Composition of antibody specifically binding to cd20

专利摘要:
A composition comprising an antibody molecule that specifically binds to CD20 and has an N-glycoside-binding complex sugar chain Fc region, a cell or a transgenic non-human animal or plant producing the composition, a method for producing the antibody composition, and the antibody Provided is a medicament containing the composition.
公开号:KR20040071254A
申请号:KR10-2004-7010074
申请日:2002-12-25
公开日:2004-08-11
发明作者:시따라겐야；사꾸라다미끼꼬；우찌다가즈히사；신까와도요히데；사또미쯔오；나까노료스께
申请人:교와 핫꼬 고교 가부시끼가이샤；
IPC主号:

专利说明:

Antibody composition that specifically binds to CD20 {COMPOSITION OF ANTIBODY SPECIFICALLY BINDING TO CD20}
[2] In view of the high binding activity, binding specificity and high stability in blood, antibodies have been attempted to diagnose, prevent and treat various diseases in humans. [Monoclonal Antibodies: Principles and Applications Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1 (1995). It has also been attempted to produce humanized antibodies such as humanized chimeric antibodies or humanized complementarity determining regions (hereinafter referred to as CDRs) transplanted antibodies from antibodies of non-human animals using genetic recombination techniques. The human type chimeric antibody is an antibody in which the antibody variable region (hereinafter referred to as V region) is an antibody of an animal other than human, and the normal region (hereinafter referred to as C region) is a human antibody. Human-type CDR grafted antibodies are antibodies in which CDRs of human antibodies are substituted with CDRs of antibodies of animals other than humans.
[3] In mammalian antibodies, five classes of IgM, IgD, IgG, IgA, and IgE are found to exist, and the diagnosis, prevention, and treatment of various diseases in humans have long blood half-lives and various effector functions. Antibodies of the human IgG class are mainly used from the characteristics (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)). Antibodies of the human IgG class are further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4. Many studies have been conducted on the antibody dependent cytotoxic activity (hereinafter referred to as ADCC activity) or complement dependent cytotoxic activity (hereinafter referred to as CDC activity), which are effector functions of antibodies of the IgG class. It has been reported that antibodies of the IgG1 subclass have the highest ADCC activity, CDC activity (Chemical Immunology, 65, 88 (1997)). Indeed, it has been reported that the removal of CD20 positive B cells detected when an anti-CD20 chimeric antibody of the IgG1 subclass was administered to a monkey was not detected when the IgG4 subclass was used [Biochemical Society Transaction (Biochem. Soc. Trans.), 25, 705 (1997). In view of the above, most of the anti-tumor humanized antibodies on the market for therapeutic antibodies that require high effector function for their effect expression are antibodies of the human IgG1 subclass.
[4] CD20 is a polypeptide of about 35 kDa, also called Bp35, identified as a human B lymphocyte specific antigen B1 by monoclonal antibodies (J. Immunol., 125, 1678 (1980)). In four-membrane penetrating molecules, it functions as a calcium channel and is known to be involved in the activation, proliferation, and differentiation of B cells (Immunology Today, 15, 450 (1994)). CD20 expression is limited from free B cells to mature B cells and is not expressed in undifferentiated cells or plasma cells. In addition, it is characterized by being expressed in 90% or more of B-cell non-Hodgkin's lymphoma and not translocated into cells even when the antibody binds to it, and treatment of B-cell lymphoma with anti-CD20 antibody has been attempted for a long time [ Blood, 69, 584 (1987)]. However, the antimicrobial effects of mouse monoclonal antibodies, which have been used initially, induce human antibodies to mouse antibodies (HAMA) in the human body, lack of effector function, etc. It was limited. Therefore, the production of chimeric antibodies between mouse antibodies and antibodies of the human IgG1 subclass using genetic recombination techniques has been investigated (Journal of Immunol., 139, 3521 (1987), W088 / 04936). In addition, the chimeric antibody IDEC-C2B8 of the human IgG1 subclass produced using the mouse monoclonal antibody 2B8 was found to have the activity of removing CD20 positive cells in vivo by examination with monkey [Blood, 83, 435 (1994), WO94 / 11026], marketed in November 1997 as Rituxan ™ (manufactured by IDEC / Genentech, also referred to as Rituximab, hereinafter referred to as Rituxan ™) in the US after clinical trials.
[5] Phase III testing of Rituxan ™ in the United States was conducted by 4 weeks of 375 mg / m 2 / week for 166 cases of lymphocytic and follicular lymphomas, with true efficiency of 48% (complete remission 6 %, 42% in part) (J. Clin. Oncol., 16, 2825 (1998)). The mechanism of action of Rituxan ™ is thought to induce apoptosis in cells by crosslinking CD20 in addition to ADCC activity and CDC activity. [Current Opinion in Immunology, 11, 541 (1999 )]. Since the sensitivity of CDC activity varies depending on the target B lymphoma cells, it is possible to enhance the therapeutic effect of Rituxan ™ by inhibiting the function of the complement inhibitory molecules CD55 or CD59 which are thought to be involved in the control. Has been discussed [Current Opinion in Immunology, 11, 541 (1999)]. However, it has been reported that the expression of these inhibitory molecules in patient-derived tumor cells and the sensitivity of CD activity in vitro do not necessarily correlate with clinical trials (Blood, 98, 1352 (2001)). . In addition, in a study using a model in which a human B lymphoma cell line Raji cell was transplanted into a mouse, it has been shown that ADCC activity through an antibody receptor (hereinafter referred to as FcγR) is important for antitumor effect [Nature Medison] Nature Medicine, 6, 443, (2000).
[6] Rituxan ™ and Chemotherapy [CHOP; Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone] are being considered for combination. % (Complete response 55%, partial response 45%), but side effects due to CHOP were observed (J. Clin. Oncol., 17, 268 (1999)). In addition, as other therapeutic anti-CD20 antibodies, radiolabeled antibodies Zevalin (IDEC), Bexxar (Corixa), and the like have been developed, but since they are all mouse antibodies and radioisotopes are used, they are highly toxic. The side effects that follow are concerned.
[7] Expression of ADCC activity and CDC activity of antibodies of the human IgG1 subclass requires binding of the antibody Fc region with antibody receptors and various complement components present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages. In addition, the binding suggests the importance of several amino acid residues in the hinge region of the antibody and the second domain of the C region (hereinafter referred to as the Cγ2 domain) [European Journal of Immunology (Eur. J. Immunol.), 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical Immunology, 65, 88 (1997). As for Rituxan ™, studies using amino acid substituents of the Cγ2 domain have identified amino acids that are important for CDC activity. [Journal of Immunol., 164, 4178 (2000), Journal of Immunology (J Immunol.), 166, 2571 (2001).
[8] The importance of sugar chains bound to the Cγ2 domain is also suggested (Chemical Immunology, 65, 88 (1997)). Regarding sugar chains, Boyd et al. Described the humanized CDR-grafted antibody CAMPATH-1H produced from Chinese hamster ovary cells (hereinafter referred to as CHO cells) or mouse myelomer NSO cells (hereinafter referred to as NSO cells). (Human IgG1 subclass) was treated with various glycolytic enzymes, and the effects of sugar chains on ADCC activity and CDC activity were examined. As a result, removal of sialic acid at the non-reducing end does not affect both activities. It has been reported that only CDC activity is affected by removing galactose residues, and that activity is reduced by about 50%, and complete removal of sugar chains loses both activity [Molecular Immunol., 32, 1311 (1995). Furthermore, Lifely et al., YO, as a result of analyzing the sugar chains and ADCC activity of the humanized CDR-grafted antibody CAMPATH-1H (human IgG1 subclass) produced by CHO cells, NSO cells or rat myelomer YO cells, Cell-derived CAMPTH-1H showed the highest ADCC activity, suggesting that N-acetylglucosamine (hereinafter referred to as GlcNAc) located in the bisecting is important for the activity [Glycobiology, 5, 813 ( 1995): WO 99/54342] These reports show that the structure of sugar chains plays a very important role in the effector function of antibodies of the human IgG1 subclass, and by altering the structure of sugar chains, antibodies with higher effector functions can be produced. In practice, however, the structure of sugar chains is diverse and complex, making it difficult to specify structures that are very important for effector function.
[9] An example of altering the sugar chain structure of the product by introducing an enzyme gene for sugar chain modification into a host cell is by introducing β-galactosid-α-2,6-sialyltransferase from rats into CHO cells. It has been reported that it is possible to prepare proteins in which sialic acid is added at the non-reducing end of sugar chains (J. Biol. Chem., 261, 13848 (1989)).
[10] In addition, by introducing human β-galactosid-2-α-fucosyltransferase into mouse L cells, H antigen (Fuc) to which fucose (hereinafter also referred to as Fuc) is added to the non-reducing end of the sugar chain. Expression of α1-2Galβ1-) has been confirmed (Science, 252, 1668, (1991)). Also, Umana et al. [Beta] -1,4-N-acetylglucosamine transfer based on the knowledge that the addition of N-acetylglucosamine located in the bisecting of the N-glycosidic link sugar chain is important for the ADCC activity of the antibody. CHO cells expressing enzyme III (GnTIII) were prepared and compared with the parent strain. Expression of GnTIII was not observed in CHO cells of the parent strain (Journal Biologic Chemistry, J. Bio. Chem., 259, 13370, (1984)). It was confirmed to have higher ADCC activity compared to the antibody expressed in [Nature Biotechnol., 17, 176 (1999): WO 99/54342]. At this time, Umana et al. Also prepared CHO cells into which the gene of β-1,4-N-acetylglucosamine transferase V (GnTV) was introduced, and overexpression of GnTIII or GnTV was toxic to CHO cells. Reported. Regarding Rituxan ™, antibodies produced using CHO cells incorporating GnTIII have been reported to show higher ADCC activity than antibodies expressed in parent strains, but the activity difference is about 10-20 times [Biotechnology and Biotechnology. Engineering (Biotechnol. Bioeng.), 74, 288 (2001).
[1] The present invention relates to antibody compositions useful for the treatment of diseases involving CD20 positive cells, such as B-cell lymphoma, cells for producing antibody compositions, and methods for producing antibody compositions using these cells.
[501] 1 shows the construction of plasmid pBS-2B8L.
[502] Figure 2 shows the construction of the plasmid pBS-2B8Hm.
[503] Figure 3 shows the construction of the plasmid pKANTEX2B8P.
[504] Fig. 4 shows the results obtained by measuring the binding activity of purified anti-CD20 chimeric antibody KM3065 and RituxanTM with human CD20 expressing cell Raji cells by varying antibody concentration using the fluorescent antibody method. Relative fluorescence intensity at each concentration is shown on the vertical axis and antibody concentration on the horizontal axis. RituxanTM, Represents the activity of KM3065, respectively.
