive GC-11 (A T P K D N E I S T (SEQ ID NO: 97)), GC-12 (P K D N E I S T F H (SEQ ID NO: 98)), GC-13 (D N E I S T F H N L (SEQ ID NO: 99)) and GC-14 (E I S T F H N L G N (SEQ ID NO: 100)) and evaluate them in the same way. The results show that GC-11 GC-12 GC-13 better contact GC33, therefore suggesting that the epitope recognized GC33, is located in the sequence from 544 to 553-th amino acid (P K D N E I S T F H)-the end of GPC3.

Example 24

The humanized GC33

Data on the sequences of the antibodies obtained from the open to the public Kabat database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/) and from the ImMunoGeneTics database (IMGT). Variable section H-chain and variable plot L-chain separately analyze the homology. The results show that the variable section H-chain has a high degree of homology with DN13 (Smithson et al., Mol Immunol. 1999; 36: 113-124), and variable plot L-chain has a high degree of homology with an mRNA IGK homo sapiens plot VLJ light chain Kappa immunoglobulin, partial cds, clone: K64, catalog number AB064105. The signal sequence with the number S40357, which today is a high degree of homology with AB064105, used as the signal sequence of the L-chain. To get humanitariannet antibody hypervariable segment (hereinafter referred to as CDR) GC33 transplanted in frame areas (hereinafter referred to as FR) of these human antibodies.

Specifically synthetic oligo-DNA by approximately 50 bases are manufactured so that approximately 20 bases of them hybridities, and then these synthetic oligo-DNA collected together by PCR to obtain the genes encoding all variable sites. Their split at the HindIII site, built in the end of the 5'-end of the synthetic oligo-DNA and BamHI site, built in the end of the 3'-end of the synthetic oligo-DNA. The clone fragments in the expression vector, HEFgγ1, in which clone a constant area of human IgG, or in the expression vector, HEFgκ1, in which clone a constant area of the human Kappa-chain (Sato et. al., Mol Immunol. 1994; 371-381). H-chain and L-chain gumanitarnogo GC33, constructed as described above, is called ver.a, respectively. Humanitariannet GC33, in which the H-chain and L-chain are ver.a (ver.a/ver.a), has a lower binding activity than an antibody with variable plots mouse GC33 (mouse/mouse). Design antibodies that hiberno combined sequence of mouse GC33 and sequence ver.a (m is nil/ver.a, ver.a/mouse), relative to the H-chain and L-chain and determine their binding activity. The results show that the decrease in binding is observed in the case ver.a/mouse, therefore, the decrease in binding activity due to replacement of amino acids refers to the H-chain (Fig). Then get modified H-chain, ver.c, ver.f, ver.h, ver.i, ver.j, ver.k. All of these humanized GC33 have the same binding activity as the chimeric antibody containing the variable plot murine GC33 (Fig). Nucleotide sequence of the variable regions of H chain gumanitarnogo GC33, ver.a, ver.c, ver.f, ver.h, ver.i, ver.j, ver.k described in SEQ ID NO: 77, 78, 79, 80, 81, 82 and 83, respectively, and their amino acid sequences described in SEQ ID NO: 84, 85, 86, 87, 88, 89 and 90, respectively. The nucleotide sequence and amino acid sequence of the variable segment of the L-chain gumanitarnogo GC33, ver.a described in SEQ ID NO: 91 and 92, respectively. In variable parts of N-chains gumanitarnogo GC33 ver.i, ver.j and ver.k 6th glutamic acid residue is replaced by a glutamine residue. thermostability of these antibodies increased significantly.

Example 25

Modification of the L-chain gumanitarnogo GC33

It is known that the reaction rate constant of desametasone proteins depends on the primary sequence. It is also known that the combination of Asn-Gly especially case is indeed to dezaminirovanie (Rocinson et. al., Proc. Natl. Acad. Sci. USA 2001; 98; 944-949). As for Asn33 in CDR1 ver.a L-chain gumanitarnogo GC33 described in SEQ ID NO: 91, the primary sequence is a combination of Asn-Gly, which, as predicted, is sensitive to dezaminirovanie.

