Anti-Glypican 3 Antibody Having Modified Sugar Chain

Abstract

An anti-glypican 3 antibody with modified sugar chains, more specifically, an anti-glypican 3 antibody lacking fucose is provided. The anti-glypican 3 antibody with modified sugar chains of the present invention may be produced by a process comprising introducing a nucleic acid encoding an anti-glypican 3 antibody into host cells with reduced fucose addition capability, such as YB2/0 cells and cells lacking a fucose transporter. The anti-glypican 3 antibody with modified sugar chains of the present invention has a high level of cytotoxic activity and therefore is useful as a cell growth inhibitor such as an anticancer agent.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a divisional application of and claims priority to U.S. application Ser. No. 11/577,944, filed on Aug. 8, 2007, which is the National Stage of International Application No. PCT/JP2005/020057, filed on Oct. 26, 2005, which claims the benefit of Japanese Patent Application Serial No. 2004-311356, filed on Oct. 26, 2004. The contents of all of the preceding applications are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

[0002]The present invention relates to an antibody to glypican 3 antigen (i.e., an anti-glypican 3 antibody) in which cytotoxic activity, especially antibody-dependent cellular cytotoxicity (ADCC) is enhanced, as well as a process for producing the antibody.

BACKGROUND

[0003]Glypican 3 (GPC3) is a member of the family of heparan sulfate proteoglycans present on the surface of cells. It has been suggested that GPC3 may be involved in cell division upon development and in cancer cell growth, but its function is still not well understood.

[0004]It has been discovered that a type of antibody that binds to GPC3 inhibits cell growth due to ADCC (antibody-dependent cellular cytotoxicity) activity and CDC (complement-dependent cytotoxicity) activity (WO 2003/00883). Furthermore, because GPC3 is cleaved in the body and secreted into the blood as a soluble form of GPC3, it has been suggested that cancer can be diagnosed using an antibody that can detect the soluble form of GPC3 (WO 2004/022739, WO 2003/100429, WO 2004/018667).

[0005]When developing an anticancer agent that utilizes antibody cytotoxic activity, preferably the antibody used will have a high level of ADCC activity. Therefore, an anti-GPC3 antibody with a high level of cytotoxic activity has been demanded.

[0006]Modification of antibody sugar chains are known to enhance its ADCC activity. For example, WO 99/54342 discloses that ADCC activity is enhanced by modifying antibody glycosylation. In addition, WO 00/61739 discloses that ADCC activity is regulated by controlling the presence or absence of fucose in antibody sugar chains. WO 02/31140 discloses producing an antibody having sugar chains that do not contain α-1,6 core fucose by producing that antibody in YB2/0 cells. WO 02/79255 discloses an antibody with sugar chains having bisecting GlcNAc. However, an anti-GPC3 antibody with enhanced ADCC activity due to sugar chain modification has not been disclosed so far.

SUMMARY

[0007]The present invention provides an anti-GPC3 antibody composition with enhanced ADCC activity caused by the alteration of the sugar chain component thereof, as well as a process for producing such an antibody.

[0008]After various investigations, the inventors have discovered that an antibody targeting GPC3 with sugar chains lacking α-1,6 core fucose have a high level of cytotoxic activity. Thus, the present invention provides an anti-GPC3 antibody composition wherein the sugar chain component of the antibody has been altered, and more specifically, an antibody composition with a greater fraction of fucose deficient anti-GPC3 antibodies. The sugar chain-modified anti-GPC3 antibody composition of the present invention has a high level of cytotoxic activity, and therefore is useful as a cell growth inhibitor such as an anticancer agent.

[0009]The present invention also provides a process for producing an anti-GPC3 antibody composition wherein the sugar chain of the antibody is modified, comprising the steps of: introducing a nucleic acid encoding the anti-GPC3 antibody into a host cell with reduced fucose addition capability such as YB2/0 cells, and culturing the host cell to obtain the antibody. Preferably, the cell with reduced capability of adding fucose to sugar chains is a cell lacking a fucose transporter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows the basic structure of N-glycoside linked sugar chains;

[0011]FIG. 2 shows ADCC activity of a chimeric antibody when HepG2 cells are targeted using human peripheral blood monocytes (PBMC);

[0012]FIG. 3 shows ADCC activity of a chimeric antibody when HuH-7 cells are targeted using human PBMC;

[0013]FIG. 4 shows ADCC activity of antibodies when HuH-7 cells are targeted using human PBMC;

[0014]FIG. 5 shows normal phase HPLC chromatograms of sugar chains modified by agalactosyl 2-AB prepared from antibodies (a, b, c) produced by FT-KO cells and by CHO cells;

[0015]FIG. 6 shows the predicted structures for the G(0) and G(0)-Fuc peaks shown in FIG. 5; and

[0016]FIG. 7 shows a differential scanning calorimetry (DSC) measurement plot for the antibodies produced by FT-KO cells (x) and by CHO cells (o).

DETAILED DESCRIPTION

[0017]The present invention features an anti-GPC3 antibody composition wherein the sugar chain component of the antibody has been modified. It is known that the structure of the sugar chain linked to an antibody has a significant effect on the expression of antibody cytotoxic activity. Sugar chains that is linked to an antibody include N-glycoside-linked sugar chains, which are attached to a nitrogen atom on the side chain of an asparagine residue on the antibody molecule, and O-glycoside-linked sugar chains, which are attached to a hydroxyl group on the side chain of a serine or threonine residue on the antibody molecule. The present invention is focused on the presence or absence of fucose in an N-glycoside-linked sugar chain.

[0018]FIG. 1 shows the basic structure of N-glycoside-linked sugar chains attached to an antibody. As shown in the IgG basic sugar chains (1) and (3) of FIG. 1, the N-glycoside linked sugar chains have a basic structure (core) wherein one mannose (Man) and two N-acetylglucosamine (GlcNAc) moieties are linked by β-1,4 linkages [-Man β1-4GlcNAc β1-4GlcNAc-]. The "GlcNAc" on the right side of the structure is called the reducing end and the "Man" on the left side of the structure is called the non-reducing end. When a fucose is linked to the reducing end, it usually takes the form of an α-linkage between the 6-position of the N-acetylglucosamine at the reducing end and the 1-position of the fucose. On the other hand, in the sugar chain shown in IgG basic sugar chain (2) of FIG. 1, in addition to the aforementioned two sugar chains, one N-acetylglucosamine (GlcNAc) moiety is linked to the non-reducing end of the basic structure (core) via a β1,4-linkage. This type of N-acetylglucosamine (GlcNAc) is called a "bisecting N-acetylglucosamine. "A sugar chain having a bisecting N-acetylglucosamine can be an O-glycoside-linked sugar chain or N-glycoside-linked sugar chain, and it is formed by transfer of N-acetylglucosamine to the sugar chain by N-acetylglucosamine transferase III (GnTIII). The gene encoding this enzyme has already been cloned, and both the amino acid sequence and the nucleotide sequence of the DNA encoding the enzyme have already been reported (NCBI database (ACCESSION D13789)).

[0019]In the present invention, the antibody composition with a modified or altered sugar chain component (sugar chain-modified antibody composition) refers to an antibody composition having a sugar chain component that differs from the antibody composition produced by a host cell serving as a reference standard.

[0020]In the present invention, one may determine whether the sugar chain component has been modified or not by using as a reference standard the antibody composition produced by a host cell serving as a reference standard. If an antibody composition has a sugar chain component different from the antibody composition from the reference standard, that antibody composition is considered as an antibody composition with a modified sugar chain component.

[0021]The host cell serving as a reference standard in the present invention is CHO DG44 cell. CHO DG44 cell can be obtained, for example, from the Invitrogen Corporation.

[0022]Examples of an antibody composition with a modified sugar chain component include, for example, an antibody composition with an increased ratio of fucose (e.g., α-1,6 core fucose)-deficient antibodies in the antibody composition and an antibody composition with an increased ratio of antibodies having an attached bisecting N-acetylglucosamine (GlcNAc) in the antibody composition.

[0023]In a preferred embodiment of the present invention, the antibody composition has a higher ratio of fucose-deficient antibodies than the antibody composition used as a reference standard.

[0024]Because some antibodies have a plurality of N-glycoside sugar chains, the fucose-deficient antibody of the present invention encompasses not only antibodies wherein no fucose is attached, but also antibodies wherein the number of fucose moieties attached to the antibody is reduced (an antibody having at least one or more sugar chains wherein fucose is not present).

[0025]When manufacturing a sugar chain-modified antibody with host cells, it is often difficult to obtain a composition containing uniform antibodies wherein all antibodies have identical sugar chains. Therefore, if an antibody composition with a modified sugar chain component of the invention is an antibody composition with an increased ratio of fucose-deficient antibodies, for example, then the antibody composition with the modified sugar chain component of the present invention may contain both antibodies deficient in fucose and antibodies not deficient in fucose, but the overall ratio of antibodies deficient in fucose will be higher than in the antibody composition produced by the host cells serving as a reference standard. The present invention is not particularly limited to a specific ratio of fucose-deficient antibodies in the antibody composition with a high ratio of fucose-deficient antibodies of the present invention, but preferably the ratio is not less than 20%, more preferably not less than 50%, and most preferably not less than 90%.

[0026]The present invention is not particularly limited to a specific ratio of bisecting N-acetylglucosamine-added antibodies in the antibody composition having a high ratio of bisecting N-acetylglucosamine-added antibodies of the present invention, but preferably the ratio is not less than 20%, more preferably not less than 50%, and most preferably not less than 90%.

[0027]The anti-GPC3 antibody composition with a modified sugar chain component of the present invention can be obtained by methods known to those skilled in the art.

[0028]For example, a fucose-deficient antibody can be produced by expressing the anti-GPC3 antibody in host cells either lacking capability or having lower capability to add α-1,6 core fucose.

[0029]The present invention is not particularly limited to the host cells lacking capability or having lower capability to add fucose, but host cells with no or reduced fucose transferase activity, host cells with lower fucose concentration in Golgi bodies, and the like may be used in the present invention. More specifically, examples of host cells include rat myeloma YB2/3HL.P2.G11.16Ag.20 cells (abbreviated as YB2/0 cells) (preserved as ATCC CRL 1662), FTVIII knockout CHO cells (WO 02/31140), Lec13 cells (WO 03/035835) and fucose transporter deficient cells (WO 2005/017155).

[0030]As used herein, the term "fucose transporter deficient cell" refers to a cell in which the quantity of fucose transporter in the cell is less than in normal cells, or fucose transporter function is attenuated due to an abnormality in the fucose transporter structure. Examples of fucose transporter deficient cells may include, for example, those cells wherein the fucose transporter gene is knocked out (hereinafter called FT-KO cells), those wherein part of the fucose transporter gene is either lacking or mutated, those deficient in the fucose transporter gene expression system, and the like. The nucleotide sequence of the gene encoding the Chinese hamster fucose transporter and the amino acid sequence thereof are shown in SEQ ID NOS: 126 and 127, respectively.

[0031]Moreover, it is possible to obtain the fucose transporter deficient cell of the present invention using RNA interference (RNAi) by utilizing the nucleotide sequence represented by SEQ ID NO: 126. RNAi refers to the following phenomenon: when double stranded RNA (dsRNA) is introduced into a cell, intracellular mRNA matching that RNA sequence is specifically degraded and cannot be expressed as a protein. Normally dsRNA is used with RNAi, but the present invention is not limited thereto and, for example, double stranded RNA formed by self-complementary single stranded RNA molecules can also be used. With respect to the regions forming the double stranded molecule, the molecule may be double stranded in all regions, or may be single stranded in some regions (for example, one or both ends). The present invention is not limited to a specific length of the oligo-RNA used in RNAi. The length of the oligo-RNA in the present invention can be, for example, 5 to 1000 bases (or 5 to 1000 by in a double stranded molecule), preferably 10 to 100 bases (or 10 to 100 by in a double stranded molecule), and most preferably 15 to 25 bases (15 to 25 by in a double stranded molecule); however, a length of 19 to 23 bases (19 to 23 by in a double stranded molecule) is especially preferred.

[0032]The aforementioned RNAi process utilizes the phenomenon wherein dsRNA consisting of both sense RNA and antisense RNA homologous to a specific gene will destroy the homologous part of the transcript (mRNA) of that gene. dsRNA corresponding to the entire sequence of the fucose transporter gene may be used, or shorter dsRNA (for example, 21 to 23 bp) corresponding to part of the sequence (small interfering RNA; siRNA) may be used. The dsRNA can be directly transferred into the cell, or a vector producing dsRNA can be prepared and transferred into a host cell, and the dsRNA can then be produced within the cell. For example, all or part of the DNA encoding the fucose transporter gene can be inserted into a vector so that it forms an inverted repeat sequence, and that vector can then be transferred into a host cell. The RNAi procedure can be carried out in accordance with the descriptions in the following references: Fire A. et al., Nature (1998), 391, 806-811; Montgomery M. K. et al., Proc. Natl. Acad. Sci. USA (1998), 95, 15502-15507; Timmons L. et al., Nature (1998), 395, 854; Sanchez A. et al., Proc. Natl. Acad. Sci. USA (1999), 96, 5049-5054; Misquitta L. et al., Proc. Natl. Acad. Sci. USA (1999), 96, 1451-1456; Kennerdell J. R. et al., Cell (1998), 95, 1017-1026; Waterhouse P. M. et al., Proc. Natl. Acad. Sci. USA (1998), 95 13959-13964; and Wianny F. et al., Nature Cell Biol. (2000), 2, 70-75.

[0033]The fucose transporter deficient cells obtained by the RNAi procedure may be screened as indicated by the fucose transporter activity. Screening can also be carried out based on the transcription and expression of the fucose transporter gene indicated by Western blotting or Northern blotting.

[0034]An antibody with a bisecting N-acetylglucosamine (GlcNAc) added to the sugar chain can be produced by expressing the anti-GPC3 antibody in a host cell having the capability to form a bisecting N-acetylglucosamine (GlcNAc) structure on the sugar chain.

[0035]A method for producing an antibody with a bisecting N-acetylglucosamine-added sugar chain is already known (WO 02/79255). The host cell having the capability to form a bisecting N-acetylglucosamine (GlcNAc) structure on a sugar chain is not particularly limited in the present invention, but may include, for example, a host cell having an expression vector containing DNA encoding GnTIII. Therefore, an anti-GPC3 antibody having a bisecting N-acetylglucosamine-added sugar chain can be produced using a host cell containing both an expression vector with DNA encoding GnTIII and an expression vector encoding the anti-GPC3 antibody. The DNA encoding GnTIII and the gene encoding the anti-GPC3 antibody can both be present on the same vector or can be present on different vectors.

[0036]Another method for increasing the ratio of fucose-deficient antibodies or bisecting N-acetylglucosamine-added antibodies in the antibody composition is to increase the ratio of those antibodies in the composition by purifying the fucose-deficient antibodies or bisecting N-acetylglucosamine-added antibodies.

[0037]Sugar chain analysis can be carried out by any methods known to those skilled in the art. For example, a sugar chain can be released from an antibody by reacting the antibody with N-glycosidase F (Roche) and the like. Then the sugar chains can be desalted by solid phase extraction using a cellulose cartridge (Shimizu Y. et al., Carbohydrate Research 332 (2001), 381-388), concentrated and dried, and fluorescent labeled with 2-aminopyridine (Kondo A. et al., Agricultural and Biological Chemistry 54:8 (1990), 2169-2170). The reagent is removed from the pyridylamino-sugar chains (PA-sugar chains) by solid phase extraction with a cellulose cartridge, then the sugar chains are concentrated by centrifugation to obtain purified PA-sugar chains. The sugar chains may be assayed by reverse phase HPLC analysis using an octadecyl silane (ODS) column. The PA-sugar chains thus prepared may be analyzed by two dimensional mapping utilizing a combination of reverse phase HPLC analysis with an ODS column and normal phase HPLC analysis with an amine column.