[505] FIG. 5 shows the binding activity of anti-CD20 chimeric antibody KM3065 and RituxanTM with human CD20-negative CCCC-CEM cells purified using a fluorescent antibody method.
[506] FIG. 6 shows ADCC activity against human CD20 expressing cells of purified anti-CD20 chimeric antibody KM3065 and Rituxan ^™ . FIG. A is a Raji cell, B is a Ramos cell, C is a WIL2-S cell as a target cell. Cytotoxic activity is shown on the vertical axis and antibody concentration is shown on the horizontal axis. Rituxan ^TM , Represents the activity of KM3065, respectively.
[507] FIG. 7 shows the elution obtained by preparing PA-sugar chains from purified anti-CD20 chimeric antibodies KM3065 and Rituxan ^™ and analyzing by reverse phase HPLC. The relative fluorescence intensity is shown on the vertical axis and the elution time on the horizontal axis, respectively.
[508] 8 shows the construction of plasmid CHfFUT8-pCR2.1.
[509] 9 shows the construction of the plasmid ploxPPuro.
[510] 10 shows the construction of plasmid pKOFUT8gE2-1.
[511] 11 shows the construction of plasmid pKOFUT8gE2-2.
[512] 12 shows the construction of plasmid pscFUT8gE2-3.
[513] Fig. 13 shows the construction of the plasmid pKOFUT8gE2-3.
[514] 14 shows the construction of plasmid pKOFUT8gE2-4.
[515] Figure 15 shows the construction of plasmid pKOFUT8gE2-5.
[516] Figure 16 shows the construction of the plasmid pKOFUT8Puro.
[517] Fig. 17 shows the results of measuring the binding activity of anti-CD20 chimeric antibody R92-3-1 produced by lectin resistant CHO / DG44 cells by varying antibody concentration using the fluorescent antibody method. Relative fluorescence intensity at each concentration is shown on the vertical axis and antibody concentration on the horizontal axis. Rituxan ^TM , Represents the activity of R92-3-1, respectively.
[518] Fig. 18 shows the results of evaluating ADCC activity of anti-CD20 chimeric antibody R92-3-1 produced by lectin resistant CHO / DG44 cells using Raji cells as target cells. The cytotoxic activity of the target cell is shown on the vertical axis of the graph, and the antibody concentration is shown on the horizontal axis, respectively. Rituxan ^TM , Represents the ADCC activity of R92-3-1, respectively.
[519] Fig. 19 shows solubility obtained by reverse phase HPLC analysis of PA-sugar chains prepared from anti-CD20 chimeric antibody R92-3-1 produced by lectin resistant CHO / DG44 cells. The relative fluorescence intensity is shown on the new axis and the elution time on the vertical axis, respectively. The analysis conditions of reverse phase HPLC, identification of sugar chain structures, and calculation of the ratio of sugar chain groups in which α-1,6-fucose was not bound were performed in the same manner as in Example 3.
[520] Fig. 20 shows a plasmid CHO-GMD preparation step in which the 5 'end of clone 34-2 is introduced into the 5' end of GMD cDNA clone 22-8 derived from CHO cells.
[521] Fig. 21 shows solubility obtained by analyzing reversed-phase HPLC of PA-ized sugar chains prepared from three types of anti-CD20 chimeric antibodies. The relative fluorescence intensity is shown on the vertical axis and the elution time on the horizontal axis, respectively. Analytical conditions of reverse phase HPLC, identification of sugar chain structures, and calculation of the ratio of the α-1,6-fucose unbound sugar chain group were performed in the same manner as in Example 3.
[522] Fig. 22 shows changes in the antibody concentration of the anti-CD20 chimeric antibody CD20 expressing cells having different ratios of sugar molecules bound to α-1,6-fucose by using the fluorescent antibody method. It is a drawing measured by. The vertical axis shows the binding activity with CD20, and the horizontal axis shows the antibody concentration. Gag CD20 chimeric antibody (96%), gag anti CD20 chimeric antibody (44%), gag anti CD20 chimeric antibody (35%), gag anti CD20 chimeric antibody (26%), Shows the activity of anti-CD20 chimeric antibody (6%), respectively.
[523] Fig. 23 shows ADCC activity against WIL2-S cells of anti-CD20 chimeric antibodies having different ratios of antibody molecules bound to sugar chains without α-1,6-fucose. The result measured by the 5 lCr method using the effector cell of donor A is shown, The cytotoxic activity is shown on a vertical axis, and antibody concentration is shown on a horizontal axis, respectively. Gag CD20 chimeric antibody (96%), gag anti CD20 chimeric antibody (44%), gag anti CD20 chimeric antibody (35%), gag anti CD20 chimeric antibody (26%), Shows the activity of anti-CD20 chimeric antibody (6%), respectively.
[524] Fig. 24 shows ADCC activity against Raji cells of anti-CD20 chimeric antibodies having different ratios of antibody molecules bound to sugar chains without α-1,6-fucose. The result measured by the LDH method using the effector cell of donor B is shown, The cytotoxic activity is shown on a vertical axis, and antibody concentration is shown on a horizontal axis, respectively. Gag CD20 chimeric antibody (96%), gag anti CD20 chimeric antibody (44%), gag anti CD20 chimeric antibody (35%), gag anti CD20 chimeric antibody (26%), Shows the activity of anti-CD20 chimeric antibody (6%), respectively.
[525] FIG. 25 shows an elution diagram of anti-CD20 chimeric antibody KM3065 isolated using a column in which a lectin immobilized to a sugar chain having a bisecting GlcNAc was immobilized. The absorbance at 280 nm is shown on the vertical axis and the elution time on the horizontal axis, respectively. (1) to (4) represent the elution positions of the fractions 1 to 4 respectively.
[526] Fig. 26 is a reverse phase HPLC analysis of fractions 1 to 4 separated using a lectin-immobilized column having sugar chains having a bisecting GlcNAc and PA-conjugated sugar chains prepared from anti-CD20 chimeric antibody KM3065 before separation. It shows the elution obtained by. The elution degree of KM3065 before separation in the upper left side, fraction ① in the upper right side, fraction ② in the left side of the middle, fraction ③ in the right side of the middle, and fraction ④ in the lower left side, respectively. The relative fluorescence intensity is shown on the vertical axis and the elution time on the horizontal axis, respectively. The blacked peaks in the figure represent PA-ized sugar chains derived from antibodies, and "*" indicates PA-ized sugar chains with bisecting GlcNAc.
[527] Fig. 27 shows ADCC activity against Raji cells of the fractions 1 to 4 and the anti-CD20 chimeric antibody KM3065 isolated using a column having a lectin immobilized to a sugar chain having a bisecting GlcNAc. The result measured by LDH method using normal donor-derived effector cells is shown, and the cytotoxic activity is shown on a vertical axis, and antibody concentration is shown on a horizontal axis, respectively. KM3065 before separation The fractions ①, Δ are fractions ②, ◇ are fractions ③, ◆ are fractions ④, and □ are Rituxan ^TM and × are antibody non-additive activities.
[11] Anti-CD20 antibody with enhanced effector function is expected to increase the therapeutic effect and to reduce the burden on the patient due to the decrease in the dose. In addition, side effects are expected to be reduced due to the elimination of the need for combination with chemotherapy and radioisotope labeled antibodies. An object of the present invention is to provide a cell producing an anti-CD20 antibody with enhanced effector function, an anti-CD20 antibody composition with enhanced effector function, a method for producing the antibody composition, a medicament containing the antibody composition and the like.
[12] The present invention relates to the following (1) to (48).
[13] (1) A composition comprising an antibody molecule that specifically binds to CD20 and has an N-glycosidic binding complex sugar chain in the Fc region, wherein the entire N-glycosidic binding complex that binds to the Fc region included in the composition A cell producing an antibody composition in which the proportion of sugar chains in which no fucose is bound to N-acetylglucosamine at the sugar chain reducing end is 20% or more in the sugar chains.
[14] (2) The sugar chain to which no fucose is bound is a sugar chain which is not α-bonded on 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end of the sugar chain. The cell described in.
[15] (3) sugar chains in which α-binding of the first position of fucose on 6 of N-acetylglucosamine at the N-glycoside-binding complex sugar chain reducing end or the activity of an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose A cell according to the above (1) or (2), wherein the activity of an enzyme involved in modification is reduced or deleted.
[16] (4) The cell according to the above (3), wherein the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose is an enzyme selected from the group consisting of the following (a), (b) and (c).
[17] (a) GMD (GDP-mannose 4,6-dehydratase);
[18] (b) Fx (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase);
[19] (c) GFPP (GDP-beta-L-fucose pyrophosphorylase).
[20] (5) The cell according to the above (4), wherein the GMD is a protein encoded by the DNA of the following (a) or (b).
[21] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41;
[22] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41 under stringent conditions and which encodes a protein having GMD activity.
[23] (6) The cell according to the above (4), wherein the GMD is a protein selected from the group consisting of the following (a), (b) and (c).
[24] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 61;
[25] (b) the amino acid sequence represented by SEQ ID NO: 61, wherein the at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has GMD activity;
[26] (c) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 61 and having GMD activity.
[27] (7) The cell according to the above (4), wherein Fx is a protein encoded by DNA having the following (a) or (b).
[28] (a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48;
[29] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48 under stringent conditions and which encodes a protein having Fx activity.
[30] (8) The cell according to the above (4), wherein Fx is a protein selected from the group consisting of the following (a), (b) and (c).
[31] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 62;
[32] (b) the amino acid sequence represented by SEQ ID NO: 62, wherein the protein comprises one or more amino acid sequences deleted, substituted, inserted and / or added, and further has Fx activity;
[33] (c) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 62 and having Fx activity.
[34] (9) The cell according to the above (4), wherein GFPP is a protein encoded by the DNA of the following (a) or (b).
[35] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51;
[36] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51 under stringent conditions and which encodes a protein having GFPP activity.
[37] (10) The cell according to the above (4), wherein GFPP is a protein selected from the group consisting of the following (a), (b) and (c).
[38] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 63;
[39] (b) the amino acid sequence represented by SEQ ID NO: 63, wherein the protein comprises one or more amino acid sequences deleted, substituted, inserted and / or added, and also has GFPP activity;
[40] (c) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 63 and having GFPP activity.
[41] (11) An enzyme involved in sugar chain modification in which the first position of fucose binds to 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal is α-1,6-fucosyltransferase The cell according to the above (3).
[42] (12) to (11), wherein α-1,6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a), (b), (c) and (d): Described cells.
[43] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1;
[44] (b) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2;
[45] (c) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity;
[46] (d) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[47] (13) Said α-1,6-fucosyltransferase is a protein selected from the group consisting of the following (a), (b), (c), (d), (e) and (f) The cell as described in (11).