In order to assess the impact of desametasone Asn33 on binding activity, receive a modified antibody, which Asn33 replaced by Asp. A point mutation introduced by using a kit for site-directed mutagenesis, Quick Change (Stratagene). More specifically, 50 μl of reaction mixture containing 125 ng of sense primer (CTT GTA CAC AGT GAC GGA AAC ACC TAT: SEQ ID NO: 172), 125 ng of antisense primer (ATA GGT GTT TCC GTC ACT GTG TAC AAG: SEQ ID NO: 173), 5 μl 10× reaction buffer, 1 ál dNTP mix, 10 ng HEFgκ, in which clone ver.a L-chain gumanitarnogo GC33, and 1 μl DNA polymerase Pfu Turbo subjected to PCR reaction, which includes 12 cycles consisting of 95°C for 30 seconds, 55°C for 1 minute and 68°C for 9 minutes. Then add the restriction enzyme, DpnI, the splitting is carried out at 37°C for 2 hours and the cleavage product is introduced into a competent cell XL1-Blue present in the set, the result of the transformant. Variable area cut out from a clone in which properly introduced each mutation, and clone again in HEFgκ. It is introduced into COS7 cell using Fugene 6 (Roche) together with HEFgγ1, in which clone ver.k H-chain of humanitarian the th GC33. From the culture supernatant isolated antibody, temporarily expressed in the cell. The concentration of antibodies determined by sandwich ELISA method using the antibody against human IgG. Binding activity of the modified antibody determined by ELISA method using immunoplates covered with a soluble form of the crustal GPC3 protein. As shown in Fig modified antibody (N33D), in which Asn33 replaced by Asp, loses binding activity, therefore, we can assume that desametasone Asn33 has a significant effect on binding activity.

As a method of suppressing desametasone Asn33 was described replacement Gly34 to another amino acid (international patent application WO 03057881A1). In accordance with the above method G34 replaced with any of the 17 amino acids other than Cys and Met, with the use of the kit for site-directed mutagenesis, Quick Change and get a number of modified antibodies, namely G34A, G34D, G34E, G34F, G34H, G34N, G34P, G34Q, G34I, G34K, G34L, G34V, G34W, G34Y, G34R, G34S and G34T. Provide temporary expression of antibodies in COS7 cells and in cell supernatant determine binding activity. It was found that the binding activity is retained, even if G34 replaced with another amino acid except Pro (G34P) and Val (G34V).

Amino acid sequence of light chain CDR1, Viseu manutech antibodies described in SEQ ID NO: 174 (G34A), SEQ ID NO: 175 (G34D), SEQ ID NO: 176 (G34E), SEQ ID NO: 177 (G34F), SEQ ID NO: 178 (G34H), SEQ ID NO: 179 (G34N), SEQ ID NO: 180 (G34T), SEQ ID NO: 181 (G34Q), SEQ ID NO: 182 (G34I), SEQ ID NO: 183 (G34K), SEQ ID NO: 184 (G34L), SEQ ID NO: 185 (G34S), SEQ ID NO: 186 (G34W), SEQ ID NO: 187 (G34Y), SEQ ID NO: 188 (G34R), SEQ ID NO: 189 (G34V) and SEQ ID NO: 190 (G34P), respectively. Amino acid sequences of variable regions of light chain of the above-mentioned antibodies described in SEQ ID NO: 191 (G34A), SEQ ID NO: 192 (G34D), SEQ ID NO: 193 (G34E), SEQ ID NO: 194 (G34F), SEQ ID NO: 195 (G34H), SEQ ID NO: 196 (G34N), SEQ ID NO: 197 (G34T), SEQ ID NO: 198 (G34Q), SEQ ID NO: 199 (G34I), SEQ ID NO: 200 (G34K), SEQ ID NO: 201 (G34L), SEQ ID NO: 202 (G34S), SEQ ID NO: 203 (G34W), SEQ ID NO: 204 (G34Y), SEQ ID NO: 205 (G34R), SEQ ID NO: 206 (G34V) and SEQ ID NO: 207 (G34P), respectively.