[0038]The sugar chain-modified anti-GPC3 antibody of the present invention is not limited to any specific antibodies, provided it binds to GPC3. Preferably, a binding to GPC3 can be specific. Preferred anti-GPC3 antibodies of the present invention include those antibodies that have the complementarity determining region (CDR) sequence shown in Table 1 below.

TABLE-US-00001 TABLE 1 Antibody CDR Amino acid sequence SEQ ID NO: M13B3(H) CDR1 NYAMS 5 CDR2 AINNNGDDTYYLDTVKD 6 CDR3 QGGAY 7 M3B8(H) CDR1 TYGMGVG 8 CDR2 NIWWYDAKYYNSDLKS 9 CDR3 MGLAWFAY 10 M11F1(H) CDR1 IYGMGVG 11 CDR2 NIWWNDDKYYNSALKS 12 CDR3 IGYFYFDY 13 M5B9(H) CDR1 GYWMH 14 CDR2 AIYPGNSDTNYNQKFKG 15 CDR3 SGDLTGGLAY 16 M6B1(H) CDR1 SYAMS 17 CDR2 AINSNGGTTYYPDTMKD 18 CDR3 HNGGYENYGWFAY 19 M10D2(H) CDR1 SYWMH 20 CDR2 EIDPSDSYTYYNQKFRG 21 CDR3 SNLGDGHYRFPAFPY 22 L9G11(H) CDR1 SYWMH 20 CDR2 TIDPSDSETHYNLQFKD 23 CDR3 GAFYSSYSYWAWFAY 24 GC33(H) CDR1 DYEMH 25 CDR2 ALDPKTGDTAYSQKFKG 26 CDR3 FYSYTY 27 GC179(H) CDR1 INAMN 28 CDR2 RIRSESNNYATYYGDSVKD 29 CDR3 EVTTSFAY 30 GC194(H) CDR1 ASAMN 31 CDR2 RIRSKSNNYAIYYADSVKD 32 CDR3 DPGYYGNPWFAY 33 GC199(H) CDR1 DYSMH 34 CDR2 WINTETGEPTYADDFKG 35 CDR3 LY 36 GC202(H) CDR1 TYGMGVG 8 CDR2 NIWWHDDKYYNSALKS 37 CDR3 IAPRYNKYEGFFAF 38 M13B3(L) CDR1 KSSQSLLDSDGKTYLN 39 CDR2 LVSKLDS 40 CDR3 WQGTHFPLT 41 M3B8(L) CDR1 KASQDINNYLS 42 CDR2 RANRLVD 43 CDR3 LQCDEFPPWT 44 M11F1(L) CDR1 RSSQSLVHSNGNTYLH 45 CDR2 KVSNRFS 46 CDR3 SQSTHVPWT 47 M5B9(L) CDR1 RSSKSLLHSNGITYLY 48 CDR2 QMSNLAS 49 CDR3 AQNLELPYT 50 M6B1(L) CDR1 KASQDINKNII 51 CDR2 YTSTLQP 52 CDR3 LQYDNLPRT 53 M10D2(L) CDR1 RASHSISNFLH 54 CDR2 YASQSIS 55 CDR3 QQSNIWSLT 56 L9G11(L) CDR1 RASESVEYYGTSLMQ 57 CDR2 GASNVES 58 CDR3 QQSRKVPYT 59 GC33(L) CDR1 RSSQSLVHSNGNTYLH 45 CDR2 KVSNRFS 46 CDR3 SQNTHVPPT 60 GC179(L) CDR1 KSSKSLLHSNGNTYLN 61 CDR2 WMSNLAS 62 CDR3 MQHIEYPFT 63 GC194(L)1 CDR1 RSSKSLLHSYDITYLY 64 CDR2 QMSNLAS 49 CDR3 AQNLELPPT 65 GC194(L)2 CDR1 SASSSVSYMY 66 CDR2 DTSNLAS 67 CDR3 QQWSSYPLT 68 GC199(L) CDR1 KSSQSLLHSDGKTFLN 69 CDR2 LVSRLDS 70 CDR3 CQGTHFPRT 71 GC202(L) CDR1 RSSQSIVHSNGNTYLE 72 CDR2 KVSNRFS 46 CDR3 FQGSHVPWT 73

[0039]The antibodies with the CDR sequence listed in the above table have a high level of cytotoxic activity. The antibodies with the CDR sequence listed in the above table recognize epitopes of amino acids 524-563 on GPC3. Because antibodies that recognize epitopes of amino acids 524-563 have a high level of cytotoxic activity, they are preferred as the anti-GPC3 antibody of the present invention.

[0040]In one preferred embodiment of the present invention, the antibody composition having a modified sugar chain component of the present invention is characterized by exhibiting enhanced ADCC activity. In the present invention, whether the ADCC activity is enhanced or not may be determined by comparing the ADCC activity of the antibody composition of the present invention with that of the reference standard antibody composition. If the antibody composition of the present invention shows higher ADCC activity than the reference standard, the ADCC activity is said to be enhanced.

[0041]ADCC activity can be measured by a method known to those skilled in the art, for example, by mixing the anti-GPC3 antibody with effector cells and target cells, and then determining the level of ADCC. More specifically, mouse spleen cells, human monocytes isolated from peripheral blood (PBMC) and bone marrow and the like can be used as the effector cells and human cells expressing GPC3 such as human hepatocellular carcinoma cell-line HuH-7 can be used as the target cells. First the target cells are labeled with ⁵¹Cr, anti-GPC3 antibody is added, the cells are incubated, and then effector cells in a suitable ratio to the target cells are added, and they are incubated together. After incubation, the supernatant is collected, and the ADCC activity is measured by counting the radioactivity in the supernatant.

Anti-GPC3 Antibody

[0042]The anti-GPC3 antibody can be prepared by a method known to those skilled in the art. For example, the antibody can be prepared by using GPC3 as a sensitizing antigen for immunization in accordance with a conventional immunization method, fusing the immune cells with known parent cells by a conventional cell fusion procedure, and screening for monoclonal antibody producing cells by a conventional screening method. More specifically, monoclonal antibodies can be prepared in the following manner. First, the GPC3 to be used as a sensitizing antigen for antibody production is obtained by expressing GPC3 (MXR7) based on the gene/amino acid sequence disclosed in Lage, H. et al., Gene 188 (1997), 151-156. In other words, the gene sequence encoding GPC3 is inserted into a known expression vector. After suitable host cells are transformed with the vector, the target human glycipan 3 protein is purified from the host cells or culture medium supernatant by a known method. Next the purified GPC3 protein is used as a sensitizing antigen. Alternatively, a partial peptide of GPC3 can be used as the sensitizing antigen. In such a process the partial peptide can be obtained by chemical synthesis according to the amino acid sequence of human GPC3. The epitopes on the GPC3 molecule recognized by the anti-GPC3 antibody of the present invention are not limited, but the anti-GPC3 antibody of the present invention may recognize any epitope present on the GPC3 molecule. This is because the anti-GPC antibody exhibits the cell growth inhibitory activity through its ADCC activity, CDC activity, or inhibition of growth factor activity, and because cell growth can also be inhibited by the action of a cytotoxic substance such as a radioactive isotope, chemotherapy drug, bacterial toxin attached to the anti-GPC3 antibody. Therefore, the antigen for preparing the anti-GPC3 antibody of the present invention can be any fragment of GPC3 provided it contains an epitope present on the GPC3 molecule.

[0043]In an especially preferred embodiment, a peptide containing amino acids 524-563 can be used as the sensitizing antigen to generate an antibody that recognizes an epitope of amino acids 524-563 of GPC3.

[0044]The mammal used for immunization with the sensitizing antigen is not particularly limited in the present invention, but preferably it should be selected in consideration of the compatibility with the parent cells to be used in cell fusion, and may include a rodent, for example, a mouse, rat, or hamster, or a rabbit, monkey, and the like. The animal may be immunized with the sensitizing antigen using a known method. In general, for example, the mammal can be injected intraperitoneally or subcutaneously with sensitizing antigen. More specifically, the sensitizing antigen can be diluted and suspended in a suitable amount of phosphate buffered saline (PBS) or physiological saline, mixed with a suitable amount of conventional adjuvant such as Freund's complete adjuvant if desired, emulsified, and administered to the mammal multiple times every 4 to 21 days. In addition, a suitable vehicle can be used upon immunization with the sensitizing antigen.

[0045]After the mammal is immunized in the above manner, and the desired antibody level is detected in the serum, the immune cells are collected from the mammal and subjected to cell fusion. Spleen cells are especially preferred immune cells for cell fusion. Mammalian myeloma cells are used as the partner parent cells to be fused with the immune cells. Known cell lines suitable for use as the myeloma cells include, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.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), 5194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), 8210 (Galfre, G et al., Nature (1979) 277, 131-133). The cell fusion of the immune cells and myeloma cells basically can be carried out in accordance with a known method, for example, the method described by Kohler and Milstein (Kohler. G and Milstein, C., Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out, for example, in a conventional liquid culture medium containing a cell fusion promoter. Examples of the cell fusion promoting chemicals include Polyethylene glycol (PEG) and Sendai virus (HVJ) and the like. If desired, a supplemental agent such as dimethyl sulfoxide and the like may be added to increase fusion efficiency. The ratio of immune cells to myeloma cells may be established arbitrarily. For example, setting the ratio of immune cells with respect to myeloma cells at 1-fold to 10-fold is preferred. A conventional liquid culture medium used for culturing these types of cells, such as RPMI-1640 liquid medium, MEM liquid medium, or another liquid medium suitable for the growth of the myeloma cell line may be used as the liquid medium in the cell fusion procedure. A serum supplement such as fetal calf serum (FCS) can also be used together. In the cell fusion procedure, specified amounts of the immune cells and myeloma cells are thoroughly mixed in the liquid culture medium, and then PEG solution (for example, average molecular weight of about 1000 to 6000) that has been heated to 37° C. is normally added at a concentration of 30 to 60% (w/v) and mixed to allow for forming fused cells (hybridomas). Next, a suitable liquid culture medium is added and centrifuged to remove the supernatant. By repeating this procedure, any cell fusion chemicals unfavorable for the growth of hybridomas are removed. Hybridomas obtained in this manner are selected by culturing them in a conventional liquid selection medium such as HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing in the HAT medium is continued for a sufficient period of time (normally a few days to a few weeks) until cells other than the target hybridomas (non-fused cells) die off. Then, a conventional limiting dilution procedure is carried out, followed by screening and monocloning hybridomas that produce the target antibody. In addition to immunizing a non-human animal with the antigen to obtain the hybridomas as above, desired human antibodies having GPC3 binding activity can be obtained by sensitizing human lymphocytes with GPC3 in vitro, and then fusing the sensitized lymphocytes with immortalized human myeloma cells (see Japanese Patent Publication No. H1-59878). In addition, it is possible to administer GPC3 as an antigen to a transgenic animal having the complete repertoire of human antibody genes to generate cells producing anti-GPC3 antibodies, and collect human antibodies to GPC3 from immortalized cells (see International Patent Application No. WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602). Hybridomas producing monoclonal antibodies prepared in the above manner can be subcultured in a conventional liquid culture medium, and may be preserved for a long period of time in liquid nitrogen.

Recombinant Antibodies

[0046]The monoclonal antibody used in the present invention is a recombinant monoclonal antibody, which can be produced by cloning the antibody gene from a hybridoma, inserting the gene into a suitable vector, and integrating the vector into a host cell (for example, see Vandamme, A. M. et al., Eur. J. Biochem. (1990) 192; 767-775, 1990). More specifically, the mRNA encoding the variable (V) region of the anti-GPC3 antibody is isolated from a hybridoma producing the anti-GPC3 antibody. mRNA can be isolated using a known method such as the guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299), and the AGPC method (Chomczynski, P., et al., Anal. Biochem. (1987) 162, 156-159) to prepare total RNA, and then preparing the target mRNA using an mRNA Purification Kit (Pharmacia) and the like. The mRNA can also be prepared directly by using a QuickPrep mRNA Purification Kit (Pharmacia). Then cDNA of the antibody V region is synthesized from the mRNA thus obtained using reverse transcriptase. cDNA can be synthesized using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), and the like. Also the 5'-RACE method using the 5'-AmpliFINDER RACE Kit (Clontech) and PCR can be used for cDNA synthesis and amplification (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002, Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). The target DNA fragment is purified from the PCR product and ligated to the vector DNA. The desired recombinant vector is prepared by inserting those vectors. It is introduced into E. coli and the desired colony is selected to prepare a desired recombinant vector. The nucleotide sequence of the target DNA is confirmed by a known method such as the dideoxynucleotide chain termination method. After DNA encoding the V region of the target anti-GPC3 antibody is obtained, it is inserted into an expression vector containing DNA encoding the desired antibody constant region (C region). For the production of the anti-GPC3 antibody used in the present invention, the antibody gene is inserted into the expression vector so that it will be expressed under the control of an expression control region such as an enhancer, promoter, and the like. Next, the antibody is expressed by transforming a host cell with that expression vector. The antibody gene can be expressed in the host cells by inserting DNA encoding the antibody heavy chain (H chain) and light chain (L chain) into separate expression vectors and simultaneously transforming the host cells, or by inserting DNA encoding both the H chain and L chain into a single expression vector and transforming the host cells (see WO 94/11523). In addition, the recombinant antibody can be produced not only by using the aforementioned host cells, but also by using a transgenic animal. For example, the antibody gene can be inserted into the middle of a gene encoding a protein produced specifically in milk (such as goat β-casein) to prepare a fused gene. Then the DNA fragment containing the fused gene containing the antibody gene is injected into a goat embryo, and the embryo is implanted in a female goat. The desired antibody can be obtained from the milk produced by the transgenic goat born from the goat implanted with the embryo and the offspring thereof. Furthermore, suitable hormones can be used in the transgenic goat to increase the amount of milk containing the desired antibody that is produced by the transgenic goat (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

Altered Antibodies

[0047]In addition to the antibodies as described above, an artificially altered gene recombinant antibody such as a chimeric antibody, humanized antibody, and the like can be used in the present invention for the purpose of reducing the xenoantigenicity to humans. Such modified antibodies can be produced according to known methods. A chimeric antibody can be obtained by ligating DNA encoding the antibody V region obtained as described above with DNA encoding the human antibody C region, and then inserting the DNAs into an expression vector. The vector in which the DNAs are inserted is integrated into host cells to produce the antibody. A chimeric antibody useful in the present invention can be obtained using such a conventional method. A humanized antibody, also called a reshaped human antibody, comprises the CDR of an antibody from a non-human mammal such as a mouse grafted onto a human antibody CDR. The general genetic engineering methods for obtaining humanized antibodies are known in the art (see EP 125023 and WO 96/02576). More specifically, a DNA sequence designed to link the mouse antibody CDR and the human antibody framework region (FR) is synthesized by PCR using as primers a plurality of oligonucleotides prepared such that they have overlapped CDR and FR terminal regions (the method described in WO 98/13388). The framework region of the human antibody to be linked via the CDR is selected such that the CDR will form a suitable antibody binding site. If necessary, amino acids of the framework region in the variable region of the antibody may be substituted so that the reshaped human antibody CDR will form a suitable antibody binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856). A human antibody C region is used for the C region of the chimeric antibody and the humanized antibody. For example, Cy1, Cy2, Cy3, and Cy4 can be used in the H chain, and Cκ and Cλ can be used in the L chain. In addition, the human antibody C region can be modified to improve the stability or productivity of the antibody. The chimeric antibody comprises the variable region of an antibody from a non-human mammal and the constant region from a human antibody. On the other hand, the humanized antibody comprises the CDR of an antibody from a non-human mammal and the framework region and C region from a human antibody. Because the humanized antibody has lower antigenicity in the human body, it is more useful as the active ingredient in the therapeutic agent of the present invention.