[48] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 23;
[49] (b) a protein consisting of the amino acid sequence represented by SEQ ID NO: 24;
[50] (c) the amino acid sequence represented by SEQ ID NO: 23, wherein at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has α-1,6-fucosyltransferase activity ;
[51] (d) the amino acid sequence represented by SEQ ID NO: 24, wherein at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has α-1,6-fucosyltransferase activity ;
[52] (e) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 23, and having α-1,6-fucosyltransferase activity;
[53] (f) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 24 and having α-1,6-fucosyltransferase activity.
[54] (14) In the above (3) to (13), wherein the activity of the enzyme is reduced or deleted by a method selected from the group consisting of the following (a), (b), (c), (d) and (e). The cell according to any one of claims.
[55] (a) a method of gene disruption targeting a gene of an enzyme;
[56] (b) a method of introducing a dominant negative of the gene of the enzyme;
[57] (c) a method of introducing a mutation for an enzyme;
[58] (d) techniques for inhibiting the transcription or translation of an enzyme's genes;
[59] (e) A method of selecting a strain that is resistant to a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain reduction terminal are recognized.
[60] (15) Any of the above (1) to (14), wherein at least the N-acetylglucosamine at the N-glycoside-linked sugar chain-reducing terminal and the first position of the fucose are resistant to lectins that recognize the α-linked sugar chain structure. The cell of Claim.
[61] (16) The cell according to any one of the above (1) to (15), wherein the cell is a cell selected from the group consisting of the following (a) to (j).
[62] (a) CHO cells derived from Chinese hamster ovary tissue;
[63] (b) rat myelomer cell line YB2 / 3HL. P2. G11. 16Ag. 20 cells;
[64] (c) mouse myelomer cell line NSO cells;
[65] (d) mouse myelomer cell line SP2 / O-Ag 14 cells;
[66] (e) Syrian hamster kidney tissue derived BHK cells;
[67] (f) monkey COS cells;
[68] (g) hybridoma cells producing antibodies;
[69] (h) human leukemia cell line Namalba cells;
[70] (i) embryonic stem cells;
[71] (j) fertilized egg cells.
[72] (17) A composition consisting of an antibody molecule that specifically binds to CD20 and has an N-glycoside binding complex sugar chain in the Fc region, wherein the entire N-glycoside binding complex that binds to the Fc region included in the composition. Transgenic non-human animals or plants incorporating a gene encoding the antibody molecule, which produces an antibody composition in which the proportion of sugar chains in which no fucose is bound to N-acetylglucosamine at the sugar chain reducing end is 20% or more in the sugar chains. , Or descendants thereof.
[73] (18) The sugar chain to which no fucose is bound is a sugar chain which is not α-bonded on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end of the fucose. Transgenic non-human animal or plant, or a progeny thereof.
[74] (19) To the sugar chain modification in which the first position of fucose binds to the activity of an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the 6th position of N-acetylglucosamine at the N-glycoside-linked sugar chain-reducing terminal The transgenic non-human animal or plant according to (17) or (18), or a progeny thereof, wherein the genome is modified so that the activity of an enzyme involved is reduced.
[75] (20) To a sugar chain modification in which the first position of fucose binds to the gene of an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the N-acetylglucosamine at the N-acetylglucosamine 6 at the N-glycosidic sugar chain reducing end. The transgenic non-human animal or plant according to (17) or (18), or a progeny thereof, wherein the gene of the enzyme involved is knocked out.
[76] (21) The enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose is an enzyme selected from the group consisting of the following (a), (b) and (c) described in (19) or (20). Transgenic non-human animal or plant, or progeny thereof.
[77] (a) GMD (GDP-mannose 4,6-dehydratase);
[78] (b) Fx (GDP-keto-6-deoxymannose, 3,5-epimerase, 4-reductase);
[79] (c) GFPP (GDP-beta-L-fucose pyrophosphorylase).
[80] (22) The transgenic non-human animal or plant according to (21), wherein GMD is a protein encoded by DNA having the following (a) or (b), or a progeny thereof.
[81] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41;
[82] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41 under stringent conditions and which encodes a protein having GMD activity;
[83] (23) The transgenic non-human animal or plant according to (21), or a progeny thereof, wherein Fx is a protein encoded by DNA having the following (a) or (b).
[84] (a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48;
[85] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48 under stringent conditions and which encodes a protein having Fx activity.
[86] (24) The transgenic non-human animal or plant according to (21), or a progeny thereof, wherein GFPP is a protein encoded by DNA having the following (a) or (b).
[87] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51;
[88] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51 under stringent conditions and which encodes a protein having GFPP activity.
[89] (25) The enzyme involved in the sugar chain modification, wherein the first position of the fucose on the 6th of the N-acetylglucosamine at the N-glycoside-linked sugar chain reduction terminal is α-1,6-fucosyltransferase, The transgenic non-human animal or plant according to (19) or (20), or a progeny thereof.
[90] (26) Said (25) whose (alpha) -1,6-fucosyltransferase is a protein which DNA selected from the group which consists of the following (a), (b), (c), and (d) codes. The transgenic non-human animal or plant described in, or Progeny thereof.
[91] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1;
[92] (b) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2;
[93] (c) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[94] (d) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[95] (27) The method of any one of (17) to (26), wherein the transgenic non-human animal is an animal selected from the group consisting of cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys, and rabbits. Transgenic non-human animals or plants described, or progeny thereof.
[96] (28) The cell according to any one of (1) to (16), wherein the antibody molecule is a molecule selected from the group consisting of the following (a), (b), (c), and (d).
[97] (a) human antibodies;
[98] (b) humanized antibodies;
[99] (c) an antibody fragment comprising the Fc region of (a) or (b);
[100] (d) a fusion protein having the Fc region of (a) or (b).
[101] (29) The cell according to any one of (1) to (16) and (28), wherein the class of the antibody molecule is IgG.
[102] (30) complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 of the light chain variable region of the antibody molecule are SEQ ID NOs: 5, 6, 7, and / or complementarity determining region 1, complementarity determining region 2, of heavy chain variable region; The cell according to any one of (1) to (16), (28), and (29), wherein the complementarity determining region 3 includes the amino acid sequence represented by SEQ ID NO: 8, 9, 10.
[103] (31) The above (1) to (16), (28), (29) and (30), wherein the light chain variable region of the antibody molecule comprises SEQ ID NO: 12, and / or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14. The cell of any one of).
[104] (32) The transgenic nonhuman of any one of (17) to (27), wherein the antibody molecule is a molecule selected from the group consisting of the following (a), (b), (c), and (d). Animals or plants, or their descendants.
[105] (a) human antibodies;
[106] (b) humanized antibodies;
[107] (c) an antibody fragment comprising the Fc region of (a) or (b);
[108] (d) a fusion protein having the Fc region of (a) or (b).
[109] (33) The transgenic non-human animal or plant according to any one of (17) to (27) and (32), or a progeny thereof, wherein the class of the antibody molecule is IgG.
[110] (34) Complementarity determining region 1, complementarity determining region 2, and complementarity determining region 3 of the light chain variable region of the antibody molecule are SEQ ID NO: 5, 6, 7, and / or complementarity determining region 1, complementarity determining region 2 of the heavy chain variable region, respectively , The transgenic non-human animal or the plant according to any one of (17) to (27), (32), and (33), wherein the complementarity determining region 3 comprises an amino acid sequence represented by SEQ ID NO: 8,9, 10, respectively. , Or descendants thereof.
[111] (35) Said (17)-(27), (32), (33) and (34) in which the light chain variable region of an antibody molecule contains SEQ ID NO: 12, and / or the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 14. The transgenic non-human animal or plant according to any one of), or a progeny thereof.
[112] (36) The antibody composition produced by the cell in any one of said (1)-(16), (28)-(31).
[113] (37) The antibody composition produced by the animal or plant which raised the transgenic non-human animal or plant in any one of said (17)-(27), (32)-(35), or its offspring, and was raised. .
[114] (38) A composition consisting of an antibody molecule that specifically binds to CD20 and has an N-glycoside binding complex chain in the Fc region, wherein the total N-glycoside complex sugar that binds to the Fc region included in the composition The antibody composition in which the ratio of the sugar chain in which the fucose is not bonded to N-acetylglucosamine of the sugar chain reduction terminal in the chain is 20% or more.
[115] (39) The sugar chain to which no fucose is bound is a sugar chain which is not α-bonded on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing terminal. The antibody composition described in.
[116] (40) The antibody composition according to the above (38), wherein the antibody molecule is a molecule selected from the group consisting of the following (a), (b), (c), and (d).
[117] (a) human antibodies;
[118] (b) humanized antibodies;
[119] (c) an antibody fragment comprising the Fc region of (a) or (b);
[120] (d) a fusion protein having the Fc region of (a) or (b).
[121] (41) The antibody composition according to any one of (38) to (40), wherein the class of the antibody molecule is IgG.
[122] (42) complementarity determining region 1, complementarity determining region 2, and complementarity determining region 3 of the light chain variable region of the antibody molecule are SEQ ID NO: 5, 6, 7, and / or complementarity determining region 1, complementarity determining region 2 of heavy chain variable region, respectively; The antibody composition according to any one of (38) to (41), wherein the complementarity determining region 3 includes an amino acid sequence represented by SEQ ID NOs: 8, 9, and 10, respectively.
[123] (43) The antibody composition according to any of (38) to (42), wherein the light chain variable region of the antibody molecule contains SEQ ID NO: 12 and / or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14.
[124] (44) The cells according to any one of (1) to (16) and (28) to (31) are cultured in a medium, and any of the above (36) and (38) to (43) in a culture. A method for producing the antibody composition, comprising the step of producing and accumulating the antibody composition according to claim and extracting the antibody composition from the culture.
[125] (45) Breeding the transgenic non-human animal or plant of any one of said (17)-(27), (32)-(35), or its offspring, and acquires the tissue or a bodily fluid from the animal or plant which raised The method of manufacturing this antibody composition including the process of extracting the antibody composition in any one of said (36), (38)-(43) from the acquired tissue or body fluid.
[126] (46) A pharmaceutical comprising the antibody composition according to any one of (36) to (43) as an active ingredient.
[127] (47) A drug for treating a CD20-related disease, comprising as an active ingredient the antibody composition according to any one of (36) to (43).
[128] (48) The therapeutic drug according to the above (47), wherein the CD20 related disease is cancer or an immune disease.
[129] The cell of the present invention is a composition consisting of an antibody molecule that specifically binds to CD20 and has an N-glycoside-binding complex sugar chain, and is a whole N-glycoside-binding complex that binds to the Fc region included in the composition. Any cell is included in the sugar chain as long as it produces an antibody composition in which the proportion of sugar chains in which fucose is not bonded to N-acetylglucosamine at the sugar chain reducing end is 20% or more.
[130] In the present invention, CD20 is a cell surface membrane protein of about 35 kDa, also referred to as B1 or Bp35, wherein one or several amino acids of the amino acid sequence represented by SEQ ID NO: 4 or the protein represented by the amino acid sequence represented by SEQ ID NO: 4 are substituted, deleted, Anything consisting of an inserted and / or added amino acid sequence and having substantially the same properties as CD20 is included.