The antibody of the present invention can be used as an inhibitor of cell growth, anti-cancer tool or tools for the diagnosis of cancer.

Example 26

Obtaining cell line human hepatoma (SK-03)expressing full-sized human GPC3

To assess the biological activity of the antibody against GPC3 get cell line human hepatoma expressing full-GPC3.

One microgram of expression vector containing the gene of the full sized human GPC3, processed Pvu I, is mixed with 2 μl of FuGENE (Roche)to obtain the complex. Then enter the gene obtained by adding the complex to the cells SK-HEP-1 (obtained from ATCC). After incubation in CO2-the incubator for 24 hours to carry out the selection of tile is to, expressing GPC3, by using MEM for Dulbecco (D-MEM, SIGMA)containing geneticin at a final concentration of 1 mg/ml and 10% FBS. Gather resistant geneticin colony and cell clone, using the method of limiting dilutions. The expression of human GPC3 in each cell clone analyzed by flow cytometry using chimeric antibody GC33 and FITC-labeled goat antibodies against human IgG (ICN). So get cell line SK-03 with stable expression.

Example 27

Comparison of CDC activity and ADCC activity of mouse-human chimeric antibodies

To directly compare the CDC activity and ADCC activity of mouse-human chimeric antibody GC33, M3C11 and M1E7 described in example 22, the CDC activity and ADCC activity of the three antibodies was measured in the same analytical system according to the method described in examples 16 and 17. As target cells for the measurement of CDC activity using CHO cells expressing full-GPC3, and to measure ADCC activity using cells SK-03. The results are shown in Fig and pig, respectively. It was found that in any analytical system GC33 has a higher CDC activity and ADCC activity than the other two antibodies.

Application in industry

The antibody of the present invention can be used in quality is as inhibitor of cell growth, anticancer means and tools for the diagnosis of cancer.

1. The antibody is able to connect with glypican 3 plot with amino acid residues 1-563 sequence SEQ ID NO:4, which includes variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:143, 144 and 158, respectively.

2. The antibody is able to connect with glypican 3 plot with amino acid residues 1-563 sequence SEQ ID NO:4 selected from:
(1) antibodies comprising variable plot heavy chain including amino acid sequence described in SEQ ID NO:84, and variable area light chain comprising the amino acid sequence described in SEQ ID NO:92;
(2) the antibody comprising the variable plot heavy chain including amino acid sequence described in SEQ ID NO:85, and variable area light chain comprising the amino acid sequence described in SEQ ID NO:92;
(3) the antibody comprising the variable plot heavy chain including amino acid sequence described in SEQ ID NO:86, and variable area light chain comprising aminokislot the second sequence, described in SEQ ID NO:92;
(4) the antibody comprising the variable plot heavy chain including amino acid sequence described in SEQ ID NO:87, and variable area light chain comprising the amino acid sequence described in SEQ ID NO:92;
(5) an antibody comprising the variable plot heavy chain including amino acid sequence described in SEQ ID NO:88, and variable area light chain comprising the amino acid sequence described in SEQ ID NO:92;
(6) an antibody comprising the variable plot heavy chain including amino acid sequence described in SEQ ID NO:89, and variable area light chain comprising the amino acid sequence described in SEQ ID NO:92; or
(7) an antibody comprising the variable plot heavy chain including amino acid sequence described in SEQ ID NO:90, and variable area light chain comprising the amino acid sequence described in SEQ ID NO:92.

3. The antibody according to claim 1 or 2, which is humanitariannet antibody.

4. Antibody having activity equivalent binding activity of the antibody according to claim 3, and having the amino acid sequence of the antibody according to claim 3, in which one or more amino acid residues substituted, deleted or added is/or inserted.