Modified Antibodies

[0048]The antibody used in the present invention is not limited to a whole molecule of antibody, but it may be an antibody fragment or a modified form of an antibody, provided it binds to GPC3 and inhibits the activity of GPC3. The present invention also encompasses bivalent antibodies as well as monovalent antibodies. Examples of an antibody fragment include Fab, F(ab')₂, Fv, Fab/c having one Fab and a complete Fc, or a single chain Fv (scFv) wherein the Fv of an H chain or L chain is linked by a suitable ligand. More specifically, to produce an antibody fragment, the antibody can be treated with an enzyme such as papain or pepsin, or a gene encoding such an antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669, Bird, R. E. et al., TIBTECH (1991) 9, 132-137). A scFv can be obtained by joining the H chain V region and L chain V region of an antibody. In a scFv, the H chain V region and L chain V region are joined by a linker, preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H chain V region and L chain V region in the scFv may be derived from any antibodies described herein. Any single chain peptide comprising 12 to 19 amino acid residues may be used as the peptide linker joining the V regions. DNA encoding scFv can be obtained by amplifying a fragment by PCR using as a template a DNA portion encoding all or a desired amino acid sequence of the sequences of DNA encoding the H chain or the H chain V region of the above-mentioned antibody and DNA encoding the L chain or the L chain V region of the above-mentioned antibody with a primer pair that defines the both ends thereof. Then the fragment is amplified with a combination of DNA encoding a peptide linker portion and a primer pair which defines both ends to be ligated to the H chain and the L chain. Once DNA encoding scFv is prepared, an expression vector containing the DNA and a host cell transformed with the expression vector can be obtained according to a standard method. The scFv can be obtained from such a host according to a standard method. These antibody fragments can be produced in a host by obtaining the gene thereof in the same manner as described above and by allowing it to be expressed. In the present invention, the term "antibody" also encompasses a fragment of the antibody. An anti-GPC3 antibody attached to various molecules such as PEG and the like can be used as a modified antibody. In the present invention, the term "antibody" also encompasses such a modified antibody. The modified antibody can be obtained by chemical modification of the antibody obtained as above. Methods for modifying antibodies have already been established in the art.

[0049]Furthermore, the antibody used in the present invention can be a bispecific antibody. A bispecific antibody may be an antibody having an antigen binding site that recognizes a different epitope on the GPC3 molecule, or it may be an antibody wherein one antigen binding site recognizes GPC3 and the other antigen binding site recognizes a cytotoxic substance such as a chemotherapy drug and cell-derived toxin. In such a case, the cytotoxic substance will act directly on cells expressing GPC3, and specifically lesion tumor cells, and suppress the growth of tumor cells. A bispecific antibody may be prepared by linking two types of antibody HL pairs. Also it may be obtained by preparing a bispecific antibody-producing fused cell through the fusion of hybridomas that produce different monoclonal antibodies. A bispecific antibody can also be prepared by genetic engineering methods.

Expression and Production of Recombinant Antibodies or Modified Antibodies

[0050]An antibody gene constructed as noted above can be expressed and obtained by known methods. In the case of mammalian cells, a common useful promoter, gene to be expressed, and a poly-A signal sequence downstream on the 3' end can be functionally linked together and expressed. For example, the human cytomegalovirus immediate early promoter/enhancer can be used as the promoter/enhancer. In addition, other promoters/enhancers that can be used to express the antibody of the present invention include viral promoters/enhancers of retrovirus, polyomavirus, adenovirus and simian virus 40 (SV40), or promoters/enhancers from mammalian cells such as human elongation factor 1α (HEF-1α). Antibodies can be readily expressed by the method of Mulligan et al. (Nature (1979) 277, 108) when SV40 promoter/enhancer is used, and by the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322) when HEF1α promoter/enhancer is used.

[0051]The antibody of the present invention may be produced using an eukaryotic expression system having the capability of adding a sugar chain to the expressed antibody. Eukaryotic cells include, for example, established mammalian cell lines and insect cell lines, animal cells, fungal cells, and yeast cells.

[0052]Preferably, the antibody of the present invention is expressed in mammalian cells, for example, CHO, COS, myeloma, BHK, Vero, or HeLa cells. The target antibody is produced by culturing the transformed host cells either in vitro or in vivo. The host cells may be cultured using known methods. For example, DMEM, MEM, RPMI-1640, or IMDM may be used as the culture medium, and a serum complement such as fetal calf serum (FCS) may be supplemented.

Isolation and Purification of Antibody

[0053]The antibody expressed and produced in the above manner can be separated from the host cells or host animals and purified to homogeniety. The antibody of the present invention can be separated and purified using an affinity column, for example, a protein A column such as Hyper D, POROS and Sepharose F.F. (Pharmacia). In addition, any conventional methods for protein separation and purification may be used in the invention. For example, the antibody can be isolated and purified by appropriately selecting and combining affinity columns such as Protein A column with chromatography columns, filtration, ultra filtration, salting-out and dialysis procedures (Antibodies A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). An antibody having a desired sugar chain can be separated with a lectin column by a method known in the art, and the method described in WO 02/30954.

Determination of Antibody Activity

[0054]The antigen binding activity (Antibodies: A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) and ligand receptor binding inhibition (Harada, A. et al., International Immunology (1993) 5, 681-690) of the antigen used in the present invention may be measured by known methods. ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or the fluorescent antibody technique can be used for measuring antigen binding activity of the anti-GPC3 antibody of the present invention. For example, EIA is carried out as follows. A sample containing the anti-GPC3 antibody, such as culture supernatant of anti-GPC3 antibody producing cells or purified antibody, is added to a plate coated with GPC3. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated and washed, and then the enzyme substrate, such as p-nitrophenyl phosphate, is added and the optical absorption is measured to evaluate the antigen binding activity.

Pharmaceutical Composition

[0055]The present invention provides a pharmaceutical composition comprising the anti-GPC3 antibody with a modified sugar chain component of the present invention.

[0056]A pharmaceutical composition comprising the antibody composition of the present invention is useful for the prevention and/or treatment of diseases associated with cell growth such as cancer, and is particularly useful for the prevention and/or treatment of liver cancer. The pharmaceutical composition comprising the antibody of the present invention can be formulated by methods known to those skilled in the art. The pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, the pharmaceutical composition can be formulated by suitably combining the antibody with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. The amount of active ingredient included in the pharmaceutical preparations is such that a suitable dose within the designated range is provided.

[0057]The sterile composition for injection can be formulated in accordance with conventional pharmaceutical practices using distilled water for injection as a vehicle.

[0058]For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate 80®, HCO-50 and the like.

[0059]Examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection will normally be packaged in a suitable ampule.

[0060]Route of administration is preferably parenteral, for example, administration by injection, transnasal administration, transpulmonary administration, transcutaneous administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection.

[0061]A suitable means of administration can be selected based on the age and condition of the patient. A single dose of the pharmaceutical composition containing the antibody or a polynucleotide encoding the antibody can be selected from a range of 0.001 to 1000 mg/kg of body weight. On the other hand, it is also possible to select a dose in the range of 0.001 to 100000 mg/body, but the present invention is by no means limited to such numerical ranges. The dose and method of administration will vary depending on the weight, age, condition, and the like of the patient, and can be suitably selected as needed by those skilled in the art.

[0062]The content of all patents and reference documents expressly cited in the specification of this application are hereby incorporated by reference in its entirety. In addition, the content of the specification and drawings of Japanese Patent Application 2004-311356, which is the basis for the priority claim of this application, are hereby incorporated by reference in its entirety.

EXAMPLES

[0063]The present invention is explained in detail through the following examples, but is by no means limited to these examples.

Example 1

Preparation of Mouse Anti-GPC3 Antibody

[0064]A soluble GPC3 protein lacking the hydrophobic region of the C terminus (amino acids 564 to 580) was prepared as the immunizing protein for the preparation of the anti-GPC3 antibody for immunization. The MRL/MpJUmmCrj-lpr/lpr mouse (hereinafter referred to as MRL/lpr mouse, purchased from Charles River Japan), which is an autoimmune disease mouse, was used as the immunization animal. Immunization was started when the mice were 7 or 8 weeks old, and a preparation for initial immunization was adjusted to a dose of 100 μg/head soluble GPC3. An emulsion was prepared using Freund's complete adjuvant (FCA, Becton Dickinson), and injected subcutaneously. After a series of five immunizations, the final immunization dose was diluted in PBS to 50 μg/head, and injected intravenously via the caudal vein. On day 4 after the final immunization, the spleen cells were resected, mixed with mouse myeloma cells P3-X63Ag8U1 (hereinafter referred to as P3U1, purchased from ATCC) in a 2:1 ratio, and cell fusion was carried out by gradually adding PEG-1500 (Roche Diagnostics). Hybridomas were screened by ELISA using immunoplates with immobilized soluble GPC3 core protein. Positive clones were monocloned by the limiting dilution procedure. As a result, 11 clones of antibodies with strong GPC3 binding activity were obtained (M3C11, M13B3, M1E7, M3B8, M11F1, L9G11, M19B11, M6B1, M18D4, M5B9, and M10D2).

[0065]Among the anti-GPC3 antibodies obtained, M11F1 and M3B8 exhibited particularly strong CDC activity. Thus the GST fusion protein containing the M11F1 and M3B8 epitopes (GC-3), which is the fusion protein containing a peptide from 524 Ala to 563 Lys of GPC3 and GST was used as immunogens for immunization of 3 Balb/c (Charles River Japan) mice and 3 MRL/lpr mice. For the first immunization, a preparation of GC-3 at a concentration of 100 μg/head was emulsified with FCA and was injected subcutaneously. After two weeks, a preparation of 50 μg/head was emulsified with Freund's incomplete adjuvant (FIA) and was injected subcutaneously. After five immunizations, the final immunization (50 μg/head) was injected intravenously to all mice via the caudal vein, and subjected to the cell fusion. Hybridomas were screened by ELISA using immunoplates with immobilized soluble GPC3 core protein lacking the hydrophobic region of the C terminus (amino acids 564-580). Positive clones were monocloned by the limiting dilution procedure. As a result, 5 clones of antibodies with strong GPC3 binding activity (GC199, GC202, GC33, GC179, and GC194) were obtained.

[0066]The H chain and L chain variable regions were cloned and each sequence was determined by standard method. Furthermore, the CDR regions were determined by comparison with a known antibody amino acid sequence database and checking for homology. The sequences of the CDR regions are shown in Tables 1 and 2.

Example 2

Preparation of Anti-GPC3 Antibody Mouse-Human Chimeric Antibody

[0067]The variable region sequences of the H chain and L chain of anti-GPC3 antibody GC33 were ligated to the constant region sequences of human IgG1 and κ chain. PCR was carried out using a synthetic oligonucleotide complementary to the 5' terminal nucleotide sequence of the antibody H chain variable region having a Kozak sequence and a synthetic oligonucleotide complementary to the 3' terminal nucleotide sequence having an NheI site. The PCR product thus obtained was cloned into pB-CH vector wherein the human IgG1 constant region has been inserted into pBluescript KS+ vector (Toyobo Co., Ltd.). The mouse H chain variable region and the human H chain (γ1 chain) constant region were ligated via the NheI site. The H chain gene fragment thus prepared was cloned into the expression vector pCXND3. Furthermore, PCR was carried out using a synthetic oligonucleotide complementary to the 5' terminal nucleotide sequence of the L variable region of the antibody having a Kozak sequence and a synthetic oligonucleotide complementary to the 3' terminal nucleotide sequence having a BsiWI site. The PCR product thus obtained was cloned into pB-CL vector wherein the human κ chain constant region has been inserted into pBluescript KS+ vector (Toyobo Co., Ltd.). The human L chain variable region and the constant region were ligated via the BsiWI site. The L chain gene fragment thus prepared was cloned into the expression vector pUCAG. The pUCAG vector is a vector prepared by digesting pCXN (Niwa et al., Gene, 1991, 108, 193-200) with the restriction enzyme BamHI to prepare a 2.6 kbp fragment, which was ligated to the restriction enzyme BamHI site of pUC19 vector (Toyobo Co., Ltd.)

[0068]To prepare the anti-GPC3 mouse-human chimeric antibody expression vector, a gene fragment was obtained by digesting pUCAG vector, in which the L chain gene fragment was inserted, with the restriction enzyme HindIII (Takara Shuzo Co., Ltd). This gene fragment was ligated to the restriction enzyme HindIII cleavage site of pCXND3 containing the H chain gene, and then cloned. The plasmid thus obtained expressed the neomycin resistance gene, DHFR gene, and anti-GPC3 mouse-human chimeric antibody gene in animal cells. (The amino acid sequence of the H chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the L chain variable region is shown in SEQ ID NO: 4.)

Example 3

Preparation of Low-Fucose Type Anti-GPC3 Chimeric Antibody

[0069]First YB2/0 (ATCC, CRL-1662) cells were cultured as the host cells in RPMI-1640 medium containing 10% FBS. Then 25 μg of the anti-GPC3 chimeric antibody expression vector prepared in Example 2 was introduced into the YB2/0 cells (ATCC CRT-1662) by electroporation at a concentration of 7.5×10⁶ cells/0.75 mL PBS(-) at 1.4 kV and 25 μF. After a recovery period of 10 min at room temperature, the cells treated by electroporation were suspended in 40 mL of RPMI-1640 medium containing 10% FBS. A 10-fold dilution was prepared using the same medium, and the cells were aliquoted into a 96-well culture plate at 100 μL/well. After culturing for 24 h in a CO₂ incubator (5% CO₂), Geneticin (Invitrogen Corp.) was added at a concentration of 0.5 mg/mL, and the cells were cultured for 2 weeks. Cell lines with a high level of chimeric antibody expression were screened using sandwich ELISA with anti-human IgG antibody, and cell lines stably expressing the antibody were established. Each anti-GPC3 mouse-human chimeric antibody was purified using Hi Trap ProteinG HP (Amersham).

Example 4

Measurement of ADCC Activity Using PBMC from Human Peripheral Blood

Preparation of Human Pbmc Solution

[0070]Heparin-added peripheral blood collected from a healthy adult was diluted 2-fold with PBS(-), and layered on Ficoll-Paque® PLUS (Amersham). After centrifugation (500×g, 30 min, 20° C.), the intermediate layer, which is the mononuclear cell fraction, was isolated. After the layer was washed 3 times, the cells were suspended in 10% FBS/RPMI to prepare a human PBMC solution.

Preparation of Target Cells

[0071]HepG2 cells (ATCC) and HuH-7 cells (Health Science Research Resources Bank) cultured in 10% FBS/RPMI-1640 medium were detached from the dish using Cell Dissociation Buffer (Invitrogen), aliquoted into a 96-well U-bottomed plate (Falcon) at a concentration of 1×10⁴ cells/well, and cultured for one day. After culturing, 5.55 MBq of ⁵¹Cr was added, and the cells were cultured for 1 h at 37° C. in a 5% CO₂ gas incubator. The cells were washed once with culture medium, and 50 μL of 10% FBS/RPMI-1640 medium was added to prepare the target cells.

Chromium Release Assay (ADCC Activity)

[0072]A volume of 50 μL of antibody solution prepared at various concentrations was added to the target cells, and allowed for reacting on ice for 15 min. Then 100 μL of human PBMC solution (5×10⁵ cells/well) was added, and the cells were incubated for 4 h at 37° C. in a 5% CO₂ gas incubator. After culturing, the plate was centrifuged and the radioactivity in 100 μL of culture supernatant was measured with a gamma-counter. The specific chromium release rate was determined by the following formula.