[131] In the amino acid sequence represented by SEQ ID NO: 4 in the present invention, a protein having one or a plurality of amino acids deleted, substituted, inserted, and / or added, and having a CD20 activity is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997 (hereinafter, Current Protocols in Molecular Biology) Abbreviated), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. The site-specific mutation introduction method described in Sci., USA, 82, 488 (1985) and the like can be obtained by introducing site-specific mutations into DNA encoding a protein having an amino acid sequence represented by SEQ ID NO: 4, for example. The number of amino acids to be deleted, substituted, inserted and / or added is one or more and the number thereof is not particularly limited, but may be deleted, substituted, inserted and / or added by known techniques such as the site-specific mutation introduction method. It is the number of the grade which can be used, for example, 1-10 dozen, Preferably it is 1-20, More preferably, it is 1-10, More preferably, it is 1-5.
[132] In addition, in the present invention, in order for the protein to be used to have CD20 activity, the amino acid sequence represented by SEQ ID NO: 4 and BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA [Methods in Enzymology, 183, 63 (1990)], etc., when calculated using analysis software, at least 80% or more, preferably 85% or more, more preferably Has at least 90%, more preferably at least 95%, particularly preferably at least 97%, most preferably at least 99% homology.
[133] In the present invention, the sugar chain that binds to the Fc region of the antibody molecule includes an N-glycosidic linking sugar chain, and the N-glycosidic linking sugar chain includes galactose-N- on the non-reducing end side of the core structure. Complex type having 1 to plural branches of acetylglucosamine (hereinafter referred to as Gal-GlcNAc) and a structure such as sialic acid and bisector N-acetylglucosamine on the non-reducing end side of Gal-GlcNAc. (Complex) sugar chains may be mentioned.
[134] The Fc region of the antibody molecule has a region where the N-glycosidic linkage sugar chains described below are bonded to each other, so that two sugar chains are bound to each molecule of the antibody. Since the N-glycoside-binding sugar chain that binds to the antibody includes any sugar chain represented by the following structural formula (I), a combination of a plurality of sugar chains exists in the two N-glycoside-binding sugar chains that bind to the antibody. Done. Therefore, the identity of the substance can be judged from the viewpoint of the sugar chain structure bonded to the Fc region.
[135]
[136] In the present invention, a composition consisting of an antibody molecule having an N-glycosidic conjugated sugar chain in an Fc region (hereinafter referred to as an antibody composition of the present invention) is an antibody having a single sugar chain structure as long as the effect of the present invention is obtained. It may be composed of sugar chains having a plurality of different sugar chain structure.
[137] In the present invention, the ratio of sugar chains in which no fucose is bound to N-acetylglucosamine at the sugar chain-reducing end in the total N-glycoside-linked complex sugar chains binding to the Fc region included in the antibody composition is in the composition. Regarding the total number of all N-glycoside-linked complex sugar chains that bind to the included Fc region, the ratio occupies the number of sugar chains in which fucose is not bonded to N-acetylglucosamine at the end of sugar chain reduction.
[138] In the present invention, sugar chains in which fucose is not bonded to N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal are N-glycoside-linked sugar chain-reducing N Sugar chains that are not α-linked to acetylglucosamine. Examples thereof include sugar chains in which the first position of this fucose is not α-linked to the 6th position of N-acetylglucosamine of the N-glycoside-linked complex sugar chain.
[139] In the total N-glycoside complex sugar chain that binds to the Fc region included in the antibody composition of the present invention, the ratio of the sugar chain having no fucose to N-acetylglucosamine at the sugar chain reduction terminal is preferably 20%. Or more, more preferably at least 25%, even more preferably at least 30%, particularly preferably at least 40%, most preferably at least 50%, and antibody compositions having a proportion of these sugar chains have high ADCC. Have activity.
[140] When the antibody concentration decreases, the ADCC activity decreases. When the percentage of sugar chains in which fucose is not bonded to N-acetylglucosamine at the sugar chain reducing end is 20% or more, high ADCC activity can be obtained even at low antibody concentrations. Can be.
[141] The proportion of sugar chains in which fucose is not bonded to N-acetylglucosamine at the sugar chain reduction terminus contained in the composition consisting of antibody molecules having N-glycoside-linked complex sugar chains in the Fc region is determined by hydrazine degradation from the antibody molecule. Using known methods such as enzyme digestion [Biochemistry Experiment 23-Glycoprotein Sugar Chain Study (Research Publishing Center) Takahashi Reiko Editing (1989)], the sugar chain was liberated and the released sugar chain was fluorescently labeled or isotope. It can be determined by labeling and separating the labeled sugar chains by chromatography. The liberated sugar chains can also be determined by analyzing by the HPAED-PAD method (J. Liq. Chromatogr., 6, 1577 (1983)).
[142] In addition, the cells of the present invention produce the composition of the present invention, and also N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminus of an enzyme that is involved in the synthesis of intracellular sugar nucleotide GDP-fucose. 6 includes cells in which the activity of an enzyme involved in the sugar chain modification at which the first position of the fucose binds α is deleted.
[143] Enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include any enzyme as long as it is an enzyme involved in the synthesis of sugar nucleotide GDP-fucose which is a source of fucose for sugar chains in a cell. Enzymes for the synthesis of intracellular sugar nucleotides GDP-fucose include enzymes that affect the synthesis of intracellular GDP-fucose.
[144] Enzymes that affect the synthesis of intracellular sugar nucleotides GDP-fucose include those which affect the activity of the enzymes involved in the synthesis pathway of the above-described sugar nucleotides GDP-fucose or become substrates of the enzymes. It also includes enzymes that affect the structure.
[145] Intracellular sugar nucleotides GDP-fucose are supplied by either the de novo synthesis pathway or the salvage synthesis pathway. Thus, all of the enzymes involved in these synthetic pathways are included in enzymes involved in the synthesis of intracellular GDP-fucose.
[146] Enzymes involved in the new biosynthetic pathway of sugar nucleotides GDP-fucose in cells specifically include GDP-mannose 4,6-dehydratase (hereinafter referred to as GMD), GDP-keto-deoxymannose 3,5-epimerase, 4-reductase (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase; hereinafter referred to as Fx), and the like.
[147] As an enzyme involved in the cellular synthesis pathway of sugar nucleotides GDP-fucose, GDP-beta-L-fucose pyrophosphorylase (hereinafter referred to as GFPP) ), Fucokinase and the like.
[148] In the present invention, the GMD is a protein encoded by the DNA of the following (a) or (b),
[149] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41
[150] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41 under stringent conditions and which encodes a protein having GMD activity.
[151] or
[152] (c) a protein consisting of the amino acid sequence represented by SEQ ID NO: 61
[153] (d) in the amino acid sequence represented by SEQ ID NO: 61, a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added, and which also has GMD activity
[154] (e) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 61 and further having GMD activity.
[155] As the DNA encoding the amino acid sequence of GMD, the DNA having the nucleotide sequence represented by SEQ ID NO: 41 and the DNA having the nucleotide sequence represented by SEQ ID NO: 41 are hybridized under stringent conditions. DNA which codes the amino acid sequence which has, etc. are mentioned.
[156] In the present invention, Fx is a protein encoded by the DNA of (a) or (b),
[157] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48
[158] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48 under stringent conditions and which encodes a protein having Fx activity.
[159] or
[160] (c) a protein consisting of the amino acid sequence represented by SEQ ID NO: 62
[161] (d) the amino acid sequence represented by SEQ ID NO: 62, wherein the at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has Fx activity
[162] (e) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 62 and having Fx activity.
[163] As the DNA encoding the amino acid sequence of Fx, the DNA having the nucleotide sequence represented by SEQ ID NO: 48 and the DNA having the nucleotide sequence represented by SEQ ID NO: 48 are hybridized under conditions conducive to the Fx activity. DNA which codes the amino acid sequence which has, etc. are mentioned.
[164] In the present invention, GFPP is a protein encoded by the DNA of the following (a) or (b),
[165] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51
[166] (b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51 under stringent conditions and which encodes a protein having GFPP activity.
[167] or
[168] (c) a protein consisting of the amino acid sequence represented by SEQ ID NO: 63
[169] (d) the amino acid sequence represented by SEQ ID NO: 63, wherein the at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has GFPP activity
[170] (e) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 63 and having GFPP activity.
[171] As the DNA encoding the amino acid sequence of GFPP, the DNA having the nucleotide sequence represented by SEQ ID NO: 51 and the DNA having the nucleotide sequence represented by SEQ ID NO: 51 are hybridized under stringent conditions. DNA which codes the amino acid sequence which has, etc. are mentioned.
[172] The enzyme involved in the sugar chain modification in which the 1st position of the fucose is on 6 of the N-acetylglucosamine at the N-acetylglucosamine-linked complex sugar chain reduction terminal is an N-glycoside-linked complex sugar chain reduction terminal. Any enzyme is included as long as the 6th position of N-acetylglucosamine and the 1st position of fucose are involved in the α-binding reaction. The enzyme involved in the α-binding reaction between the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked complex sugar chain reduction terminal is N-acetyl at the N-glycoside-linked complex sugar chain reduction terminal. It refers to an enzyme that affects the reaction of α binding between 6th position of glucosamine and 1st position of fucose.
[173] Examples of enzymes involved in the α-binding reaction of the 1st position of fucose on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal specifically include α-1,6-fucosyltransferase. (alpha) -L-fucosidase etc. are mentioned.
[174] Furthermore, the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked complex sugar chain-reducing terminal described above affect the activity of the enzyme involved in the α-binding reaction or the substrate of the enzyme. Enzymes that affect the structure of these substances are also included.
[175] In the present invention, α-1,6-fucosyltransferase is a protein encoded by the DNA of the following (a), (b), (c) or (d),
[176] (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1
[177] (b) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2
[178] (c) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[179] (d) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[180] or
[181] (e) a protein consisting of the amino acid sequence represented by SEQ ID NO: 23
[182] (f) a protein consisting of the amino acid sequence represented by SEQ ID NO: 24
[183] (g) the amino acid sequence represented by SEQ ID NO: 23, wherein at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has α-1,6-fucosyltransferase activity
[184] (h) the amino acid sequence represented by SEQ ID NO: 24, wherein at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has α-1,6-fucosyltransferase activity
[185] (i) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 23 and having α-1,6-fucosyltransferase activity
[186] (j) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 24 and having α-1,6-fucosyltransferase activity.
[187] In addition, DNA encoding the amino acid sequence of α-1,6-fucosyltransferase includes DNA having a nucleotide sequence represented by SEQ ID NO: 1 or 2, DNA DNA having a nucleotide sequence represented by SEQ ID NO: 1 or 2; DNA encoding the protein which hybridizes under the condition of a condition, and which has alpha-1,6-fucosyltransferase activity, etc. are mentioned.