5. The antibody is able to connect with glypican 3 plot with amino acid residues 1-563 sequence SEQ ID NO:4 selected from:
(1) antibodies comprising variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:174, 144 and 158, respectively;
(2) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:175, 144 and 158, respectively;
(3) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:176, 144 and 158, respectively;
(4) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and varied the plot light chain, containing CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:177, 144 and 158, respectively;
(5) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:178, 144 and 158, respectively;
(6) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:179, 144 and 158, respectively;
(7) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:180, 144 and 158, respectively;
(8) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain, with the containing a series of CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:181, 144 and 158, respectively;
(9) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:182, 144 and 158, respectively;
(10) the antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:183, 144 and 158, respectively;
(11) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:184, 144 and 158, respectively;
(12) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2, 3, comprising the amino acid sequence described in SEQ ID NO:185, 144 and 158, respectively;
(13) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:186, 144 and 158, respectively;
(14) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:187, 144 and 158, respectively;
or
(15) an antibody comprising the variable plot heavy chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:123, 124 and 125, respectively, and variable area light chain contains the CDR 1, 2 and 3 comprising the amino acid sequence described in SEQ ID NO:188, 144 and 158, respectively.

6. The antibody is able to connect with glypican 3 plot with amino acid residues 1-563 sequence SEQ ID NO:4 that contains the variable area light chain selected from:
(1) the variable segment light chain including the corresponding amino acid sequence, described in SEQ ID NO:191;
(2) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:192;
(3) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:193;
(4) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:194;
(5) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:195;
(6) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:196;
(7) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:197;
(8) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:198;
(9) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:199;
(10) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:200;
(11) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:201;
(12) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:202;
(13) the variable segment light chain comprising the amino acid serial is lnost, described in SEQ ID NO:203;
(14) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:204; or
(15) the variable segment light chain comprising the amino acid sequence described in SEQ ID NO:205;
and variable plot heavy chain selected from:
(1) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:84;
(2) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:85;
(3) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:86;
(4) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:87;
(5) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:88;
(6) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:89; or
(7) the variable segment of a heavy chain including amino acid sequence described in SEQ ID NO:90.

7. The antibody according to any one of claims 4 to 6, which is humanitariannet antibody.

8. Polynucleotide encoding variable plot heavy chain and variable area light chain of the antibody according to claim 3 or 7.

9. Polynucleotide on the .8, having any of the sequences described in SEQ ID NO:57 and 77-83 encoding the variable plot heavy chain and SEQ ID NO:67 or 91 encoding the variable area light chain.

10. The vector for the production of antibodies against glypican 3, including polynucleotide of claim 8 or 9.

11. A host cell producing the antibody against glypican 3, containing the vector of claim 10.

12. A method of obtaining antibodies against glypican 3, including:
(a) culturing the host cell according to claim 11,
(b) isolation of antibody from the culture of (a), and
(c) purification of antibodies.

13. Inhibitor of cell growth, comprising as active ingredient an antibody according to any one of claims 1 to 7, where the specified inhibitor of cell growth inhibits the growth of animal cells.

14. The use of antibodies according to any one of claims 1 to 7 for the treatment of cancer.

15. The use of antibodies according to any one of claims 1 to 7 for the treatment of hepatoma.

16. Peptide for the production of antibodies against glypican 3, comprising the amino acid sequence of amino acid residues 546-551 glypican 3.



 

Same patents:

FIELD: medicine.

SUBSTANCE: polypeptide contains extracellular and transmembrane domains of a human tissue factor. A DNA fragment coding this polypeptide, and a plasmid pET28a(+) are used to produce a genetic make-up enabling biosynthesis of the recombinant human tissue factor. Also, the E coli BL21 [DE3]/p6E-tTF strain that is a producer of the recombinant human tissue factor is offered.

EFFECT: high-yield recombinant protein and simplified recovery and purification procedures.

2 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is described in a cartridge genetic maker based on GeneClip-Ul-neo vector producing three siRNA - reproduction inhibitors of human immunodeficiency virus type 1 and human CCR5 gene. The maker contains a fragment of the length 27 bp related to a conservative region of a HIV-1 genome reverse transcriptase domain, a fragment of the length 27 bp related to the other conservative region of the HIV-1 genome reverse transcriptase domain, as well as a fragment 19 bp related to CCR5 gene mRNA. The invention can be used in medicine and scientific research.