Specific chromium release rate (%)=(A-C)×100/(B-C)

In this formula, A represents the mean value of radioactivity (cpm) in each well; B represents the mean value of radioactivity (cpm) in a well wherein 100 μL of 2% NP-40 aqueous solution (Nonidet P-40, Code No. 252-23, Nacalai Tesque) and 50 μL of 10% FBS/RPMI medium were added to the target cells; and C represents the mean value of radioactivity (cpm) in a well wherein 150 μL of 10% FBS/RPMI medium was added to the target cells. The assay was conducted in triplicate, and the means and standard deviations of ADCC activity (%) were calculated.

[0073]FIGS. 2 and 3 show the ADCC activity of the anti-GPC3 chimeric antibody measured using PBMC. In the figures, the vertical axis represents cytotoxic activity (%) and the horizontal axis represents the concentration (μg/mL) of antibody added. FIG. 2 shows the results when HepG2 cells are used as the target cells and FIG. 3 shows the results for HuH-7 cells. The open circles show the activity of chimeric GC33 antibody produced by CHO cells, and the filled circles show the activity of chimeric GC33 antibody produced by YB2/0 cells. The low fucose type GC33 chimeric antibody produced by the YB2/0 cells shows stronger ADCC activity than the GC33 chimeric antibody produced by CHO cells, clearly indicating that ADCC activity of the anti-GPC3 antibody is enhanced by sugar chain modification.

Example 5

Establishment of Antibody Producing Cells

[0074]Hygromycin B was added to SFMII(+) medium at a final concentration of 1 mg/mL, and a fucose transporter deficient cell line (clone 3F2) was subcultured in the medium. A suspension of 3F2 cells in Dulbecco phosphate buffer (8×10⁶ cells/0.8 mL) was prepared. To the cell suspension, 25 μg of antibody expression vector was added (Reference Examples 1 and 2), and the cell suspension was transferred to a Gene Pulser Cuvette. After the cuvette was let stand on ice for 10 min, the vector was introduced into the cells by electroporation using a GENE-PULSER II at 1.5 kV and 25 μFD. The cells were suspended in 40 mL of SFMII(+) medium and transferred to a 96-well flat bottom plate (Iwaki) at 100 μL/well. After the plate was incubated in a CO₂ incubator for 24 h at 37° C., Geneticin (Invitrogen, Cat. No. 10131-027) was added at a final concentration of 0.5 mg/mL. The amount of antibody produced by the drug-resistant cells was measured to establish humanized anti-GPC3 antibody producing cell lines.

Example 6

Antibody Purification

[0075]The supernatant from the antibody expressing cell line was collected and loaded on Hitrap® rProtein A column (Pharmacia Cat. No. 17-5080-01) using a P-1 pump (Pharmacia). After the column was washed with a binding buffer (20 mM sodium phosphate (pH 7.0)), and the protein was eluted with an elution buffer (0.1 M Glycin-HCl (pH 2.7)). The eluate was immediately neutralized with neutralizing buffer (1 M Tris-HCl (pH 9.0)). The antibody elution fractions were selected by DC protein assay (BIO-RAD Cat. No. 500-0111) and pooled, and were concentrated to about 2 mL with a Centriprep-YM10 (Millipore Cat. No. 4304). Next, the antibodies were separated by gel filtration using a Superdex 200 26/60 column (Pharmacia) equilibrated with 20 mM acetate buffer with 150 mM NaCl (pH 6.0). The monomer fraction peaks were collected, concentrated with Centriprep-YM10, and filtered through MILLEX-GW 0.22 μm Filter Unit (Millipore Cat. No. SLGV 013SL), and then preserved at 4° C. The absorption at 280 nm was measured and the concentration of purified antibody was calculated from the molar absorption coefficient.

Example 7

In vitro ADCC Activity of Humanized Anti-GPC3 Antibody Produced by FT-KO Cells

[0076]FIG. 4 shows the in vitro ADCC activity of anti-GPC3 antibody produced by FT-KO cells when human PBMC is used. The method is as described in Example 4. In the figure, the vertical axis represents cytotoxic activity (%) and the horizontal axis represents the concentration (μg/mL) of antibody added. HuH-7 cells were used as the target cells. The open circles show the activity of anti-GPC3 antibody produced by wild type CHO cells, and the filled circles show the activity of anti-GPC3 antibody produced by FT-KO cells. The low fucose type anti-GPC3 antibody produced by the FT-KO cells shows stronger ADCC activity than the anti-GPC3 antibody produced by wild type CHO cells, clearly indicating that ADCC activity of the anti-GPC3 antibody produced by FT-KO cells is enhanced.

Example 8

Analysis of Sugar Chains of Humanized Anti-GPC3 Antibody Produced by FT-KO Cells

1. Preparation of 2-Aminobenzamide-Labeled Sugar Chains (2-AB Labeled Sugar Chains)

[0077]The antibodies produced by the FT-KO cells of the present invention and antibodies produced by CHO cells as a control sample were treated with N-Glycosidase F (Roche Diagnostics) to release the sugar chains from the protein (Weitzhandler M. et al., Journal of Pharmaceutical Sciences 83:12 (1994), 1670-1675). After removing the protein with ethanol (Schenk B. et al., The Journal of Clinical Investigation 108:11 (2001), 1687-1695), the sugar chains were concentrated and dried, and fluorescent labeled with 2-aminopyridine (Bigge J. C. et al., Analytical Biochemistry 230:2 (1995), 229-238). The reagent was removed from the 2-AB labeled sugar chains by solid phase extraction using a cellulose cartridge, and after concentration by centrifugation, purified 2-AB labeled sugar chains were obtained. Next, the purified 2-AB labeled sugar chains were treated with β-galactosidase (Seikagaku Corp.) to obtain agalactosyl 2-AB labeled sugar chains.

2. Analysis of Agalactosyl 2-AB Labeled Sugar Chains by Normal Phase HPLC

[0078]The antibodies produced by the FT-KO cells of the present invention and the antibodies produced by the CHO cells as a control sample were prepared as agalactosyl 2-AB labeled sugar chains according to the above method, and analyzed by normal phase HPLC using an amide column (Tosoh Corp. TSKgel Amide-80), and the chromatograms were compared. In the antibodies produced by the CHO cells, the main component is G(0), and G(0)-Fuc accounts for about 4% of the peak area. On the other hand, in the antibodies produced by the FT-KO cells, G(0)-Fuc is the main component, and is present at not less than 90% of the peak area in each of the cell lines (FIG. 5 and Table 2). FIG. 6 shows the putative structures for peaks G(0) and G(0)-Fuc.

TABLE-US-00002 TABLE 2 Relative ratio of sugar chains estimated from normal phase HPLC analysis of agalactosyl 2-AB sugar chains Sugar chain CHO FT-KO-a FT-KO-b FT-KO-c G(0)-Fuc 4.0% 92.4% 92.5% 93.2% G(0) 96.0% 7.6% 7.5% 6.8%

Example 9

Thermal Stability Analysis of Humanized Anti-GPC3 Antibody Produced by FT-KO Cells

1. Preparation of Sample Solution for DSC Measurement

[0079]The external dialysis solution was 20 mol/L sodium acetate buffer (pH 6.0) containing 200 mmol/L sodium chloride. A dialysis membrane filled with 700 μg equivalents of antibody solution was dialyzed by immersing in the external dialysis solution overnight to prepare a sample solution.

2. Measurement of Thermal Degradation Temperature by DSC

[0080]After both the sample solution and reference solution (external dialysis solution) were thoroughly degassed, they were each placed in the calorimeter and thermally equilibrated at 20° C. Next DSC measurement was carried out from 20° C. to 100° C. at a scan rate of approximately 1 K/min. The result is represented by the tip of the degradation peak as a function of temperature (FIG. 7). The thermal degradation temperature of the antibodies produced by CHO cells and the antibodies produced by FT-KO cells were found to be equivalent.

Reference Example 1

Humanization of GC33

[0081]Antibody sequence data were obtained from the publicly disclosed Kabat Database located at ftp.ebi.ac.uk/pub/databases/kabat/ and the ImMunoGeneTics Database (IMGT), and the H chain variable region and L chain variable region were separately subjected to a homology search. It was found that the H chain variable region has a high level of homology with DN13 (Smithson et al., Mol. Immunol. 1999; 36: 113-124). It was also found that the L chain variable region has a high level of homology with the Homo sapiens IGK mRNA for immunoglobulin kappa light chain VLJ region, partial cds, clone:K64 of Accession Number AB064105. The signal sequence of Accession Number 540357, which has a high level of homology with AB064105, was used as the L chain signal sequence. Then the CDR was grafted to the FR of these antibodies to prepare a humanized antibody.

[0082]More specifically, synthetic oligo-DNAs of approximately 50 base were designed in such a manner that approximately 20 bases of them were hybridized each other, and these synthetic oligo-DNAs were was assembled by PCR to prepare a gene encoding each variable region. They were digested at the HindIII sequence inserted at the terminus on the 5' end of the synthetic oligo-DNA and the BamHI sequence inserted at the terminus on the 3' end of the synthetic oligo-DNA, and the synthetic oligo-DNA was cloned into an expression vector HEFgyl where the human IgG1 constant region was cloned, or to the expression vector HEFgγ1 where the human κ-chain constant region was cloned (Sato et al., Mol. Immunol., 1994; 371-381). The H chain and L chain of the humanized GC33 constructed as above were each designated ver.a. The humanized GC33 (ver.a/ver.a) wherein both the H chain and L chain were ver.a had lower binding activity than an antibody with the mouse GC33 variable regions (mouse/mouse). Chimeric antibodies were prepared by combining mouse GC33 sequences and ver.a sequences for the H chains and L chains (mouse/ver.a, ver.a/mouse), and the binding activity was evaluated. Lower binding activity was found with ver.a/mouse antibody, indicating that the decrease in binding activity is due to amino acid replacement was attributed to the H chain. Then modified H chains designated ver.c, ver.f, ver.h, ver.i, ver.j, and ver.k were prepared. All humanized GC33 antibodies exhibited the same level of binding activity as the chimeric antibody having mouse GC33 variable regions. The nucleotide sequences of the humanized GC33H chain variable regions ver.a, ver.c, ver.f, ver.h, ver.i, ver.j, and ver.k are shown in SEQ ID NOS:74, 75, 76, 77, 78, 79, and 80, and the amino acid sequences thereof are shown in SEQ ID NOS: 81, 82, 83, 84, 85, 86, and 87, respectively. The nucleotide sequence of the humanized GC33 L chain variable region ver.a is shown in SEQ ID NO: 88, and the amino acid sequence thereof is shown in SEQ ID NO: 89, respectively. In the humanized GC33H chain variable regions ver.i, ver.j, and ver.k, the sixth glutamic acid was replaced by a glutamine. These antibodies exhibited markedly increased thermal stability.

Reference Example 2

Alteration of Humanized GC33 L Chain

[0083]With respect to protein deamidation, the reaction rate constant of deamidation was known to be dependent on the primary sequence. It is also known that Asn-Gly is particularly susceptible to deamidation (Rocinson et al., Proc. Natl. Acad. Sci. USA 2001; 98: 944-949). Because Asn 33 within CDR1 of the humanized GC33 L chain ver.a variable region of SEQ ID NO: 88 has the primary sequence Asn-Gly, this residue is predicted to be susceptible to deamidation.

[0084]To evaluate the effect of deamidation of Asn 33 on binding activity of the antibody, a modified antibody was prepared wherein Asn 33 was replaced with Asp. Quick Change Site-Directed Mutagenesis Kit (Stratagene) was used for introducing a point mutation. More specifically, 50 μL of reaction solution containing 125 ng of sense primer (CTT GTA CAC AGT GAC GGAAAC ACC TAT: SEQ ID NO: 124), 125 ng of antisense primer (ATA GGT GTT TCC GTC ACT GTG TAC AAG: SEQ ID NO: 125), 5 μL of 10× reaction buffer, 1 μL of dNTP mix, 10 ng of HEFgκ to which humanized GC33 L chain ver.a had been cloned, and 1 μL of Pfu Turbo DNA Polymerase was run through 12 cycles consisting of 30 sec at 95° C., 1 min at 55° C., and 9 min at 68° C. The reaction product was digested with the restriction enzyme DpnI for 2 h at 37° C., and introduced into XL1-Blue competent cells to obtain transformants. The variable region was cut out from the clones containing the correct mutation and cloned again into the expression vector HEFgx. The expression vector HEFgyl containing humanized GC33H chain ver.k was introduced into COS7 cells by using Fugene 6 (Roche). Culture medium supernatant was collected from the cells transiently expressing the modified antibody. The antibody concentration was quantitated by sandwich ELISA using anti-human IgG antibody, and binding activity of the modified antibody was evaluated by ELISA using a plate coated with a soluble GPC3 core protein. Binding activity was lost in the modified antibody (N33D) in which Asn 33 was replaced by Asp, suggesting that the binding activity is significantly affected by deamidation at Asn 33.

[0085]Deamidation of Asn 33 was reported to be suppressed by replacing Gly 34 with another amino acid residue (WO 03057881 A1). In accordance with that method, a series of modified antibodies were prepared by replacing G34 with 17 other amino acid residues except for Cys and Met using the Quick Chane Site-Directed Mutagenesis Kit to prepare G34A, G34D, G34E, G34F, G34H, G34N, G34P, G34Q, G34I, G34K, G34L, G34V, G34W, G34Y, G34R, G34S, and G34T. The binding activity of the antibodies was evaluated using culture supernatant of COS7 cells transiently expressing the antibodies. It was revealed that binding activity is maintained even if G34 is replaced with another amino acid residues other than Pro (G34P), and Val (G34V).

[0086]The amino acid sequences of the L chain CDR1 of the modified antibodies are represented by SEQ ID NO: 90 (G34A), SEQ ID NO: 91 (G34D), SEQ ID NO: 92 (G34E), SEQ ID NO: 93 (G34F), SEQ ID NO: 94 (G34H), SEQ ID NO: 95 (G34N), SEQ ID NO: 96 (G34T), SEQ ID NO: 97 (G34Q), SEQ ID NO: 98 (G34I), SEQ ID NO: 99 (G34K), SEQ ID NO: 100 (G34L), SEQ ID NO: 101 (G34S), SEQ ID NO: 102 (G34W), SEQ ID NO: 103 (G34Y), SEQ ID NO: 104 (G34R), SEQ ID NO: 105 (G34V), and SEQ ID NO: 106 (G34P), respectively. The amino acid sequences of the L chain variable regions of the modified antibodies are represented by SEQ ID NO: 107 (G34A), SEQ ID NO: 108 (G34D), SEQ ID NO: 109 (G34E), SEQ ID NO: 110 (G34F), SEQ ID NO: 111 (G34H), SEQ ID NO: 112 (G34N), SEQ ID NO: 113 (G34T), SEQ ID NO: 114 (G34Q), SEQ ID NO: 115 (G34I), SEQ ID NO: 116 (G34K), SEQ ID NO: 117 (G34L), SEQ ID NO: 118 (G34S), SEQ ID NO: 119 (G34W), SEQ ID NO: 120 (G34Y), SEQ ID NO: 121 (G34R), SEQ ID NO: 122 (G34V), and SEQ ID NO: 123 (G34P), respectively.

Reference Example 3

Destruction of Fucose Transporter Gene in CHO Cells

1. Construction of Targeting Vector

[0087](1) Preparation of KO1 vector

[0088]The hygromycin resistance gene (Hygr) was constructed by PCR with Hyg5-BH and Hyg3-NT primers from pcDNA3.1/Hygro (Invitrogen), which has a sequence identical to the 5' portion of the fucose transporter gene start codon by attaching a BamHI site and TGCGC sequence to the 5' portion of the start codon and a NotI site added to the 3' portion containing the region up to the SV40 polyA addition signal, and the Hygr fragment was cut off.