[188] In the present invention, the DNA hybridized under stringent conditions is a colony hive using DNA or a fragment of DNA, such as a DNA consisting of a nucleotide sequence represented by SEQ ID NO: 1, 2, 48, 51 or 41 as a probe. DNA obtained by using the redidation method, the plaque hybridization method, the Southern blot hybridization method, or the like, specifically, in the presence of 0.7 to 1.0 M sodium chloride using a filter immobilized with DNA derived from colonies or plaques. After hybridization at 65 ° C., the filter was washed under 65 ° C. conditions by using an SSC solution having a concentration of 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 × consists of 150 mM sodium chloride and 15 mM sodium citrate). DNA that can be identified. Hybridization can be performed in accordance with methods described in Molecular Cloning Second Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), and the like. have. A hybridizable DNA, specifically DNA having at least 60% homology with at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, 48, 51 or 41, preferably 70% or more, more preferably 80% or more More preferably at least 90%, particularly preferably at least 95% and most preferably at least 98%.
[189] In the present invention, in the amino acid sequence represented by SEQ ID NO: 23, 24, 61, 62 or 63, at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also α-1,6-fuco Proteins with siltransferase activity, GMD activity, Fx activity or GFPP activity are described in Molecular Cloning Second Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc, Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. The site-specific mutation introduction method described in Sci USA, 82, 488 (1985), etc. can be used to introduce site-specific mutations into DNA having a nucleotide sequence represented by SEQ ID NO: 1, 2, 41, 48 or 51, for example. have. The number of amino acids to be deleted, substituted, inserted and / or added is one or more and the number thereof is not particularly limited, but the number of deletions, substitutions or additions by known techniques such as the site-specific mutation introduction method. For example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5 pieces.
[190] In addition, in the present invention, in order for the protein to be used to have α-1,6-fucosyltransferase activity, GMD activity, Fx activity or GFPP activity, the amino acid sequence represented by SEQ ID NO: 23, 24, 61, 62 or 63, respectively And BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA [Methods in Enzymology, 183, 63 (1990)], etc., when calculated using analysis software, at least 80% or more, preferably 85% or more, more preferably Has at least 90%, more preferably at least 95%, particularly preferably at least 97%, most preferably at least 99% homology.
[191] In addition, the No. 1 position of fucose is above 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing end of the cell of the present invention, ie, the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose. As a method of obtaining the cells whose activity of the enzyme involved in the α-linked sugar chain modification is reduced or deleted, any method can be used as long as it can reduce the target enzyme activity. As a method of lowering or deleting the enzyme activity described above,
[192] (a) a method of gene destruction targeting the gene of an enzyme;
[193] (b) a method of introducing a dominant negative of the gene of the enzyme;
[194] (c) a method of introducing a mutation for an enzyme;
[195] (d) techniques for inhibiting the transcription or translation of an enzyme's genes;
[196] (e) a method of selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain-reducing terminal are recognized.
[197] As a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain-reducing terminal are α-bonded, any lectin can be used as long as the lectin can recognize the sugar chain structure. have. Specific examples include lentil lectin LCA [Lentil Agglutinin derived from Lens Culinaris], pea lectin PSA [Pea Lectin derived from Pisum sativum], silkworm lectin VFA Agulutinine derived from Vicia faba, Wild mushroom lectin AAL (Lectin derived from Aleuria aurantia), etc. are mentioned.
[198] The host cell which produces the antibody composition of this invention includes any cell as long as it is a host cell which can express an anti CD20 antibody molecule, ie, the host cell which inserted the gene which codes an anti CD20 antibody molecule. Examples thereof include yeast, animal cells, insect cells, plant cells, and the like. These cells include those described below in 1, and especially among animal cells, CHO cells derived from Chinese hamster ovary tissue, and rat myelomer cell line YB2 / 3HL. P2. Cll. 16Ag. 20 cells, mouse myelomer cell line NSO cells, mouse myelomer cell line SP2 / O-Ag14 cells, BHK cells derived from Syrian hamster kidney tissue, hybridoma cells producing antibodies, human leukemia cell line Namalba cells, embryonic stem cells, Fertilized egg cells and the like are preferable. Specifically, the rat myelomer cell line YB2 / 3HL into which the gene of the anti-CD20 antibody of the present invention is inserted. P2. Gll. 16Ag. 20 cell transformed clone KM3056 (FERM BP-7834).
[199] A human antibody-producing transgenic non-human animal or plant of the present invention, or a progeny thereof, comprising a antibody molecule that specifically binds to CD20 and has an N-glycoside-binding complex sugar chain in the Fc region. Of the total N-glycoside-linked complex sugar chains that bind to the Fc region included, the antibody composition which produces an antibody composition having a ratio of 20% or more of sugar chains in which no fucose is bound to N-acetylglucosamine at the sugar chain reducing end thereof It includes any transgenic non-human animal or plant in which the gene encoding the antibody molecule is introduced, or any descendant thereof. Specifically, this antibody-producing transgenic animal can be produced by introducing a gene of an antibody that specifically binds CD20 to mouse ES cells, and generating the ES cells after transplantation into early embryos of other mice. The antibody-producing transgenic animal can also be produced by introducing a gene of an antibody that specifically binds CD20 into the fertilized egg of an animal and generating the fertilized egg.
[200] Transgenic non-human animals include cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys or rabbits.
[201] In the present invention, the antibody molecule includes any molecule including the Fc region of the antibody, but examples thereof include antibodies, fragments of the antibody, and fusion proteins including the Fc region.
[202] Antibodies are proteins produced in vivo by an immune response as a result of foreign antigen stimulation, and have an activity that specifically binds to the antigen. As an antibody, an antibody produced by gene recombination technology, that is, an antibody expression vector containing an antibody gene, is inserted into a host cell, in addition to an antibody that immunizes an antigen to an animal and secretes hybridoma cells produced as spleen cells of an immune animal. The antibody acquired by introducing, etc. are mentioned. Specific examples include antibodies produced by hybridomas, humanized antibodies, human antibodies, and the like.
[203] Hybridomas include cells producing monoclonal antibodies having desired antigen specificity obtained by cell fusion of B cells obtained by immunizing antigens with mammals other than humans and myelomer cells derived from mice and the like. .
[204] Examples of humanized antibodies include humanized chimeric antibodies, humanized complementarity determining regions (hereinafter referred to as complementarity determining regions are also referred to as CDRs), and the like.
[205] Humanized chimeric antibodies are also known as antibody heavy chain variable regions (hereinafter referred to as V regions, heavy chains referred to as HV or VH as H chains) and antibody light chain variable regions (hereinafter referred to as LV or VL as L chains) in non-human animals. And the heavy chain normal region (hereinafter also referred to as CH) of the human antibody and the light chain normal region (hereinafter also referred to as CL) of the human antibody. Any animal other than human can be used as long as hybridomas such as mice, rats, hamsters, and rabbits can be produced.
[206] The human type chimeric antibody obtains cDNA encoding VH and VL from Hamilbrioma producing monoclonal antibody, and inserts into human expression cell expression vector having a gene encoding human antibody CH and human antibody CL, respectively. Chimeric antibody expression vectors can be constructed and expressed by introducing them into host cells to produce them.
[207] As the CH of the human type chimeric antibody, any one belonging to human immunoglobulin (hereinafter referred to as hIg) can be used, but the hIg class is preferable, and the hIgG1, hIgG2, hIgG3, and hIgG4 subclasses belong to the hIg class. Any can be used. As the CL of the human-type chimeric antibody, any one belonging to hIg can be used, and any of κ class or λ class can be used.
[208] Human-type CDR grafted antibody means the antibody which transplanted the amino acid sequence of CDR of VH and VL of the antibody of an animal other than a human to the suitable position of VH and VL of a human antibody.
[209] Human-type CDR grafted antibodies construct cDNA encoding the V region in which the CDR sequences of VH and VL of antibodies of non-human animals are implanted into the CDR sequences of VH and VL of any human antibody, and the CH and human antibodies of human antibodies. The humanized CDR grafted antibody expression vector can be constructed by inserting into an expression vector for a host cell each having a gene encoding CL, thereby constructing the humanized CDR grafted antibody by introducing the expression vector into a host cell.
[210] Any of the subtypes of hIgG class belonging to the hIgG class is preferable, and any of subclasses of hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class can be used. As the CL of the humanized CDR-grafted antibody, any one belonging to hIg can be used, and any of κ class or λ class can be used.
[211] Human antibodies originally mean antibodies naturally present in the human body, but human antibody phage libraries and human antibody-producing transgenic animals or human antibodies produced by recent advances in genetic, cellular, and developmental engineering techniques. And antibodies obtained from transgenic plants.
[212] Antibodies present in human cells can be cultured, for example, by isolating human peripheral blood lymphocytes, infecting with EB virus or the like and immortalizing and cloning to produce the lymphocytes producing the antibody and purifying the antibody in culture. .
[213] The human antibody phage library is a library in which antibody fragments such as Fab (Fragment of antigen binding) and 1 main chain antibody are expressed on the phage surface by inserting an antibody gene prepared from human B cells into the phage gene. From this library, phage expressing the antibody fragment having the desired antigen-binding activity can be recovered using the binding activity to the substrate on which the antigen is immobilized. This antibody fragment can in turn be converted into human antibody molecules consisting of two complete H chains and two complete L chains by genetic engineering techniques.
[214] The fragment of an antibody means the fragment containing the Fc region of the said antibody. Examples of the fragment of the antibody include monomers of the H chain, dimers of the H chain, and the like.
[215] The fusion protein including the Fc region includes a substance in which an antibody or fragment of the antibody including the Fc region of the antibody is fused with a protein such as an enzyme or a cytokine.
[216] As the antibody molecule of the present invention, any antibody molecule that specifically binds to CD20 can be used, but preferably, complementarity determining region 1, complementarity determining region 2, and complementarity determining region 3 of the light chain variable region are SEQ ID NO: 5, 6, 7, and / or complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 of the heavy chain variable region specifically bind to CD20 comprising amino acid sequences represented by SEQ ID NOs: 8, 9, 10, respectively Antibody molecules, more preferably antibody molecules that specifically bind to CD20, wherein the light chain variable region comprises SEQ ID NO: 12, and / or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14;
[217] As a medicine of this invention, the pharmaceutical composition containing the antibody composition of this invention mentioned above, ie, the composition of an anti CD20 antibody molecule, as an active ingredient is mentioned.
[218] CD20-related diseases include cancers such as B cell lymphoma, immune diseases such as inflammatory diseases and autoimmune diseases.
[219] In the present invention, ADCC activity means that in vivo, antibodies bound to cell surface antigens, such as tumor cells, activate effector cells through binding of the antibody Fc region to the Fc receptor present on the effector cell surface. Impaired activity [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 2.1 (1995)]. Effector cells include killer cells, natural killer cells, activated macrophages and the like.