EFFECT: invention allows producing more effective anti-HIV preparations based on interfering RNA produced in cells by means of the introduced cartridge genetic maker containing palindromes for three siRNA production.

3 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: recovered polynucleotide coding aminopeptidase containing a nucleotide sequence presented in the description is presented. Also, polynucleotide hybridised with said polynucleotide in the high stiffness environment is presented. An expression vector containing said polynucleotide, and an applicable host cell are described. Polypeptide related to the sequence presented in the description and being aminopeptidase is presented. A method of producing said polypeptide involving the stages of applicable host cell transformation by said polynucleotide or vector, cell cultivations in the medium adequate for said polynucleotide expression, and optional polypeptide purification from said cell or culture medium is offered. Besides, there has been described diagnostic technique for Aspergillus-infection in an organism involving the stages: a) recovery of a biological sample from said organism which is supposed to be Aspergillus infected, b) recovery of nucleic acid from said sample, c) determination whether said recovered nucleic acid contains polynucleotides hybridised with polynucleotide recovered from Aspergillus niger.

EFFECT: invention allows diagnosing Aspergillus-infection in the organism.

13 cl, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Versions of a human CD37 specific antibody are presented, each containing a variable site of a light and heavy chain. A coding nucleic acid and a based expression vector are described. There are disclosed: a host cell containing a vector, a method of producing the antibody with using the cell, and also a composition for decreasing the B-cell count and a method of treating diseases associated with aberrant B-cell activity, with using the CD37-specific antibodies.

EFFECT: use of the invention provides specific loss of 80% BJAB cells at the antibody concentration 10 mcg/ml, and also increases the survival rate of Daudi mice as compared with Rhithuximab therapy that can find application for treating various tumours.

39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with conjugates "caliheamicin derivative-carrier". Essence of invention includes methods of obtaining conjugates "monomer cytotoxic medication-caliheamicin/carrier" with considerably higher loading with medication than in earlier described methods, with low aggregation degree and with low content of low-conjugated fraction (LCF), as well as conjugates "cytotoxic medication-caliheamicin derivative/anti-CD22-antibody". Invention also includes compositions, which contain conjugate with CD22 antibody and application of said conjugates.

EFFECT: invention makes it possible to create conjugates with high loading of cytotoxic medication.

181 cl, 11 ex, 13 tbl, 29 dwg

FIELD: chemistry.

SUBSTANCE: described is a polypeptide, having phytase activity, selected from: a polypeptide with an amino acid sequence given in the description of the invention, a fragment of that polypeptide, and a polypeptide which exhibits at least 90% identity with the said polypeptide. Described also is a polynucleotide which codes the said polypeptide. An expression cassette and an expression vector which contain the described polynucleotides are disclosed. A host organism producing the described polypeptide is disclosed. Furthermore, a feed additive and animal feed containing the said polypeptide, host organism or metabolites thereof is obtained. The invention discloses use of the said polypeptide to prepare a feed additive or animal feed and for hydrolysis of myoinositol hexakisphosphate to inorganic monophosphate, to myoinositol with lower degree of phosphorylation and to free myoinositol.

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14 cl, 11 dwg, 9 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: recombinant technique is used to produce proteins consisting of the module SEQ ID NO: 3, involving replacement of serine and/or glutamate with lysine and/or cysteine and/or addition of the amino-terminal TAG according to SEQ ID NO:20-24 or carboxy-terminal TAG according to SEQ ID NO:25-28, which contain lysine or cysteine along with other amino acids.

EFFECT: invention enables to obtain modified spider silk protein, capable of binding with other substances through a lysine or cysteine residue with preservation of structural integrity of the spider silk protein.

21 cl, 8 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

Il2 antibodies // 2425054

FIELD: medicine, pharmaceutics.