TABLE-US-00003 Forward primer (SEQ ID NO: 128) Hyg5-BH 5'-GGA TCC TGC GCA TGA AAA AGC CTG AAC TCA CC-3' Reverse primer (SEQ ID NO: 129) Hyg3-NT 5'-GCG GCC GCC TAT TCC TTT GCC CTC GGA CG-3'

[0089]The fucose transporter targeting vector ver.1 (hereinafter designated the KO1 vector) was constructed by inserting the 5' portion of the fucose transporter (from the SmaI at base No. 2780 to the BamHI at base No. 4323 of the nucleotide sequence shown in SEQ ID NO: 126), the 3' portion (from base No. 4284 to the Sad at base No. 10934), and an Hygr fragment into pMC1DT-A vector (Yagi T, Proc. Natl. Acad. Sci. USA, Vol. 87, p. 9918-9922, 1990). The characteristic of the KO1 vector is that Hygr will be expressed from the fucose transporter promoter when homologous recombination takes place because no promoter is attached to the Hygr fragment. However, Hygr is not always expressed to the extent that resistance to hygromycin B is acquired if only one copy of the vector is inserted into a cell by homologous recombination. The KO1 vector was cleaved by NotI and introduced into the cell. It is expected that the fucose transporter will lose 41 base pairs of exon 1 including the start codon by introduction of the KO1 vector, which will result in the loss of its function.

(2) Preparation of pBSK-pgk-1-Hygr

[0090]The pBSK-pgk-1 vector was prepared by cutting off the mouse pgk-1 gene promoter from pKJ2 vector (Popo H, Biochemical Genetics, Vol. 28, p. 299-308, 1990) with EcoRI-PstI, and cloning it into the EcoRI-PstI site of pBluescript (Stratagene). By PCR with the HygS-AV and Hyg3-BH primers from pcDNA3.1/Hygro, an EcoT22I site and Kozak sequence were attached to the 5' portion of Hygr, and a BamHI site was added to the 3' portion containing the region up to the SV40 poly A addition signal, and then the Hygr fragment was cut off

TABLE-US-00004 Forward primer (SEQ ID NO: 130) Hyg5-AV 5'-ATG CAT GCC ACC ATG AAA AAG CCT GAA CTC ACC-3' Reverse primer (SEQ ID NO: 131) Hyg3-BH 5'-GGA TCC CAG GCT TTA CAC TTT ATG CTT C-3'

[0091]The pBSK-pgk-1-Hygr vector was prepared by inserting the Hygr (EcoT22I-BamHI) fragment into the PstI-BamHI site of pBSK-pgk-1.

(3) Preparation of the KO2 Vector

[0092]The fucose transporter targeting vector ver.2 (hereinafter designated the KO2 vector) was constructed by inserting the 5' portion of the fucose transporter (from the SmaI at base No. 2780 to the BamHI at base No. 4323 of the nucleotide sequence shown in SEQ ID NO: 126), the 3' portion (from base No. 4284 to the Sad at base No. 10934), and pgk-1Hygr fragment into pMC1DT-A vector. Unlike the KO1 vector, KO2 vector will confer resistance to hygromycin B even if only one copy of the vector is inserted by homologous recombination because the pgk-1 gene promoter is attached to Hygr. The KO2 vector was cleaved by NotI and inserted into the cells. It is expected that the fucose transporter will lose 46 base pairs of exon 1 including the start codon by the introduction of the KO2 vector, which will result in the loss of its function.

(4) Preparation of pBSK-pgk-1-Puror

[0093]The pBSK-pgk-1-Puror vector was prepared by cleaving pPUR vector (BD Biosciences) with PstI and BamHI, and inserting the digested fragment (Puror) into the PstI-BamHI site of pBSK-pgk-1.

(5) Preparation of the KO3 Vector

[0094]The fucose transporter targeting vector ver.3 (hereinafter designated the KO3 vector) was constructed by inserting the 5' portion of the fucose transporter (from the SmaI at base No. 2780 to the BamHI at base No. 4323 of the nucleotide sequence shown in SEQ ID NO: 126), the 3' portion (from base No. 4284 to the Sad at base No. 10934), and pgk-1-Puror fragment into pMC1DT-A vector. In addition, a sequence for binding with the primer for screening shown below was attached to the 3' end of pgk-1-Puror. The KO3 vector was cleaved by NotI and inserted into the cells. It is expected that the fucose transporter will lose 46 base pairs of exon 1 including the start codon by the introduction of the KO3 vector, which will result in the loss of its function.

TABLE-US-00005 Reverse primer (SEQ ID NO: 132) RSGR-A 5'-GCT GTC TGG AGT ACT GTG CAT CTG C-3'

[0095]The above three species of targeting vectors were used to knock out the fucose transporter gene.

2. Introduction of Vectors into CHO Cells

[0096]HT Supplement (100×) (Invitrogen Cat. No. 11067-030) and penicillin-streptomycin (Invitrogen Cat. No. 15140-122) were added to CHO-S-FMII HT (Invitrogen Cat. No. 12052-098), each at a volume of 1/100 with respect to the volume of CHO-S-SFMII HT. CHO DXB11 cells were subcultured in the culture medium (hereinafter designated SFMII(+)), and this SFMII(+) medium also used for culturing the cells after gene transfer. The CHO cells were suspended in Dulbecco phosphate buffer (hereinafter designated PBS, Invitrogen Cat. No. 14190-144) at a concentration of 8×10⁶ cells/0.8 mL. Then 30 μg of the targeting vector was added to the cell suspension, and the cell suspension was transferred to a Gene Pulser Cuvette (4 mm) (Bio-Rad, Cat. No. 1652088). After the cuvette was let stand on ice for 10 min, the vector was introduced into the cells by electroporation with a GENE-PULSER II (Bio-Rad, Code No. 340BR) at 1.5 kV and 25 μFD. After introduction of the vector, the cells were suspended in 200 mL of SFMII(+) medium and transferred to twenty 96-well flat bottomed plates (Iwaki, Cat. No. 1860-096) at 100 μL/well. The plates were incubated in a CO₂ incubator for 24 h at 37° C., and then the reagent was added.

3. Knockout Step 1

[0097]Either the KO1 or KO2 vector was introduced into the CHO cells, and after 24 h the cells were selected using hygromycin B (Invitrogen, Cat. No. 10687-010). Hygromycin B was dissolved in the SFMII(+) up to a concentration of 0.3 mg/mL and was added at 100 μL/well.

4. Screening for Homologous Recombinants by PCR

(1) Preparation of PCR Sample

[0098]Screening for homologous recombinants was carried out by PCR. The CHO cells used in screening were cultured in 96-well plates. After the supernatant was removed, 50 μL/well of buffer for cytolysis was added, and the cells were first heated at 55° C. for 2 h and then 95° C. for 15 min to inactivate protease K to prepare PCR template. The buffer for cytolysis consisted of 5 μL of 10×LA buffer II (Takara Bio Inc., LA Taq added), 2.5 μL of 10% NP-40 (Roche, Cat. No. 1 332 473), 4 μL of proteinase K (20 mg/mL, Takara Bio, Inc. Cat. No. 9033), and 38.5 μL of distilled water (Nacalai Tesque Cat. No. 36421-35) per well.

(2) PCR Conditions

[0099]PCR reaction mixture contained 1 μL of the above PCR sample, 5 μL of 10×LA buffer II, 5 μL of MgCl₂ (25 mM), 5 μL of dNTP (2.5 mM), 2 μL of primer (10 μM each), 0.5 μL of La Taq (5 IU/μL, Cat. No. RR002B) and 29.5 μL of distilled water (50 μL in total). For screening of cells containing the KO1 vector, TP-F4 and THygro-R1 were used as PCR primers, and for screening of cells containing the KO2 vector, TP-F4 and THygro-F1 were used as PCR primers.

[0100]PCR conditions for the cells containing the KO1 vector consisted of preheating at 95° C. for 1 min, conducting 40 cycles of the amplification cycle of 30 sec at 95° C., 30 sec at 60° C., and 2 min at 60° C., and then reheating at 72° C. for 7 min. PCR conditions for the cells containing the KO2 vector consisted of preheating at 95° C. for 1 min, 40 cycles of the amplification cycle of 30 sec at 95° C. and 3 min at 70° C., and then reheating at 70° C. for 7 min.

[0101]The primers used are listed below. In cell samples wherein homologous recombination occurred with the KO1 vector or the KO2 vector, DNA of approximately 1.6 kb or 2.0 kb will be amplified, respectively. The primer TP-F4 was designed for the 5' genome region of the fucose transporter outside of the vector, and THygro-F1 and THygro-R1 were designed for the Hygr gene inside of the vector.

TABLE-US-00006 Forward primer (KO1, KO2) (SEQ ID NO: 133) TP-F4 5'-GGA ATG CAG CTT CCT CAA GGG ACT CGC-3' Reverse primer (KO1) (SEQ ID NO: 134) THygro-R1 5'-TGC ATC AGG TCG GAG ACG CTG TCG AAC-3' Reverse primer (KO2) (SEQ ID NO: 135) THygro-F1 5'-GCA CTC GTC CGA GGG CAA AGG AAT AGC-3'

5. PCR Screening Results

[0102]A total of 918 cells containing the KO1 vector were analyzed, and 1 cell was appeared to be a homologous recombinant (homologous recombination rate: approximately 0.1%). A total of 537 cells containing the KO2 vector were analyzed and 17 cells were appeared to be homologous recombinants (homologous recombination rate: approximately 3.2%).

6. Southern Blot Analysis

[0103]Homologous recombination was further confirmed by Southern blot. A total of 10 μg of genomic DNA was prepared from the cultured cells by the standard method to be analyzed in Southern blotting. PCR was conducted using the two primers listed below to prepare a 387 by probe corresponding to the region of base Nos. 2113 to 2500 of the nucleotide sequence shown in SEQ ID NO: 126, which was used in Southern blotting. The genomic DNA was cleaved by BglII.

TABLE-US-00007 Forward primer (SEQ ID NO: 136) Bgl-F: 5'-TGT GCT GGG AAT TGA ACC CAG GAC-3' Reverse primer (SEQ ID NO: 137) Bgl-R: 5'-CTA CTT GTC TGT GCT TTC TTC C-3'

[0104]As a result of cleavage with BglII, the blot will show a band of approximately 30 kb from the fucose transporter chromosome, approximately 4.6 kb from the chromosome wherein homologous recombination with the KO1 vector occurred, and approximately 5.0 kb from the chromosome wherein homologous recombination with the KO2 vector occurred. The experiment comprised 1 cell from homologous recombination with the KO1 vector and 7 cells from homologous recombination with the KO2 vector. The only cell obtained from the homologous recombination with the KO1 vector was first designated 5C1, but later analysis revealed that this cell consisted of multiple cell populations. Therefore the cells were cloned by limiting dilution before used in the experiment. One of the cells obtained with the KO2 vector was designated 6E2.

7. Knockout Step 2

[0105]The three vectors were used to establish cell lines completely defective of fucose transporter gene from the cells wherein homologous recombination with the KO1 and KO2 vectors took place. The combinations of vectors and cells was as follows: Method 1 combined the KO2 vector and 5C1 cells (KO1), Method 2 combined the KO2 vector and 6E2 cells (KO2), and Method 3 combined the KO3 vector and 6E2 cells (KO2). Each vector was introduced into the appropriate cells, and after 24 h selection was started using hygromycin B and puromycin (Nacalai Tesque, Cat. No. 29455-12). The final concentration of hygromycin B was set to 1 mg/mL in Method 1, and 7 mg/mL in Method 2. In Method 3 hygromycin B was added at a final concentration of 0.15 mg/mL and puromycin at a final concentration of 8 ng/mL.

8. Screening for Homologous Recombinants by PCR

[0106]For screening of cells from Method 1, PCR was carried out to detect cells having homologous recombination with both KO1 and KO2 vectors. For screening of cells from Method 2 the following PCR primers were designed: TPS-F1 was configured in the region from base Nos. 3924 to 3950 of SEQ ID NO: 126, and SHygro-R1 was configured in the region from base Nos. 4248 to 4274. These primers will amplify 350 by of the fucose transporter gene region containing a deletion due to the KO2 vector. Therefore, in the PCR screening in Method 2, those dells providing no amplification product of 350 bp are considered to be completely lacking the fucose transporter gene. The PCR conditions consisted of preheating for 1 min at 95° C., 35 cycles of the amplification cycle of 30 sec at 95° C. and 1 min at 70° C., and reheating for 7 min at 70° C.

TABLE-US-00008 Forward primer (SEQ ID NO: 138) TPS-F1: 5'-CTC GAC TCG TCC CTA TTA GGC AAC AGC-3' Reverse primer (SEQ ID NO: 139) SHygro-R1: 5'-TCA GAG GCA GTG GAG CCT CCA GTC AGC-3'

[0107]For Method 3, TP-F4 was used as the forward primer and RSGR-A was used as the reverse primer. The PCR conditions consisted of preheating for 1 min at 95° C., 35 cycles of the amplification cycle of 30 sec at 95° C., 30 sec at 60° C., and 2 min at 72° C., and reheating for 7 min at 72° C. In the sample of cells having homologous recombination with the KO3 vector, DNA of approximately 1.6 kb will be amplified. This PCR procedure will detect those cells having homologous recombination with the KO3 vector and also those still having homologous recombination with the KO2 vector.

9. PCR Screening Results

[0108]In Method 1, a total of 616 cells were analyzed, and 18 cells were appeared to be homologous recombinants (homologous recombination rate: 2.9%). In Method 2, a total of 524 cells were analyzed, and 2 cells were appeared to be homologous recombinants (homologous recombination rate: 0.4%). In addition, in Method 3, a total of 382 cells were analyzed, and 7 cells were appeared to be homologous recombinants (homologous recombination rate: 1.8%).

10. Southern Blot Analysis

[0109]Southern blotting was carried out according to the method described above. Among the cells analyzed, 1 cell completely lacking the fucose transporter gene was found. In knockout step 1, the analysis results of PCR and Southern blotting were consistent, but not in knockout step 2. Possible causes are as follows: 1. In Method 1 cells having homologous recombination independently with either KO1 or KO2 were mixed together; 2. The fucose transporter gene is present not as one pair (2 genes) but as multiple pairs (or not less than 3 genes); and 3. During the culture of the cell lines established in the knockout step 1, the copy number of the fucose transporter gene remaining in the subcultured cells increased.

11. Analysis of Fucose Expression

[0110]Fucose expression was analyzed by PCR in 26 cells found to be homologous recombinants. A total of 1×10⁶ cells were stained on ice for 1 h with 100 μL of PBS containing 5 mg/mL of Lens culinaris Agglutinin, FITC conjugate (Vector Laboratories, Cat. No. FL-1041) 2.5% FBS, and 0.02% sodium azide (hereinafter designated as FACS solution). Then the cells were rinsed 3 times with FACS solution and analyzed with FACSCalibur (Becton Dickinson). The results clearly showed that fucose expression is decreased only in the cells found to be completely lacking the fucose transporter gene in the Southern blot analysis.

[0111]The above results have revealed the following:

[0112]From the fact that only one cell out of 616 cells screened has complete deletion of the fucose transporter gene, the frequency of homologous recombination was very low at approximately 0.16%. As noted above, there are several possible reasons why the results of PCR and Southern blot analysis in knockout step 2 was not consistent. However, the cell lines obtained in Method 3 may not comprise a mixture of cells having homologous recombination independently with the KO2 and KO3 vectors, because selection was made using two types of drugs. In addition, it is unlikely that all of the other cell lines having homologous recombination by PCR comprise multiple cell populations. As noted above, if there are 3 or more fucose transporter genes present, targeting of the gene in cells would be particularly difficult. Homologous recombinants could be obtained only by using a vector such as the KO1 vector where Hyger is hardly expressed and by screening a large number of cells.