[220] Hereinafter, the present invention will be described in detail.
[221] 1. Preparation of Cells Producing the Antibody Composition of the Present Invention
[222] A host cell for use in producing the antibody composition of the present invention is produced by the method described below for the antibody composition of the present invention, and the host cell is provided with a gene encoding an anti-CD20 antibody by the method described below. It can produce by introducing.
[223] (1) Gene destruction technique that targeted gene of enzyme
[224] The host cell used for the production of the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine on N-acetylglucosamine 6 at the N-glycosidic bond complex sugar chain reducing end. The gene can be produced by targeting genes of enzymes involved in sugar chain modification to which α is bound by α and using gene destruction methods. Specific enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD, Fx, GFPP, and fucokinase. Examples of enzymes involved in sugar chain modification, in which the 1st position of fucose is on 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal, specifically α-1,6-fucosyltransferra An azee, (alpha) -L-fucositase, etc. are mentioned.
[225] The gene here includes DNA or RNA.
[226] Gene destroying methods include any method as long as the method can destroy the gene of the target enzyme. Examples include antisense method, ribozyme method, homologous recombination method, RNA-DNA oligonucleotide method (hereinafter referred to as RDO method), RNA interface method (hereinafter referred to as RNAi method), method using retrovirus, transposon The method using this etc. are mentioned. Hereinafter, these are demonstrated concretely.
[227] (a) Preparation of host cell for producing cells of the present invention by antisense method or ribozyme
[228] The host cell used for the production of the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine on N-acetylglucosamine 6 at the N-glycosidic bond complex sugar chain reducing end. Targeting enzyme genes involved in the sugar chain modification to which α 1 is bound, Cell Engineering, 12, 239 (1993), Bio / Technology, 17, 1097 (1999), Human Molecular Genetics (Hum) Mol. Genet., 5, 1083 (1995), Cell Engineering, 13, 255 (1994), Prod. Natl. Acad. Sci. USA, 96, 1886 (1999. The antisense method or ribozyme method described in the above) can be used, for example, as follows.
[229] Enzyme involved in the synthesis of intracellular glyconucleotide GDP-fucose or N-acetylglucosamine at the N-acetylglucosamine at the end of N-glycosidic conjugated sugar chain is involved in the sugar chain modification in which the first position of fucose is CDNA or genomic DNA encoding an enzyme is prepared.
[230] The base sequence of the prepared cDNA or genomic DNA is determined.
[231] The first position of α-fucose binds to 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal or the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose based on the determined DNA sequence. Constructs of appropriate length antisense genes or ribozymes comprising DNA portions, untranslated region portions or intron portions encoding enzymes involved in sugar chain modification.
[232] In order to express this antisense gene or ribozyme in a cell, the recombinant vector is produced by inserting the fragment or full length of the prepared DNA downstream of the promoter of a suitable expression vector.
[233] A transformant is obtained by introducing this recombinant vector into a host cell suitable for this expression vector.
[234] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal The cell of the present invention can be obtained by selecting a transformant based on the activity of the enzyme. In addition, a host cell used for the production of the cell of the present invention can be obtained by selecting a transformant based on the sugar chain structure of the glycoprotein on the cell membrane or the sugar chain structure of the production antibody molecule.
[235] Host cells used to make the cells of the invention include yeasts, animal cells, insect cells, plant cells, etc., enzymes or N-glycosidic binding complexes involved in the synthesis of the target intracellular sugar nucleotide GDP fucose. Any one can be used as long as the first position of the fucose on the 6th of the N-acetylglucosamine at the sugar chain reducing end has an enzyme gene involved in the sugar chain modification to which α is bound. Specifically, the expression vector of 3 mentioned later is mentioned.
[236] As the expression vector, one containing a promoter at a position capable of autonomous replication or insertion in a chromosome in the host cell and capable of transcription of the designed antisense gene or ribozyme is used. Specifically, the expression vector of 3 mentioned later is mentioned.
[237] As a method of introducing a gene into various host cells, a method of introducing a recombinant vector suitable for various host cells described in 3 below can be given.
[238] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal As a method of selecting a transformant using the activity of an enzyme as an index, the following method is mentioned, for example.
[239] How to choose a transformant
[240] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal As a method for selecting cells in which the activity of the enzyme is reduced or deleted, [Chemical Chemistry Experiment Course 3-Glucose I, Glycoprotein (Tokyo Kagaku Co., Ltd.), Japan Biochemistry Society (1988)], [Cell Engineering, Separate Book] , Experimental Protocol Series, Glycobiology Experimental Protocol, Glycoprotein Glycolipid Proteoglycan (manufactured by Shujun Corporation) Naoyuki Suzuki Akemi Furukawa Kiyoshi Sugahara Supervision of Kazuyuki (1996)], Molecular Cloning Second Edition, Current Protocols in Molecular Biol The biochemical method, genetic engineering method, etc. which were described in the lodge etc. are mentioned. Biochemical methods include, for example, methods for assessing enzyme activity using enzyme specific substrates. As a genetic engineering method, the Northern analysis, RT-PCR method, etc. which measure mRNA amount of an enzyme gene, etc. are mentioned, for example.
[241] As a method of selecting a transformant using the sugar chain structure of the glycoprotein on the cell membrane as an index, for example, the method described in (5) of I described later is mentioned. As a method of selecting a transformant based on the sugar chain structure of a production antibody molecule, the method as described in 4 mentioned later or 5 mentioned later is mentioned, for example.
[242] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. As a method of preparing the cDNA which codes for, the method described below is mentioned, for example.
[243] DNA preparation method
[244] Total RNA or mRNA is prepared from tissues or cells of various host cells.
[245] CDNA library is prepared from the prepared total RNA or mRNA.
[246] Enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or enzymes involved in the sugar chain modification in which the first place of fucose is on the 6th of N-glycoside-linked complex sugar chain reducing terminal N-acetylglucosamine. Degenerate primers were prepared based on the amino acid sequence, and the enzyme or N-glycosidic conjugated sugar chains involved in the synthesis of intracellular sugar nucleotide GDP-fucose by PCR using the prepared cDNA library as a template. A gene fragment encoding an enzyme involved in the sugar chain modification at which the 1st position of the fucose binds to the 6th position of N-acetylglucosamine at the reducing end is obtained.
[247] Using the obtained gene fragment as a probe, screening the cDNA library and enzymatically involved in the synthesis of intracellular glyconucleotide GDP-fucose or N-acetylglucosamine at the end of N-glycoside binding complex sugar chain reduction terminal 6 DNA encoding the enzyme involved in the sugar chain modification to which the first position of the fucose binds can be obtained.
[248] The mRNA of the tissue or cell of a human or non-human animal may be commercially available (for example, Clontech), or may be prepared from the tissue or cell of a human or non-human animal as follows. Methods for preparing total RNA from tissues or cells of human or non-human animals include guanidine-trifluoroacetate acetate method (Methods in Enzymology, 154, 3 (1987)), Acidic guanidine phenol chloroform (AGPC) method [Analytical Biochemistry, 162, 156 (1987); Experimental medicine, 9, 1937 (1991)].
[249] Moreover, the oligo (dT) immobilized cellulose column method (molecular cloning 2nd edition) etc. are mentioned as a method of preparing mRNA as poly (A) ⁺ RNA from all RNA.
[250] In addition, mRNA can be prepared by using kits such as a Fast Track mRNA Isolation Kit (Invitrogen) and a Quick Prep mRNA Purification Kit (Pharmacia).
[251] CDNA libraries are prepared from tissue or cellular mRNA of prepared human or non-human animals. For cDNA library construction, see Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2nd Ed. (1989) or the like, or commercially available kits such as SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies), ZAP-cDNA Synthesis Kit (STRATAGENE).
[252] As a cloning vector for producing a cDNA library, any phage vector, plasmid vector, or the like can be used as long as it can be autonomously replicated in E. coli K12 strain. Specifically, ZAP Express [STRATAGENE, Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)] , Lambda ZAP II (STRATAGENE), λgt10, λgt11 [DNA cloning, A Practical Approach, 1, 49, (1985), λTriplEx (Clontech), λExCell (Pharmacia), pT7T318U ( Pharmacia), pcD2 (Mole. Cell. Biol., 3, 280 (1983)) and pUC18 (Gene, 33, 103 (1985)).
[253] Any host microorganism may be used as the host microorganism, but Escherichia coli is preferably used. Specifically, Escherichia coli XL1-Blue MRF '[STRATAGENE, Strategies, 5, 81, (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], E. coli Y1088 [Science, 222, 778 (1983)], E. coli Y1090 (Science, 222, 778 (1983)], E. coli NM522 [ Journal of Molecular Biology (J. Mol. Biol.), 166, 1 (1983)], E. coli K802 [Journal of Molecular Biol., 16, 118 (1966)]. ] And Escherichia coli JM105 (Gene, 38, 275 (1985)) and the like.
[254] The cDNA library may be used for subsequent analysis as it is, but the oligocap method developed by Sugahara et al. [Gene, 138, 171 (1994) in order to reduce the ratio of incomplete length cDNA and to efficiently obtain full length cDNA. ); Gene, 200, 149 (1997); Protein nucleic acid enzymes, 41, 603 (1996); Experimental medicine, 11, 2491 (1993); cDNA cloning (urethosa) (1996); Production Method of Gene Library (Urinary Co., Ltd.) (1994)] may be used for the following analysis.
[255] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. Degenerating primers specific for the 5 'and 3' nucleotide sequences of the nucleotide sequence predicted to encode this amino acid sequence based on the amino acid sequence of the nucleotide sequence were prepared, and the PCR method using the produced cDNA library as a template. DNA amplification using [PCR Protocols, Academic Press (1990)], an enzyme or N-glycosidic conjugated sugar chain involved in the synthesis of intracellular sugar nucleotide GDP-fucose. The gene fragment encoding the enzyme involved in the sugar chain modification that α-binding of the first position of the fucose on 6 of the N-acetylglucosamine at the reducing end is obtained. Can be.
[256] A sugar chain modification in which the first gene of the fucose binds to the enzyme that is involved in the synthesis of the intracellular sugar nucleotide GDP-fucose or the N-acetylglucosamine at the N-acetylglucosamine at the end of N-glycosidic conjugated sugar chain reduction. The DNA encoding the enzyme involved in is commonly used sequencing method, such as dideoxy method such as Sanger (Procedings of the National Academy of Sciences, 74) , 5463 (1977) or an ABIPRISM 377 DNA sequencer (manufactured by Applied Biosystems) can be confirmed by analysis using a sequencing device.