SUBSTANCE: humanised monoclonal antibody and its active fragment under the invention neutralises human IL2 activity by binding with said human IL2 prior to, during and/or after binding of said human IL2 with human IL2 receptor. A variable light chain region of said antibody contains an adherent amino acid KAPKA sequence in its second frame region, and in addition, in the CDR1-CDR3 regions, contains the amino acid sequences presented in SEQ ID NO 1-3 disclosed in the description, while a variable heavy chain region contains in the CDR1 - CDR3 regions, amino acid sequences presented in SEQ ID NO 4-6 disclosed in the description. The invention describes a polynecleotide molecule coding the antibody under the invention or its active fragment, a pharmaceutical composition based on said antibody or its active fragment exhibiting human IL2 neutralising action, and also an application of said antibody or its active fragment or the polynecleotide molecule coding it for preparing a drug which optionally contains an auxiliary anti-inflammatory or anticancer agent for treating inflammatory diseases or tumours, respectively.

EFFECT: production of the alternative specific IL2 activity inhibitors which directly bind with human IL2.

24 cl, 18 dwg, 4 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Versions of a human CD37 specific antibody are presented, each containing a variable site of a light and heavy chain. A coding nucleic acid and a based expression vector are described. There are disclosed: a host cell containing a vector, a method of producing the antibody with using the cell, and also a composition for decreasing the B-cell count and a method of treating diseases associated with aberrant B-cell activity, with using the CD37-specific antibodies.

EFFECT: use of the invention provides specific loss of 80% BJAB cells at the antibody concentration 10 mcg/ml, and also increases the survival rate of Daudi mice as compared with Rhithuximab therapy that can find application for treating various tumours.

39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.

EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.

29 cl, 15 dwg, 6 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: binding molecule represents a CD45RO and CD45RB chimeric antibody. The molecule contains two domains with consistent hypervariable sites CDR1, CDR2 and CDR3, and CDR1', CDR2' and CDR3', CDR1 has amino acid sequence NYIIH, CDR2 has amino acid sequence YFNPYNHGTKYNEKFKG, and CDR3 has amino acid sequence SGPYAWFDT. CDR1' has amino acid sequence RASQNIGTSIQ, CDR2' has amino acid sequence SSSESIS, and CDR3' has amino acid sequence QQSNTWPFT. Related coding polynucleotide is described.

EFFECT: use of the invention allows to induce immunosuppression, to inhibit T-cell response and primary lymphocyte reaction in the mixed culture, to prolong survival time in mice with severe combined immunodeficiency SCID.

6 cl, 5 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of an angiopoietin-2 (Ang-2) specific antibody and a pharmaceutical antibody composition for treatment of various diseases associated with angiopoietin-2 overexpression. Also there are described methods of inhibition, modulation and treatment of various diseases mediated by angiopoietin-2 activity. There are offered: coding nucleic acid, an expression vector and a vector-transformed cell, as well as a method for producing antibodies.

EFFECT: use of the invention ensures new high-cytotoxicity antibodies (according to ELISA analysis IC50=0,35 nM) comparable with a common antibody Ab536 that further can find application in medicine.

22 cl, 2 dwg, 11 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is claimed isolated human antibody or its fragment, which binds to human EGFR. Antibody contains corresponding CDR areas of light and heavy chain. Its conjugate with anti-neoplastic means or marker is described. Also described are: coding nucleic acid, expression vector, recombinant cell-host for obtaining antibodies and method of inhibiting growth of tumor, expressing EGFR on the basis of antibody.

EFFECT: application of invention provides antibodies with affinity comparable or higher, than in IMC-C225, which neutralises EGFR activation, what can be applied in medicine for treatment of tumours.

36 cl, 14 dwg, 6 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine. What is offered is ProThrThrLysThrTyrPheProHisPhe peptide, a based pharmaceutical composition which is used for antitumour immune response stimulation, and also to methods of treating a mammal and immune response modulation.

EFFECT: range of products for cancer treatment is extended.

19 cl, 49 tbl, 3 ex

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