Sequence CWU 1

13911743DNAHomo sapiens 1atggccggga ccgtgcgcac cgcgtgcttg gtggtggcga tgctgctcag cttggacttc 60ccgggacagg cgcagccccc gccgccgccg ccggacgcca cctgtcacca agtccgctcc 120ttcttccaga gactgcagcc cggactcaag tgggtgccag aaactcccgt gccaggatca 180gatttgcaag tatgtctccc taagggccca acatgctgct caagaaagat ggaagaaaaa 240taccaactaa cagcacgatt gaacatggaa cagctgcttc agtctgcaag tatggagctc 300aagttcttaa ttattcagaa tgctgcggtt ttccaagagg cctttgaaat tgttgttcgc 360catgccaaga actacaccaa tgccatgttc aagaacaact acccaagcct gactccacaa 420gcttttgagt ttgtgggtga atttttcaca gatgtgtctc tctacatctt gggttctgac 480atcaatgtag atgacatggt caatgaattg tttgacagcc tgtttccagt catctatacc 540cagctaatga acccaggcct gcctgattca gccttggaca tcaatgagtg cctccgagga 600gcaagacgtg acctgaaagt atttgggaat ttccccaagc ttattatgac ccaggtttcc 660aagtcactgc aagtcactag gatcttcctt caggctctga atcttggaat tgaagtgatc 720aacacaactg atcacctgaa gttcagtaag gactgtggcc gaatgctcac cagaatgtgg 780tactgctctt actgccaggg actgatgatg gttaaaccct gtggcggtta ctgcaatgtg 840gtcatgcaag gctgtatggc aggtgtggtg gagattgaca agtactggag agaatacatt 900ctgtcccttg aagaacttgt gaatggcatg tacagaatct atgacatgga gaacgtactg 960cttggtctct tttcaacaat ccatgattct atccagtatg tccagaagaa tgcaggaaag 1020ctgaccacca ctattggcaa gttatgtgcc cattctcaac aacgccaata tagatctgct 1080tattatcctg aagatctctt tattgacaag aaagtattaa aagttgctca tgtagaacat 1140gaagaaacct tatccagccg aagaagggaa ctaattcaga agttgaagtc tttcatcagc 1200ttctatagtg ctttgcctgg ctacatctgc agccatagcc ctgtggcgga aaacgacacc 1260ctttgctgga atggacaaga actcgtggag agatacagcc aaaaggcagc aaggaatgga 1320atgaaaaacc agttcaatct ccatgagctg aaaatgaagg gccctgagcc agtggtcagt 1380caaattattg acaaactgaa gcacattaac cagctcctga gaaccatgtc tatgcccaaa 1440ggtagagttc tggataaaaa cctggatgag gaagggtttg aaagtggaga ctgcggtgat 1500gatgaagatg agtgcattgg aggctctggt gatggaatga taaaagtgaa gaatcagctc 1560cgcttccttg cagaactggc ctatgatctg gatgtggatg atgcgcctgg aaacagtcag 1620caggcaactc cgaaggacaa cgagataagc acctttcaca acctcgggaa cgttcattcc 1680ccgctgaagc ttctcaccag catggccatc tcggtggtgt gcttcttctt cctggtgcac 1740tga 17432580PRTHomo sapiens 2Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu1 5 10 15Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp 20 25 30Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly 35 40 45Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val 50 55 60Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys65 70 75 80Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala 85 90 95Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln 100 105 110Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala 115 120 125Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe 130 135 140Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp145 150 155 160Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro 165 170 175Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu 180 185 190Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe 195 200 205Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln 210 215 220Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile225 230 235 240Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 245 250 255Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys 260 265 270Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly 275 280 285Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu 290 295 300Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu305 310 315 320Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys 325 330 335Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser 340 345 350Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile 355 360 365Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu 370 375 380Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser385 390 395 400Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala 405 410 415Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr 420 425 430Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His 435 440 445Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp 450 455 460Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys465 470 475 480Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly 485 490 495Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly 500 505 510Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr 515 520 525Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro 530 535 540Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser545 550 555 560Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe 565 570 575Phe Leu Val His 5803115PRTMus musculus 3Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Lys Trp Ile 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ala 1154112PRTMus musculus 4Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 11055PRTMus musculus 5Asn Tyr Ala Met Ser1 5617PRTMus musculus 6Ala Ile Asn Asn Asn Gly Asp Asp Thr Tyr Tyr Leu Asp Thr Val Lys1 5 10 15Asp75PRTMus musculus 7Gln Gly Gly Ala Tyr1 587PRTMus musculus 8Thr Tyr Gly Met Gly Val Gly1 5916PRTMus musculus 9Asn Ile Trp Trp Tyr Asp Ala Lys Tyr Tyr Asn Ser Asp Leu Lys Ser1 5 10 15108PRTMus musculus 10Met Gly Leu Ala Trp Phe Ala Tyr1 5117PRTMus musculus 11Ile Tyr Gly Met Gly Val Gly1 51216PRTMus musculus 12Asn Ile Trp Trp Asn Asp Asp Lys Tyr Tyr Asn Ser Ala Leu Lys Ser1 5 10 15138PRTMus musculus 13Ile Gly Tyr Phe Tyr Phe Asp Tyr1 5145PRTMus musculus 14Gly Tyr Trp Met His1 51517PRTMus musculus 15Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asn Tyr Asn Gln Lys Phe Lys1 5 10 15Gly1610PRTMus musculus 16Ser Gly Asp Leu Thr Gly Gly Leu Ala Tyr1 5 10175PRTMus musculus 17Ser Tyr Ala Met Ser1 51817PRTMus musculus 18Ala Ile Asn Ser Asn Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Met Lys1 5 10 15Asp1913PRTMus musculus 19His Asn Gly Gly Tyr Glu Asn Tyr Gly Trp Phe Ala Tyr1 5 10205PRTMus musculus 20Ser Tyr Trp Met His1 52117PRTMus musculus 21Glu Ile Asp Pro Ser Asp Ser Tyr Thr Tyr Tyr Asn Gln Lys Phe Arg1 5 10 15Gly2215PRTMus musculus 22Ser Asn Leu Gly Asp Gly His Tyr Arg Phe Pro Ala Phe Pro Tyr1 5 10 152317PRTMus musculus 23Thr Ile Asp Pro Ser Asp Ser Glu Thr His Tyr Asn Leu Gln Phe Lys1 5 10 15Asp2415PRTMus musculus 24Gly Ala Phe Tyr Ser Ser Tyr Ser Tyr Trp Ala Trp Phe Ala Tyr1 5 10 15255PRTMus musculus 25Asp Tyr Glu Met His1 52617PRTMus musculus 26Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys1 5 10 15Gly276PRTMus musculus 27Phe Tyr Ser Tyr Thr Tyr1 5285PRTMus musculus 28Ile Asn Ala Met Asn1 52919PRTMus musculus 29Arg Ile Arg Ser Glu Ser Asn Asn Tyr Ala Thr Tyr Tyr Gly Asp Ser1 5 10 15Val Lys Asp308PRTMus musculus 30Glu Val Thr Thr Ser Phe Ala Tyr1 5315PRTMus musculus 31Ala Ser Ala Met Asn1 53219PRTMus musculus 32Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp Ser1 5 10 15Val Lys Asp3312PRTMus musculus 33Asp Pro Gly Tyr Tyr Gly Asn Pro Trp Phe Ala Tyr1 5 10345PRTMus musculus 34Asp Tyr Ser Met His1 53517PRTMus musculus 35Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys1 5 10 15Gly362PRTMus musculus 36Leu Tyr13716PRTMus musculus 37Asn Ile Trp Trp His Asp Asp Lys Tyr Tyr Asn Ser Ala Leu Lys Ser1 5 10 153814PRTMus musculus 38Ile Ala Pro Arg Tyr Asn Lys Tyr Glu Gly Phe Phe Ala Phe1 5 103916PRTMus musculus 39Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn1 5 10 15407PRTMus musculus 40Leu Val Ser Lys Leu Asp Ser1 5419PRTMus musculus 41Trp Gln Gly Thr His Phe Pro Leu Thr1 54211PRTMus musculus 42Lys Ala Ser Gln Asp Ile Asn Asn Tyr Leu Ser1 5 10437PRTMus musculus 43Arg Ala Asn Arg Leu Val Asp1 54410PRTMus musculus 44Leu Gln Cys Asp Glu Phe Pro Pro Trp Thr1 5 104516PRTMus musculus 45Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His1 5 10 15467PRTMus musculus 46Lys Val Ser Asn Arg Phe Ser1 5479PRTMus musculus 47Ser Gln Ser Thr His Val Pro Trp Thr1 54816PRTMus musculus 48Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr1 5 10 15497PRTMus musculus 49Gln Met Ser Asn Leu Ala Ser1 5509PRTMus musculus 50Ala Gln Asn Leu Glu Leu Pro Tyr Thr1 55111PRTMus musculus 51Lys Ala Ser Gln Asp Ile Asn Lys Asn Ile Ile1 5 10527PRTMus musculus 52Tyr Thr Ser Thr Leu Gln Pro1 5539PRTMus musculus 53Leu Gln Tyr Asp Asn Leu Pro Arg Thr1 55411PRTMus musculus 54Arg Ala Ser His Ser Ile Ser Asn Phe Leu His1 5 10557PRTMus musculus 55Tyr Ala Ser Gln Ser Ile Ser1 5569PRTMus musculus 56Gln Gln Ser Asn Ile Trp Ser Leu Thr1 55715PRTMus musculus 57Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln1 5 10 15587PRTMus musculus 58Gly Ala Ser Asn Val Glu Ser1 5599PRTMus musculus 59Gln Gln Ser Arg Lys Val Pro Tyr Thr1 5609PRTMus musculus 60Ser Gln Asn Thr His Val Pro Pro Thr1 56116PRTMus musculus 61Lys Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Asn1 5 10 15627PRTMus musculus 62Trp Met Ser Asn Leu Ala Ser1 5639PRTMus musculus 63Met Gln His Ile Glu Tyr Pro Phe Thr1 56416PRTMus musculus 64Arg Ser Ser Lys Ser Leu Leu His Ser Tyr Asp Ile Thr Tyr Leu Tyr1 5 10 15659PRTMus musculus 65Ala Gln Asn Leu Glu Leu Pro Pro Thr1 56610PRTMus musculus 66Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr1 5 10677PRTMus musculus 67Asp Thr Ser Asn Leu Ala Ser1 5689PRTMus musculus 68Gln Gln Trp Ser Ser Tyr Pro Leu Thr1 56916PRTMus musculus 69Lys Ser Ser Gln Ser Leu Leu His Ser Asp Gly Lys Thr Phe Leu Asn1 5 10 15707PRTMus musculus 70Leu Val Ser Arg Leu Asp Ser1 5719PRTMus musculus 71Cys Gln Gly Thr His Phe Pro Arg Thr1 57216PRTMus musculus 72Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu1 5 10 15739PRTMus musculus 73Phe Gln Gly Ser His Val Pro Trp Thr1 574345DNAArtificial SequenceMouse-human chimeric antibody H chain 74caggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34575345DNAArtificial SequenceMouse-human chimeric antibody H chain 75caggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggacg aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34576345DNAArtificial SequenceMouse-human chimeric antibody H chain 76caggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34577345DNAArtificial SequenceMouse-human chimeric antibody H chain 77caggtgcagc tggtggagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgac atctgaggac acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34578345DNAArtificial SequenceMouse-human chimeric antibody H chain 78caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggacg aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34579345DNAArtificial SequenceMouse-human chimeric antibody H chain 79caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34580345DNAArtificial SequenceMouse-human chimeric antibody H chain 80caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc gactatgaaa tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggagct cttgatccta aaactggtga tactgcctac 180agtcagaagt tcaagggcag agtcacgctg accgcggaca aatccacgag cacagcctac

240atggagctga gcagcctgac atctgaggac acggccgtgt attactgtac aagattctac 300tcctatactt actggggcca gggaaccctg gtcaccgtct cctca 34581115PRTArtificial SequenceMouse-human chimeric antibody H chain 81Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11582115PRTArtificial SequenceMouse-human chimeric antibody H chain 82Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11583115PRTArtificial SequenceMouse-human chimeric antibody H chain 83Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11584115PRTArtificial SequenceMouse-human chimeric antibody H chain 84Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11585115PRTArtificial SequenceMouse-human chimeric antibody H chain 85Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11586115PRTArtificial SequenceMouse-human chimeric antibody H chain 86Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11587115PRTArtificial SequenceMouse-human chimeric antibody H chain 87Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser 11588336DNAArtificial SequenceMouse-human chimeric antibody L chain 88gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaaatac acatgttcct 300cctacgtttg gccaggggac caagctggag atcaaa 33689112PRTArtificial SequenceMouse-human chimeric antibody L chain 89Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 1109016PRTArtificial Sequencemutant antibody L chain 90Arg Ser Ser Gln Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His1 5 10 159116PRTArtificial Sequencemutant antibody L chain 91Arg Ser Ser Gln Ser Leu Val His Ser Asn Asp Asn Thr Tyr Leu His1 5 10 159216PRTArtificial Sequencemutant antibody L chain 92Arg Ser Ser Gln Ser Leu Val His Ser Asn Glu Asn Thr Tyr Leu His1 5 10 159316PRTArtificial Sequencemutant antibody L chain 93Arg Ser Ser Gln Ser Leu Val His Ser Asn Phe Asn Thr Tyr Leu His1 5 10 159416PRTArtificial Sequencemutant antibody L chain 94Arg Ser Ser Gln Ser Leu Val His Ser Asn His Asn Thr Tyr Leu His1 5 10 159516PRTArtificial Sequencemutant antibody L chain 95Arg Ser Ser Gln Ser Leu Val His Ser Asn Asn Asn Thr Tyr Leu His1 5 10 159616PRTArtificial Sequencemutant antibody L chain 96Arg Ser Ser Gln Ser Leu Val His Ser Asn Thr Asn Thr Tyr Leu His1 5 10 159716PRTArtificial Sequencemutant antibody L chain 97Arg Ser Ser Gln Ser Leu Val His Ser Asn Gln Asn Thr Tyr Leu His1 5 10 159816PRTArtificial Sequencemutant antibody L chain 98Arg Ser Ser Gln Ser Leu Val His Ser Asn Ile Asn Thr Tyr Leu His1 5 10 159916PRTArtificial Sequencemutant antibody L chain 99Arg Ser Ser Gln Ser Leu Val His Ser Asn Lys Asn Thr Tyr Leu His1 5 10 1510016PRTArtificial Sequencemutant antibody L chain 100Arg Ser Ser Gln Ser Leu Val His Ser Asn Leu Asn Thr Tyr Leu His1 5 10 1510116PRTArtificial Sequencemutant antibody L chain 101Arg Ser Ser Gln Ser Leu Val His Ser Asn Ser Asn Thr Tyr Leu His1 5 10 1510216PRTArtificial Sequencemutant antibody L chain 102Arg Ser Ser Gln Ser Leu Val His Ser Asn Trp Asn Thr Tyr Leu His1 5 10 1510316PRTArtificial Sequencemutant antibody L chain 103Arg Ser Ser Gln Ser Leu Val His Ser Asn Tyr Asn Thr Tyr Leu His1 5 10 1510416PRTArtificial Sequencemutant antibody L chain 104Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His1 5 10 1510516PRTArtificial Sequencemutant antibody L chain 105Arg Ser Ser Gln Ser Leu Val His Ser Asn Val Asn Thr Tyr Leu His1 5 10 1510616PRTArtificial Sequencemutant antibody L chain 106Arg Ser Ser Gln Ser Leu Val His Ser Asn Pro Asn Thr Tyr Leu His1 5 10 15107112PRTArtificial Sequencemutant antibody L chain 107Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110108112PRTArtificial Sequencemutant antibody L chain 108Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Asp Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110109112PRTArtificial Sequencemutant antibody L chain 109Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Glu Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110110112PRTArtificial Sequencemutant antibody L chain 110Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Phe Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110111112PRTArtificial Sequencemutant antibody L chain 111Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn His Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110112112PRTArtificial Sequencemutant antibody L chain 112Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Asn Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110113112PRTArtificial Sequencemutant antibody L chain 113Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Thr Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110114112PRTArtificial Sequencemutant antibody L chain 114Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gln Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110115112PRTArtificial Sequencemutant antibody L chain 115Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser

Gln Ser Leu Val His Ser 20 25 30Asn Ile Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110116112PRTArtificial Sequencemutant antibody L chain 116Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Lys Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110117112PRTArtificial Sequencemutant antibody L chain 117Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Leu Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110118112PRTArtificial Sequencemutant antibody L chain 118Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Ser Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110119112PRTArtificial Sequencemutant antibody L chain 119Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Trp Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110120112PRTArtificial Sequencemutant antibody L chain 120Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Tyr Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110121112PRTArtificial Sequencemutant antibody L chain 121Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110122112PRTArtificial Sequencemutant antibody L chain 122Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Val Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110123112PRTArtificial Sequencemutant antibody L chain 123Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Pro Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 11012427DNAArtificial SequenceArtificial Primer 124cttgtacaca gtgacggaaa cacctat 2712527DNAArtificial SequenceArtificial Primer 125ataggtgttt ccgtcactgt gtacaag 2712610939DNACricetulus griseus 126gagctcaatt aaccctcact aaagggagtc gactcgatcc tttacagaaa acttgcaaac 60cctcttggag tagaaaagta gtagtatctg acacaagtat cagcaaaatg caaacttctc 120cccatcccca gaaaaccatt ataaaaaccc ccatatctta tgcccaactg tagtgatata 180ttatttatga tttattaaaa cttgcttaag gattcagaaa gcaaagtcag ccttaagcta 240tagagaccag gcagtcagtg gtggtacaca cctttaatcc caggactcag gattaagaag 300tagacggacc tctgttagtt caagtctacc attacctaca caagagtgaa gagtaaccga 360tctcatgcct ttgatcccag cagctgggat catgtgcatt caatcccagc attcgggagt 420tatataagac aggagcaagg tctcagagct ggcattcatt ctccagccac attgaggata 480ggaaaacatt gaagtgtcag gatgctgagg agaggcagca gtttgaggtt tggtagaacc 540aggatcacct tttggtctga ggtagagtaa gaactgtggc tggctgcttt gcttttctga 600tcttcagctt gaagcttgaa ctccaatatt tgtctctggg tctattatta tcatgttaca 660cctaacttta aagctgattt acgcaagaca gttgtaggtg gacctttctt tcctgcccac 720cagttcccaa ataactgaca cggagactca atattaatta taaatgattg gttaatagct 780cagtcttgtt actggctaac tcttacattt taaattaact catttccatc cctttacttg 840ctgccatgtg gttcatggct tgttcaagtc ctgcttcttc tgtctctggc tggtgatgcc 900tctggttctg ccctttatcc cagaattctc ctagtctggc tctcctgccc agctataggc 960cagtcagctg tttattaacc aatgagaata atacatattt atagtgtaca aagattgctc 1020ctcaacaccc aattttttat gtgcaacctg agaatctgga ctcattgccc tcatgcttgc 1080agaggcggca cccttaccca ctaagccacc tttctagccc tgttgctttt gttttttgag 1140acaggttcca ctatgtagcc caggctggcc tcaaactgac cattctcctg cctaaacctc 1200ccgaacactg gaattatagt caaggcctac ctgccctggc attttcacac ttttatttcc 1260tggctgagtc cattgacttt acactcatca aggttgaacc agttggagtt taattacagt 1320gccaatcgca ctgaatccca cataatcaaa caacttcaag gaagcaaaaa accagttttt 1380cctgaagatc aatgtcagct tgcctgattc agaatagacc cccgaaaaaa ggcaaatgct 1440tgataaccaa tttcttctta ttgttcaatc ccctgctgct gtgtgtaagc tcctgagaaa 1500ggacagtaag gggacattca tgatcagaga aagagcccca actccccccc cagccccacc 1560cccaccctgt ccacagtctg ttggtttggt ttccccctgg ctgacaccca gaaatcacaa 1620cataatcacc taggtcactg taacaagttc ctttctggaa aatgctacaa atgatattgg 1680taacatgagt aatgaataat gcctggagtc caactccctt gtgacccagc aatgttttcc 1740gtgggtgctc ccttccccag ctgcaggcct gacatgtacc ttaaaaagcc tcccctggag 1800gacagaattt tgtgggtact atagtgttct cacaaatact tcccctaata cccttactta 1860gttaccataa ataacatgca gcccctggtg aggcacacag ggctccaatg tacagcttct 1920cagacactgc aggaaccttc ctctcctaat gcagcactgg tctcttcagg ctggacagca 1980ggaacccata ccactccaat cctagtgtgg agtagagctg tctacgaaaa ccagcagatc 2040tatagctaaa tgtgtttcaa ttttatgctt tgacaaattg tactgacccc acccccaccc 2100cttccccctt gctgtgctgg gaattgaacc caggaccttg tgcatgccag gcaagtactc 2160taacactgag ctatagcccc aatctttcat ccaagtctct atgtgtgccc acactcgctt 2220tttattttga gacaaaaggt tcttattttg agataaggtc tcactatgtt gccttgactt 2280tttttttttt ttttttttga acttttgacc ttcctacctc agctgagact acaagtcttt 2340taccatcagg cccggctgat ggtaaaataa cagtatttga aatagtttaa acacatcatc 2400ttaatggtca accacacaat ttccgaaatg ttgctggctc agtctggggc aaacctgtcc 2460gccccaacat tggtgctagg aagaaagcac agacaagtag ccctcccagc tcaggagtaa 2520aagacctgga gggggtggcc cacttcggtc aagttcacgg gatggggagg ggtaccctcc 2580tccagtagtg gtggtatttg gcagttcctc caccgacgcc ctctggaagc acctgcttgg 2640acccgcaaag ccaggaatgc agcttcctca agggactcgc cagcgagggt aacaggacag 2700aggcgtccca agagggctgg ggcggaaggg ggaagacagg gtcggcctta gatagggcaa 2760agggccttct ggctgtgttc ccggggtaac cgccccacca cgcctggagc ccgacgtggc 2820gagcgatggg gacagcgagc aggaagtcgt actggggagg gccgcgtagc agatgcagcc 2880gagggcggcg ctgccaggta cacccgaggg caccgcgggg gtgagcgcca ggtccctgaa 2940ccagccaggc ctccagagcc gagtccggcg gaccgacggt acgttctgga atgggaaggg 3000atccgggaca ccgaattgct gcattgaggg gctcagaggt tctgatgtgg gagtccagaa 3060agggttttat ctaccggagg tgatgtgact tccggcctct ggaagtgctg ttggagtctc 3120tgggaccttg ggtcctctcg actaggtttg gaaggggtga aataggggta gggagaaagg 3180agaggactgc agcaatgtct tcccgaacga cctgggttcg ggaggggtcg aaggacaagg 3240ggctgttgtg gggggtcttc agacgcggag gggtggtatt ctattttctg ggaagatggt 3300gtcgatgcac ttgaccaagt ctagtcgatc tgaagaggct aggggaacag acagtgagag 3360aggatggtgg agggagtggc agaacccttc cagaaactgg gagaggctct agcacctgca 3420accccttccc tggcctccgg ggagtcccag aagagggcag gaccatggac acaggtgcat 3480tcgtgccggc gcgctccggc ctggcgaagg tgcgcgctct tggaggccgc gggagggcca 3540gacgcgcgcc cggagagctg gccctttaag gctacccgga ggcgtgtcag gaaatgcgcc 3600ctgagcccgc ccctcccgga acgcggcccg agacctggca agctgagacg gaactcggaa 3660ctagcactcg gctcgcggcc tcggtgaggc cttgcgcccg ccatgcctct gtcattgccc 3720ctcgggccgc ctccctgaac ctccgtgacc gccctgcagt cctccctccc ccccttcgac 3780tcggcgggcg cttccgggcg ctcccgcagc ccgccctcca cgtagcccac acctccctct 3840cggcgctccg cttcccacgc ggtccccgac ctgttctttc ctcctccacc ctgcccttct 3900gtccctctcc cttcctttct cccctcgact cgtccctatt aggcaacagc ccctgtggtc 3960cagccggcca tggctgtcaa ggctcacacc cttagctagg ccccttctcc cttccctggg 4020tcttgtctca tgaccccctg ccccgcccgg gagcgagcgc gatgtggagc agtgcctctg 4080gcaagcagaa cttcacccaa gccatgtgac aattgaaggc tgtaccccca gaccctaaca 4140tcttggagcc ctgtagacca gggagtgctt ctggccgtgg ggtgacctag ctcttctacc 4200accatgaaca gggcccctct gaagcggtcc aggatcctgc gcatggcgct gactggaggc 4260tccactgcct ctgaggaggc agatgaagac agcaggaaca agccgtttct gctgcgggcg 4320ctgcagatcg cgctggtcgt ctctctctac tgggtcacct ccatctccat ggtattcctc 4380aacaagtacc tgctggacag cccctccctg cagctggata cccctatctt cgtcactttc 4440taccaatgcc tggtgacctc tctgctgtgc aagggcctca gcactctggc cacctgctgc 4500cctggcaccg ttgacttccc caccctgaac ctggacctta aggtggcccg cagcgtgctg 4560ccactgtcgg tagtcttcat tggcatgata agtttcaata acctctgcct caagtacgta 4620ggggtggcct tctacaacgt ggggcgctcg ctcaccaccg tgttcaatgt gcttctgtcc 4680tacctgctgc tcaaacagac cacttccttc tatgccctgc tcacatgtgg catcatcatt 4740ggtgagtggg gcccgggggc tgtgggagca ggatgggcat cgaactgaag ccctaaaggt 4800caacactgta ggtaccttta cttactgtcc caggtccctt gcatcagcag ttacaggaag 4860agccctgtag aaaacaaata acttccttat ggtcattcaa caagttaggg acccagccag 4920ggtgaaaata atgttagcag caactacagc aaagatggct ctcgccactt gcatgattaa 4980aatgtgccag gtactcagat ctaagcattg gatccacatt aactcaacta atccctatta 5040caaggtaaaa tatatccgaa ttttacagag ggaaaaccaa ggcacagaga ggctaagtag 5100cttgaccagg atcacacagc taataatcac tgacatagct gggatttaaa cataagcagt 5160tacctccata gatcacacta tgaccaccat gccactgttc cttctcaaga gttccaggat 5220cctgtctgtc cagttctctt taaagaggac aacacatctg acattgctac cttgaggtaa 5280catttgaaat agtgggtaga catatgtttt aagttttatt cttacttttt atgtgtgtgt 5340gtttgggggg ccaccacagt gtatgggtgg agataagggg acaacttaag aattggtcct 5400ttctcccacc acatgggtgc tgaggtctga actcaggtca tcaggattgg cacaaatccc 5460tttacccact gagccatttc actggtccaa tatatgtgtg cttttaagag gctttaacta 5520ttttcccaga tgtgaatgtc ctgctgatca ttatcccctt ttacccggaa gccctctggg 5580aggtgccatc cctgtggtcg tctgcataca aatggggaaa ctgcaactca gagaaacaag 5640gctacttgcc agggccccac aagtaagata ggctgggatg ccatcccaga ctggccacac 5700tccctggcct gtgcttcaag ccagtttact ttgttcctgc ccattggaag ttagcatgtt 5760gcagtcaaac acaataacta caggccaaaa gtgcttttaa attaaagtca gatgaacttt 5820taaacatcca gagctcctca actgcaggag ttacaacctg attctgcaac catctttgca 5880gtgcccggta gtcatatgta gctagaggct cttggctagg acagcatgtg ttaggaaaca 5940tctggccctg agatcattga attgagtgac tgctgggtga caaagaccaa ggcatccgtt 6000ccctgagagt cctgggcaag cagcaatgtg accttcattt gtacctactc aggttcttta 6060tctgtcctgt ttgacctact tagtctcctc tggtgtctca gaggcccagg ctgggtactc 6120tggatgtcag gatcaggcca atgcgcacat ctgccctaga aatgtccccc tggttgagca 6180gctcctgaat ccatcggtaa agggtctgga ccagggagga gtcagataaa aagctgacag 6240cactggggga ctccatgggg aactcccacc tgcccccaca catccatcct aagagaactg 6300gtattccttg tttcctcttt gtcctacaag gcaccctggg atcccacttc agtctcccag 6360ccttgccagg gttagagggc atgagcctcc ttgtggggaa tttagatgca agaaggtaca 6420gtcactagag aacctgagct cagatcccca aagtaaccag tacctgatag tgaggcagct 6480gagaaccgca gcagcctgcc tgagtggctg aactctgcgg cctccggaac tggccccaac 6540tgttgggtct cctcttcctt cctcctgtga gggagggccc atctctgata agtgctgtgg 6600ggactctaga gtagggagga ggaggagcaa tctaagcagg ccttactgag aagtccttgc 6660tggcatgtgg ctgcctgagg agtacagact gggaacaccc atttgaatga gtaaggtttt 6720tcctgaaggc catggggagc cacggaggaa aatcatttta gttacaagac aaagagtaga 6780ttggttaaca tgggagcaag gacatggccc caattttcat agatgaagga aattggaact 6840cagagaggtt aagtaacttc tcccaaatag ctcagcttca aaatcacaga acagtcagag 6900tctagatctc tctgatgcct gtgatggtcc tgccattcca tgttgctgat ccctgtggca 6960tcagtaagcc tctaccttgt gggaatgcag gatctaaatg aagagaggaa gtgctggccc 7020catgctgtgg tctggaaagc tatgcaggct ctttgagcag agagtgaccc acaagtgaat 7080agagtcctat gagactcaaa gcaacatcca cccttaagca gctctaacca aatgctcaca 7140ctgagggagc caaagccaag ttagagtcct gtgcttgccc aaggtcactt tgcctggccc 7200tcctcctata gcacccgtgt tatcttatag ccctcattac agtgattaca attataatta 7260gagaggtaac agggccacac tgtccttaca cattcccctg ctagattgta gctgggagag 7320ggggagatgt aggtggctgg gggagtggga gggaagatgc agattttcat tctgggctct 7380actccctcag ccattttttg gtgtgggagt tagactttgg atatgttgat gatgaggtaa 7440gggccacaga acagtctgaa ctgtggtatc agaatcctgt ccctctccct ctctcctcat 7500ccctcttcac cttgtcactc ctctgtctgc tacaggtggt ttctggctgg gtatagacca 7560agagggagct gagggcaccc tgtccctcat aggcaccatc ttcggggtgc tggccagcct 7620ctgcgtctcc ctcaatgcca tctataccaa gaaggtgctc ccagcagtgg acaacagcat 7680ctggcgccta accttctata acaatgtcaa tgcctgtgtg ctcttcttgc ccctgatggt 7740tctgctgggt gagctccgtg ccctccttga ctttgctcat ctgtacagtg cccacttctg 7800gctcatgatg acgctgggtg gcctcttcgg ctttgccatt ggctatgtga caggactgca 7860gatcaaattc accagtcccc tgacccacaa tgtatcaggc acagccaagg cctgtgcgca 7920gacagtgctg gccgtgctct actatgaaga gactaagagc ttcctgtggt ggacaagcaa 7980cctgatggtg ctgggtggct cctcagccta tacctgggtc aggggctggg agatgcagaa 8040gacccaagag gaccccagct ccaaagaggg tgagaagagt gctattgggg tgtgagcttc 8100ttcagggacc tgggactgaa cccaagtggg gcctacacag cactgaaggc ttcccatgga 8160gctagccagt gtggccctga gcaatactgt ttacatcctc cttggaatat gatctaagag 8220gagccagggt ctttcctggt aatgtcagaa agctgccaaa tctcctgtct gccccatctt 8280gttttgggaa aaccctacca ggaatggcac ccctacctgc ctcctcctag agcctgtcta 8340cctccatatc atctctgggg ttgggaccag ctgcagcctt aaggggctgg attgatgaag 8400tgatgtcttc tacacaaggg agatgggttg tgatcccact aattgaaggg atttgggtga 8460ccccacacct ctgggatcca gggcaggtag agtagtagct taggtgctat taacatcagg 8520aacacctcag