[257] Using this gene fragment DNA as a probe, colony hybridization or plaque hybridization (molecular cloning second edition) is performed on cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. , Enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine at the N-acetylglucosamine at the N-glycosidic conjugated sugar chain reducing terminal, which is involved in the sugar chain modification in which the first place of fucose binds DNA of the enzyme can be obtained.
[258] In addition, enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine at the N-acetylglucosamine at the end of N-glycoside-linked complex sugar chains are involved in sugar chain modification in which the first place of fucose binds. Intracellular sugars are screened by PCR using cDNA or cDNA libraries synthesized from mRNAs contained in tissues or cells of human or non-human animals using PCR as a template, using primers used to obtain gene fragments encoding enzymes. DNAs of enzymes involved in the sugar chain modification in which the first position of fucose binds to 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end of the enzyme involved in the synthesis of nucleotide GDP-fucose. It can also be acquired.
[259] Enzymes involved in the synthesis of the obtained intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine at the N-acetylglucosamine at the end of N-glycosidic linkage complex sugar chain reduction is involved in sugar chain modification in which the first position of fucose binds The base sequence of the DNA encoding the enzyme is converted from the terminal into a commonly used sequencing method such as Sanger et al. [Decidings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) , 74, 5463 (1977) or a base sequence analysis device such as an ABIPRISM 377 DNA sequencer (manufactured by Applied Biosystems) is used to determine the base sequence of this DNA.
[260] Synthesis of intracellular glyconucleotide GDP-fucose from genes in the database by searching for base sequence databases such as GenBank, EMBL, and DDBL using homology search programs such as BLAST method based on the determined base sequence of cDNA The gene encoding the enzyme involved in the sugar chain modification in which the first position of the fucose on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal is determined.
[261] Examples of the nucleotide sequence of the gene encoding the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose obtained by the above method include the nucleotide sequence set forth in SEQ ID NO: 48, 51 or 41. Examples of the nucleotide sequence of a gene encoding an enzyme involved in a sugar chain modification in which the 1st position of fucose is on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal are, for example, SEQ ID NO: 1 or 2 The base sequence described in the following is mentioned.
[262] Enzymes or N- involved in the synthesis of intracellular sugar nucleotide GDP-fucose by chemical synthesis using a DNA synthesizer such as Parkin Elma's DNA synthesizer model 392 using the phosphoramidite method based on the determined base sequence of DNA. The cDNA of the enzyme which is involved in the sugar chain modification in which the 1st position of the fucose is alpha 6 on the N-acetylglucosamine at the glycoside-linked complex sugar chain reduction terminal can also be obtained.
[263] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. The method described below can be mentioned as a method of preparing genomic DNA of the above.
[264] Method of preparing genomic DNA
[265] As a method for producing genomic DNA, known methods described in Molecular Cloning Second Edition, Current Protocols, Molecular Biology, and the like can be given. In addition, by using a genomic DNA library screening system (Genome Systems Inc.) or Universal GenomeWalker ™ Kits (CLONTECH Corp.), the enzyme or N-glycosidic conjugated sugar chain reduction involved in the synthesis of intracellular sugar nucleotide GDP-fucose It is also possible to isolate genomic DNA of an enzyme involved in sugar chain modification, wherein the first position of fucose binds to 6 of terminal N-acetylglucosamine.
[266] As a base sequence of genomic DNA of the enzyme which participates in the synthesis | combination of the intracellular sugar nucleotide GDP-fucose obtained by the said method, the base sequence of sequence number 57 or 60 is mentioned, for example. As the base sequence of the genomic DNA of the enzyme which is involved in the sugar chain modification in which the first position of the fucose is α-linked on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal, the base sequence described in SEQ ID NO: 3, for example. Can be mentioned.
[267] In addition, the first position of fucose is α-binding on 6 of N-acetylglucosamine at the end of N-glycosidic binding complex sugar chain reduction terminal or enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose without using an expression vector. The host cell of the present invention can also be obtained by directly introducing into the host cell an antisense oligonucleotide or ribozyme designed based on the base sequence of the enzyme for sugar chain modification.
[268] Antisense oligonucleotides or ribozymes can be prepared by conventional methods or by using DNA synthesizers. Specifically, an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or a sugar chain modification in which the first position of fucose is α-binding on 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal. Sequence information of oligomeric nucleotides having a sequence corresponding to 5 to 150 bases, preferably 5 to 60 bases, more preferably 10 to 40 bases, among the nucleotide sequences of the cDNA and genomic DNA encoding the enzymes involved. It can be prepared by synthesizing an oligomeric nucleotide (antisense oligomeric nucleotide) corresponding to a sequence complementary to this oligomeric nucleotide based on or a ribozyme comprising a sequence of this oligomeric nucleotide.
[269] Oligomeric nucleotides include oligo RNAs and derivatives of these oligomeric nucleotides (hereinafter referred to as oligomeric nucleotide derivatives).
[270] Oligomeric nucleotide derivatives include oligomeric nucleotide derivatives in which the phosphate diester bonds in oligomeric nucleotides are converted to phosphorothioate bonds, oligomeric nucleotide derivatives in which the phosphate diester bonds in oligomeric nucleotides are converted to N3'-P5 'phosphoramidate bonds, Oligomeric nucleotide derivatives in which ribose and phosphodiester bonds in oligomeric nucleotides are converted to peptide nucleic acid bonds, oligomeric nucleotide derivatives in which uracil in oligomeric nucleotides is replaced with C-5 propynyluracil, and uracil in oligomeric nucleotides into C-5 thiazoluracil Substituted derivative oligomeric nucleotides, cytosine in oligomeric nucleotides oligonucleotide derivatives substituted with C-5 propynylcytosine, cytosine in oligomeric nucleotides Oligomeric nucleotide derivatives substituted with phenoxazine-modified cytosine, oligomeric nucleotide derivatives with ribose in oligomeric nucleotides substituted with 2'-0-propylribose, or ribose in oligomeric nucleotides with 2'-methoxyethoxyribose Oligomeric nucleotide derivatives substituted with the like. [Cell engineering, 16, 1463 (1997)].
[271] (b) Preparation of host cells used for the production of cells of the invention by homologous recombination
[272] The host cell used for the production of the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or N-acetylglucosamine on N-acetylglucosamine 6 at the N-glycosidic bond complex sugar chain reducing end. The gene can be produced by targeting a gene of an enzyme involved in sugar chain modification to which the first position binds α and modifying a target gene on a chromosome using homologous recombination.
[273] Modification of the target gene on the chromosome is referred to as Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter abbreviated as the Manipulating the Mouse Embryo Laboratories Manual). Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Bio-Manual Series Octal Targeting, Construction of Mutant Mice Using ES Cells, Urethosa (1995) (hereinafter, "Creation of Mutant Mice Using ES Cells") For example, it can carry out as follows using the method as described in the above.
[274] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. Prepare genomic DNA of.
[275] Based on the nucleotide sequence of the genomic DNA, the target gene to be modified (e.g., N-acetylglucosamine at the end of N-glycoside binding complex sugar chain reducing enzyme or enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose) A target vector for homologous recombination of the enzyme (structural gene or promoter gene) involved in the sugar chain modification to which α-fucose is bound to α6 is prepared.
[276] By introducing the produced target vector into a host cell and selecting a cell that has undergone homologous recombination between the target gene and the target vector, the host cell used for cell production of the present invention can be produced.
[277] As host cells, yeasts, animal cells, insect cells, plant cells, etc., N-acetyl at the N-glycoside-linked complex sugar chain reduction terminal or enzyme involved in the synthesis of the target intracellular sugar nucleotide GDP-fucose As long as the first place of the fucose on the 6th place of glucosamine has the gene of the enzyme which is involved in the sugar chain modification which binds to α, it can be used. Specifically, the host cell of the following 3 is mentioned.
[278] Enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the first position of fucose binds to 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. As a method of preparing genomic DNA of the above, a method for preparing genomic DNA according to (a) of (1) may be mentioned.
[279] As a base sequence of genomic DNA of the enzyme which participates in the synthesis | combination of the intracellular sugar nucleotide GDP-fucose obtained by the said method, the base sequence of SEQ ID NO: 57 or 60 is mentioned, for example. As the base sequence of the genomic DNA of the enzyme involved in the sugar chain modification in which the first position of the fucose is α-linked on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal, for example, SEQ ID NO: The base sequence described is mentioned.
[280] Target vectors for homologous recombination of target genes include Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Bio-Manual Series Octal Targeting, Construction of Mutant Mice Using ES Cells (Urinary History) (1995) It can produce according to the method as described. The target vector can be both a replacement type and an insertion type.
[281] In order to introduce target vectors into various host cells, a method of introducing a recombinant vector suitable for various host cells described below can be used.
[282] As a method for efficiently screening homologous recombinants, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Bio Manual Series Octal Targeting, Construction of Mutant Mice Using ES Cells (Urinary History ), Such as positive selection, promoter selection, negative selection, poly A selection, and the like, can be used. As a method of selecting a homologous recombinant of interest from the selected cell lines, Southern hybridization method (molecular cloning second edition) or PCR method for genomic DNA [PCR Protocols, Academic Press (1990) )], And the like.
[283] (c) preparation of host cells used to produce cells of the invention by the RDO method
[284] The host cell used to prepare the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or fucose on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end. By targeting the gene of the enzyme involved in the sugar chain modification to which the first position of α is bound, the RDO method can be used, for example, as follows.
[285] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. CDNA or genomic DNA of is prepared.
[286] The base sequence of the prepared cDNA or genomic DNA is determined.
[287] Based on the determined DNA sequence, the first position of fucose is α-binding on 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal or enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose. A construct of a suitable length of RDO including a portion encoding an enzyme involved in sugar chain modification, a portion of an untranslated region, or an intron portion is designed and synthesized.
[288] Fuco on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end or the enzyme involved in the synthesis of the targeted RDO, the intracellular sugar nucleotide GDP-fucose, was introduced into the host cell. The host cell of the present invention can be produced by selecting a transformant in which a mutation has occurred in an enzyme involved in sugar chain modification to which the first place of the os is bound.
[289] As host cells, yeasts, animal cells, insect cells, plant cells, etc., N-acetyl at the N-glycoside-linked complex sugar chain reduction terminal or enzyme involved in the synthesis of the target intracellular sugar nucleotide GDP-fucose As long as the first place of the fucose on the 6th place of glucosamine has the gene of the enzyme which is involved in the sugar chain modification which binds to α, it can be used. Specifically, the host cell of the following 3 is mentioned.
[290] In order to introduce RDO into various host cells, a method of introducing a recombinant vector suitable for various host cells described below can be used.
[291] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. As a method of preparing cDNA of "the preparation method of DNA" as described in said (1) of (1), etc. are mentioned, for example.
[292] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. As a method of preparing genomic DNA of the above, for example, a method of preparing genomic DNA according to (1) of (1) may be mentioned.