cctgcctttg aagggaagtg ggagcttggc caagggagga aatggccatt 8580ctgccctctt cagtgtggat gagtatggca gacctgttca tggcagctgc accctggggt 8640ggctgataag aaaacattca cctctgcatt tcatatttgc agctctagaa cgggggagag 8700ccacacatct tttacgggtt aagtagggtg atgagctcct ccgcagtccc taaccccagc 8760tttacctgcc tggcttccct tggcccagct acctagctgt actccctttc tgtactcttc 8820tcttctccgt catggcctcc cccaacacct ccatctgcag gcaggaagtg gagtccactt 8880gtaacctctg ttcccatgac agagcccttt gaatacctga acccctcatg acagtaagag 8940acatttatgt tctctggggc tggggctgaa ggagcccact ggttctcact tagcctatct 9000ggctcctgtc acaaaaaaaa aaaaagaaaa aaaaaaagca taaaccaagt tactaagaac 9060agaagttggt ttataacgtt ctggggcagc aaagcccaga tgaagggacc catcgaccct 9120ctctgtccat atcctcatgc tgcagaagta caggcaagct cctttaagcc tcatatagga 9180acactagcct cactcatgag ggttttactc catgacctgt caacctcaaa gccttcaaca 9240tgaggactcc aacgtaaatt tggggacaga agcactcaga ccatacccca gcaccacacc 9300ctcctaacct cagggtagct gtcattctcc tagtctcctc tcttgggcct ttagaacccc 9360catttccttg gggtaatgtc tgatgttttt gtccctgtca taaaaagatg gagagactgt 9420gtccagcctt tgattcctac ttcctacaat cccaggttct aatgaagttt gtggggcctg 9480atgccctgag ttgtatgtga tttaataata aaaaagcaag atacagcatg tgtgtggact 9540gagtgagggc cacagggatc taaaagccaa gtgtgagggg acccagctac agcaggcagc 9600atcctgagcc tggaatctct tcaggacaag aattctccat atacctacct actctgggga 9660gtaggtggcc agagttcaag cttcccttag taccaactac cactggctgt gctcttactg 9720aaggcagaca tggcactgag tgctgtccat ctgtcactca tctccacagc cattcctaat 9780gtgtggggtg ggagccatca ccaaacccca ttttcagata aggacacagg ctcagagagg 9840cttgtgtgga gaaaagtagc agcagaattc agagagctgg gtctcctgca gcaccttgga 9900ctgccagcag ccacagtgct tgtcacacag cacatactca aaagaatgcc agccccctca 9960gcctagagtg cctggccttt ctttcagatg aggaagaggg tcaaagctgt tagcttgccc 10020accatatgac cacatacatg accaacagct tgagggaggg aggattactg tggctcccag 10080cctgagaggt gggacaccca aatgtattag gtccttgaat cagggctgac cttgtgattc 10140agtcactcct accagaatgc tggggaatgg ggatgccaaa ggcaaaggag gctttctaag 10200gtgtggtgta agataggcat ttctgcttcc atgtacacct gtgagcagag taggaaggcc 10260ctgtggagaa tatatcccac aaaccagtag cccttcctgg cagtgggtga atactgccac 10320cctatacccc tatgcaaggc cagtagaacc acccaaccca caacatctag agaaattaca 10380ggtcatctta agcctctaaa ttgtggagaa actcgacatg cgcacgattc ctaacctgct 10440agcctagggt gcggggtgga taatttaagg aaactggggt ttcttataga atcggaggct 10500ccatgaagtc accctgacaa gaggtcagca atagccagca gcagtggcta ctcctaagcc 10560tccagacaga gcaccctgtg aatgtacctt attctcacat ctgggtgtct ataggtgtga 10620ctgggtcaga tgtcacccag gccattgcaa tgggccctta gccccatggg gtgttgggat 10680agcagccaag cagctcccat gctgagatac tgcctgcagt agactgatgg ataagaaaac 10740aaggcccaaa atgttttctt tccagacttg atctttcttt gttcaaaaat gctgttttcc 10800cttaaacttg cccaaaccca ttgttttgca gttgaggaaa ataaggcata gaaagattaa 10860aggaagtttc tgaggttaca gagcaaagta ctggcttcac ctgaaataga caggtgtgcc 10920ctgatcctga tttgagctc 10939127352PRTCricetulus griseus 127Met Ala Leu Thr Gly Gly Ser Thr Ala Ser Glu Glu Ala Asp Glu Asp1 5 10 15Ser Arg Asn Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu Val 20 25 30Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu Asn Lys 35 40 45Tyr Leu Leu Asp Ser Pro Ser Leu Gln Leu Asp Thr Pro Ile Phe Val 50 55 60Thr Phe Tyr Gln Cys Leu Val Thr Ser Leu Leu Cys Lys Gly Leu Ser65 70 75 80Thr Leu Ala Thr Cys Cys Pro Gly Thr Val Asp Phe Pro Thr Leu Asn 85 90 95Leu Asp Leu Lys Val Ala Arg Ser Val Leu Pro Leu Ser Val Val Phe 100 105 110Ile Gly Met Ile Ser Phe Asn Asn Leu Cys Leu Lys Tyr Val Gly Val 115 120 125Ala Phe Tyr Asn Val Gly Arg Ser Leu Thr Thr Val Phe Asn Val Leu 130 135 140Leu Ser Tyr Leu Leu Leu Lys Gln Thr Thr Ser Phe Tyr Ala Leu Leu145 150 155 160Thr Cys Gly Ile Ile Ile Gly Gly Phe Trp Leu Gly Ile Asp Gln Glu 165 170 175Gly Ala Glu Gly Thr Leu Ser Leu Ile Gly Thr Ile Phe Gly Val Leu 180 185 190Ala Ser Leu Cys Val Ser Leu Asn Ala Ile Tyr Thr Lys Lys Val Leu 195 200 205Pro Ala Val Asp Asn Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val 210 215 220Asn Ala Cys Val Leu Phe Leu Pro Leu Met Val Leu Leu Gly Glu Leu225 230 235 240Arg Ala Leu Leu Asp Phe Ala His Leu Tyr Ser Ala His Phe Trp Leu 245 250 255Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val Thr 260 265 270Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val Ser Gly 275 280 285Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu Tyr Tyr Glu 290 295 300Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser Asn Leu Met Val Leu Gly305 310 315 320Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp Glu Met Gln Lys Thr 325 330 335Gln Glu Asp Pro Ser Ser Lys Glu Gly Glu Lys Ser Ala Ile Gly Val 340 345 35012832DNAArtificial SequenceArtificial Primer 128ggatcctgcg catgaaaaag cctgaactca cc 3212929DNAArtificial SequenceArtificial Primer 129gcggccgcct attcctttgc cctcggacg 2913033DNAArtificial SequenceArtificial Primer 130atgcatgcca ccatgaaaaa gcctgaactc acc 3313128DNAArtificial SequenceArtificial Primer 131ggatcccagg ctttacactt tatgcttc 2813225DNAArtificial SequenceArtificial Primer 132gctgtctgga gtactgtgca tctgc 2513327DNAArtificial SequenceArtificial Primer 133ggaatgcagc ttcctcaagg gactcgc 2713427DNAArtificial SequenceArtificial Primer 134tgcatcaggt cggagacgct gtcgaac 2713527DNAArtificial SequenceArtificial Primer 135gcactcgtcc gagggcaaag gaatagc 2713624DNAArtificial SequenceArtificial Primer 136tgtgctggga attgaaccca ggac 2413722DNAArtificial SequenceArtificial Primer 137ctacttgtct gtgctttctt cc 2213827DNAArtificial SequenceArtificial Primer 138ctcgactcgt ccctattagg caacagc 2713927DNAArtificial SequenceArtificial Primer 139tcagaggcag tggagcctcc agtcagc 27

Claims

1. A composition comprising an anti-glypican 3 antibody, wherein the anti-glypican 3 antibody comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:25, a CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a CDR3 comprising the amino acid sequence of SEQ ID NO:27, wherein the sugar chain component of the antibody has been modified.

2. A composition comprising an anti-glypican 3 antibody, wherein the anti-glypican 3 antibody comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:60, wherein the sugar chain component of the antibody has been modified.

3. The composition of claim 1, wherein the anti-glypican 3 antibody further comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:60.

4. A composition comprising an anti-glypican 3 antibody, wherein the anti-glypican 3 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, and wherein the sugar chain component of the antibody has been modified.

5. A composition comprising an anti-glypican 3 antibody, wherein the anti-glypican 3 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:109, and wherein the sugar chain component of the antibody has been modified.

6. The composition of claim 4, wherein the anti-glypican 3 antibody further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:109.

7. A composition comprising an anti-glypican 3 antibody, wherein the anti-glypican 3 antibody comprises a light chain variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:46, a CDR3 comprising the amino acid sequence of SEQ ID NO:60, and a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104.

8. The composition of claim 7, wherein the anti-glypican 3 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121.

9. The composition of claim 1, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

10. The composition of claim 2, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

11. The composition of claim 3, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

12. The composition of claim 4, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

13. The composition of claim 5, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

14. The composition of claim 6, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

15. The composition of claim 7, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

16. The composition of claim 8, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 20% of all anti-glypican 3 antibodies in the composition.

17. The composition of claim 1, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

18. The composition of claim 2, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

19. The composition of claim 3, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

20. The composition of claim 4, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

21. The composition of claim 5, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

22. The composition of claim 6, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

23. The composition of claim 7, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

24. The composition of claim 8, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 50% of all anti-glypican 3 antibodies in the composition.

25. The composition of claim 1, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

26. The composition of claim 2, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

27. The composition of claim 3, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

28. The composition of claim 4, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

29. The composition of claim 5, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

30. The composition of claim 6, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

31. The composition of claim 7, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

32. The composition of claim 8, wherein the sugar chain component has been modified to be fucose-deficient and wherein fucose-deficient anti-glypican 3 antibodies comprise more than 90% of all anti-glypican 3 antibodies in the composition.

33. A method for producing an anti-glypican 3 antibody with a modified sugar chain component, the method comprising:(a) providing a cell expressing a nucleic acid encoding an anti-glypican 3 antibody, wherein the cell is a mutant cell that has a reduced capability of adding fucose to sugar chains as compared to a corresponding wild type cell; and(b) culturing the mutant cell, thereby producing the anti-glypican 3 antibody with a modified sugar chain component, wherein the anti-glypican 3 antibody comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:25, a CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a CDR3 comprising the amino acid sequence of SEQ ID NO:27.

34. A method for producing an anti-glypican 3 antibody with a modified sugar chain component, the method comprising:(a) providing a cell expressing a nucleic acid encoding an anti-glypican 3 antibody, wherein the cell is a mutant cell that has a reduced capability of adding fucose to sugar chains as compared to a corresponding wild type cell; and(b) culturing the mutant cell, thereby producing the anti-glypican 3 antibody with a modified sugar chain component, wherein the anti-glypican 3 antibody comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:45, a CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a CDR3 comprising the amino acid sequence of SEQ ID NO:60.

35. A method for producing an anti-glypican 3 antibody with a modified sugar chain component, the method comprising:(a) providing a cell expressing a nucleic acid encoding an anti-glypican 3 antibody, wherein the cell is a mutant cell that has a reduced capability of adding fucose to sugar chains as compared to a corresponding wild type cell; and(b) culturing the mutant cell, thereby producing the anti-glypican 3 antibody with a modified sugar chain component, wherein the anti-glypican 3 antibody comprises a light chain variable region comprising a CDR2 comprising the amino acid sequence of SEQ ID NO:46, a CDR3 comprising the amino acid sequence of SEQ ID NO:60, and a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104.

36. The method of claim 33, wherein the mutant cell lacks a fucose transporter.

37. The method of claim 34, wherein the mutant cell lacks a fucose transporter.

38. The method of claim 35, wherein the mutant cell lacks a fucose transporter.

39. The method of claim 33, further comprising introducing the nucleic acid encoding the anti-glypican 3 antibody into the mutant cell.

40. The method of claim 34, further comprising introducing the nucleic acid encoding the anti-glypican 3 antibody into the mutant cell.

41. The method of claim 35, further comprising introducing the nucleic acid encoding the anti-glypican 3 antibody into the mutant cell.

42. The method of claim 33, further comprising collecting and purifying the antibody.

43. The method of claim 34, further comprising collecting and purifying the antibody.

44. The method of claim 35, further comprising collecting and purifying the antibody.

45. A method of inhibiting cell growth, the method comprising contacting the composition of claim 1 with mammalian cells in an amount sufficient to inhibit growth of the cells.

46. A method of inhibiting cell growth, the method comprising contacting the composition of claim 2 with mammalian cells in an amount sufficient to inhibit growth of the cells.

47. A method of inhibiting cell growth, the method comprising contacting the composition of claim 3 with mammalian cells in an amount sufficient to inhibit growth of the cells.

48. A method of inhibiting cell growth, the method comprising contacting the composition of claim 4 with mammalian cells in an amount sufficient to inhibit growth of the cells.

49. A method of inhibiting cell growth, the method comprising contacting the composition of claim 5 with mammalian cells in an amount sufficient to inhibit growth of the cells.

50. A method of inhibiting cell growth, the method comprising contacting the composition of claim 6 with mammalian cells in an amount sufficient to inhibit growth of the cells.

51. A method of inhibiting cell growth, the method comprising contacting the composition of claim 7 with mammalian cells in an amount sufficient to inhibit growth of the cells.

52. A method of inhibiting cell growth, the method comprising contacting the composition of claim 8 with mammalian cells in an amount sufficient to inhibit growth of the cells.

53. A method of treating cancer, the method comprising administering the composition of claim 1 to a patient in need of treatment for cancer.

54. A method of treating cancer, the method comprising administering the composition of claim 2 to a patient in need of treatment for cancer.

55. A method of treating cancer, the method comprising administering the composition of claim 3 to a patient in need of treatment for cancer.

56. A method of treating cancer, the method comprising administering the composition of claim 4 to a patient in need of treatment for cancer.

57. A method of treating cancer, the method comprising administering the composition of claim 5 to a patient in need of treatment for cancer.

58. A method of treating cancer, the method comprising administering the composition of claim 6 to a patient in need of treatment for cancer.

59. A method of treating cancer, the method comprising administering the composition of claim 7 to a patient in need of treatment for cancer.

60. A method of treating cancer, the method comprising administering the composition of claim 8 to a patient in need of treatment for cancer.

61. The method of claim 53, wherein the cancer is liver cancer.

62. The method of claim 54, wherein the cancer is liver cancer.

63. The method of claim 55, wherein the cancer is liver cancer.

64. The method of claim 56, wherein the cancer is liver cancer.

65. The method of claim 57, wherein the cancer is liver cancer.

66. The method of claim 58, wherein the cancer is liver cancer.

67. The method of claim 59, wherein the cancer is liver cancer.

68. The method of claim 60, wherein the cancer is liver cancer.

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