[293] The base sequence of the DNA is cleaved with a suitable restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and a commonly used nucleotide sequence analysis method such as Sanger Oxymethod (Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)) and the like, and an automatic sequence analysis device such as an ALF DNA sequencer (Manufactured by Pharmacia) and the like can be used to determine the base sequence of the DNA.
[294] RDO can be prepared by using a conventional method or a DNA synthesizer.
[295] RDO is introduced into the host cell, and the enzyme is involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-acetylglucosamine on the N-acetylglucosamine 6 at the N-glycosidic conjugated sugar chain reducing terminal. As a method of selecting a cell in which a mutation has occurred in a gene of an enzyme involved in the sugar chain modification to which α is bound, the mutation of a gene on a chromosome described in Molecular Cloning 2nd Edition, Current Protocols, Molecular Biology, etc. The method of detecting directly is mentioned.
[296] In addition, the N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal of the enzyme involved in the synthesis of the introduced intracellular sugar nucleotide GDP-fucose described in (1) of (1) above. A method for selecting a transformant using the activity of an enzyme involved in the sugar chain modification to which α-fucose is bound to α6 is selected as the index, and the sugar chain structure of the glycoprotein on the cell membrane described in (5) below. The method of selecting a transformant as an index, or the method of selecting a transformant using the sugar chain structure of the produced antibody molecule described in 4 or 5 below as an index, can also be used.
[297] Constructs of RDOs are described in Science, 273 , 1386 (1996): Nature Medicine, 4 , 285 (1998); Hepatology, 25 , 1462 (1997); Gene Therapy, 5 , 1960 (1999); Gene Therapy, 5 , 1960 (1999); Journal of Molecular Medicine (J. Mol. Med.), 75 , 829 (1997); Procedures of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 96 , 8774 (1999); Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 96 , 8768 (1999); Nucleic Acid Research (Nuc. Acids. Res.), 27 , 1323 (1999); Investigation of Dematolology (Invest. Dematol.), 111 , 1172 (1998); Nature Biotech., 16 , 1343 (1998); Nature Biotech., 18 , 43 (2000); It can be designed according to the description of Nature Biotech., 18 , 555 (2000) and the like.
[298] (d) Preparation of host cell used for producing cell of the present invention by RNAi method
[299] The host cell used to prepare the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or fucose on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end. The target of the enzyme involved in the sugar chain modification to which the first position of α is bound can be targeted, and the RNAi method can be used, for example, as follows.
[300] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. CDNA is prepared.
[301] The base sequence of the prepared cDNA is determined.
[302] Based on the DNA sequence determined, an enzyme or α-binding sugar on which the 1st position of fucose binds to 6 of N-acetylglucosamine at the end of the chain-reducing sugar chain or the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose. Construct a construct of an RNAi gene of appropriate length that includes a portion encoding an enzyme involved in chain modification or a portion of an untranslated region.
[303] In order to express the RNAi gene in a cell, a recombinant vector is produced by inserting a fragment or full length of the prepared DNA downstream of a promoter of a suitable expression vector.
[304] The transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
[305] A sugar chain modification in which the first position of the fucose α-bonds on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal or the activity of an enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose. By selecting a transformant based on the activity of the enzyme involved in the production or the sugar chain structure of the production antibody molecule or the glycoprotein on the cell surface, the host cell used for producing the cell of the present invention can be obtained.
[306] As host cells, yeasts, animal cells, insect cells, plant cells, etc., N-acetyl at the N-glycoside-linked complex sugar chain reduction terminal or enzyme involved in the synthesis of the target intracellular sugar nucleotide GDP-fucose As long as the first place of the fucose on the 6th place of glucosamine has the gene of the enzyme which is involved in the sugar chain modification which binds to α, it can be used. Specifically, the host cell of the following 3 is mentioned.
[307] As the expression vector, one containing a promoter at a position capable of autonomous replication or insertion into a chromosome in the host cell and capable of transferring the designed RNAi gene is used. Specifically, the expression vector of 3 mentioned later is mentioned.
[308] For introduction of genes into various host cells, a method of introducing a recombinant vector suitable for various host cells described below can be used.
[309] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal As a method of selecting a transformant based on the activity of the enzyme to be mentioned, the method as described in (a) of (1) of this paragraph 1 is mentioned, for example.
[310] As a method of selecting a transformant based on the sugar chain structure of the sugar protein on a cell membrane, the method as described in (5) of this paragraph 1 is mentioned, for example. As a method of selecting a transformant using the sugar chain structure of the produced antibody molecule as an index, for example, the method described in 4 below or 5 below may be mentioned.
[311] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. As a method of preparing the cDNA of "the preparation method of DNA" as described in (a) of (1) of this paragraph 1, etc. are mentioned, for example.
[312] In addition, the 1st position of fucose is on the 6th position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal or the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose without using an expression vector. The host cell used for producing the cell of this invention can also be obtained by introduce | transducing the RNAi gene designed based on the nucleotide sequence of the enzyme which participates in the sugar chain binding to directly into a host cell.
[313] RNAi gene can be prepared by a conventional method or a DNA synthesizer.
[314] Constructs of RNAi genes are described in Nature, 391 , 806 (1998); Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 95 , 15502 (l998); Nature, 395 , 854 (l998): Procedures of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 96 , 5049 (1999); Cell, 95 , 1017 (l998); Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 96 , 1451 (1999); Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 95 , 13959 (l998); Nature Cell Biol., 2 , 70 (2000), etc. can be designed according to description.
[315] (e) Preparation of the host cell used for producing the cell of this invention by the method using a transposon
[316] The host cell used for producing the cell of the present invention is involved in the synthesis of intracellular sugar nucleotide GDP-fucose using the transposon system described in Nature Genet., 25 , 35 (2000) and the like. Activity of the enzyme involved in the sugar chain modification that α-binding of fucose on 6 of N-acetylglucosamine at the N-acetylglucosamine-linked complex sugar chain-reducing terminal, or sugar on the production antibody molecule or cell membrane By selecting a mutant as an index of the sugar chain structure of the protein, a host cell used for producing the cell of the present invention can be produced.
[317] The transposon system is a system for inducing mutations by randomly inserting a foreign gene onto a chromosome. A transposon system is generally used to randomly insert a foreign gene inserted into a transposon as a mutation-producing vector. Expression vectors of transposes are simultaneously introduced into cells.
[318] Traposase can be used as long as it is suitable for the sequence of the transposon to be used.
[319] As a foreign gene, any gene can be used as long as it induces mutation in the DNA of the host cell.
[320] As host cells, yeast, animal cells, insect cells, plant cells, etc., N-acetyl at the N-glycoside-linked complex sugar chain reducing end or an enzyme involved in the synthesis of sugar nucleotides GDP-fucose in the target cells As long as the first place of the fucose on the 6th place of glucosamine has the gene of the enzyme which is involved in the sugar chain modification which binds to α, it can be used. Specifically, the host cell of the following 3 is mentioned. For introduction of genes into various host cells, a method of introducing a recombinant vector suitable for various host cells described below can be used.
[321] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal As a method of selecting a mutant based on the activity of the enzyme to be mentioned, the method as described in (a) of (1) of this paragraph 1 is mentioned, for example.
[322] As a method of selecting a mutant based on the sugar chain structure of the glycoprotein on a cell membrane, the method as described in (5) of this paragraph 1 is mentioned, for example. As a method of selecting a mutant based on the sugar chain structure of a production antibody molecule, the method as described later in 4 or 5 later is mentioned, for example.
[323] (2) Technique to introduce dominant negative body of gene of enzyme
[324] The host cell used to prepare the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or fucose on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end. It can produce by using the technique of targeting the gene of the enzyme which participates in the sugar chain modification which the 1st position of (alpha) couple | bonds, and introduce | transduces the dominant negative body of the enzyme. Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD, Fx, GFPP, and fucokinase. As an enzyme involved in the sugar chain modification in which the 1st position of the fucose is on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal, specifically, α-1,6-fucosyltrans Perase, alpha-L-fucosidase, etc. are mentioned.
[325] These enzymes are enzymes that catalyze specific reactions having substrate specificities, and dominant negatives of these enzymes can be produced by destroying the active centers of enzymes having such catalytic specificities. Among the target enzymes, the production of the dominant negative body will be described in detail below using GMD as an example.
[326] Analysis of the three-dimensional structure of GMD derived from Escherichia coli revealed that four amino acids (133 threonine, 135 th glutamic acid, 157 th tyrosine, and 161 lysine) play an important role in enzyme activity. Structure, 8 , 2, 2000). That is, as a result of producing a variant in which these four amino acids were substituted with other other amino acids based on the three-dimensional information, the enzyme activity was significantly reduced in all the variants. On the other hand, little change was observed in all the variants regarding the coenzyme NADP of GMD and GDP-mannose, which is a substrate. Therefore, a dominant negative body can be produced by substituting these four amino acids which are responsible for the enzyme activity of GMD. Based on the result of GMD derived from Escherichia coli, homology comparison and stereoscopic prediction based on amino acid sequence information are performed. For example, in GMD derived from CHO cells (SEQ ID NO: 41), the 155th threonine, 157 A dominant negative body can be produced by substituting the first glutamic acid, the 179th tyrosine, and the 183th lysine with other amino acids. Production of a gene incorporating such amino acid substitution can be carried out using a site-specific mutation introduction method described in Molecular Cloning Second Edition, Current Protocols, Molecular Biology, and the like.
[327] The host cell used for producing the cell of the present invention is a molecular cloning second edition, Current Protocols Molecular Biology and Manifold, using the dominant negative gene of the target enzyme produced as described above. According to the gene introduction method described in Ting Mouse Embroidery 2nd Edition and the like, for example, it can be produced as follows.
[328] Enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or enzymes involved in the sugar chain modification at which the 1st position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. A gene encoding a dominant negative body of (hereinafter, abbreviated as a dominant negative body gene) is prepared.
[329] Based on the full-length DNA of the prepared dominant negative body gene, the DNA fragment of the appropriate length containing the part which codes the protein as needed is prepared.
[330] The recombinant vector is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of the appropriate expression vector.
[331] The transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
[332] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal By selecting a transformant as an indicator based on the activity of an enzyme or a sugar chain structure of a sugar protein on a production antibody molecule or a cell membrane, a host cell used for producing a cell of the present invention can be produced.
[333] Examples of host cells include yeasts, animal cells, insect cells, and plant cells, such as N-acetylglucosamine at the end of N-glycoside binding complex sugar chain or enzymes involved in the synthesis of the target intracellular sugar nucleotide GDP-fucose. If the first position of the fucose on the 6th has a gene of an enzyme involved in the sugar chain modification that α-linked, it can be used. Specifically, the host cell of the following 3 is mentioned.
[334] As the expression vector, a promoter containing a promoter at a position capable of autonomous replication or insertion into a chromosome in the host cell and capable of transferring a DNA encoding a target dominant negative may be used. Specifically, the expression vector of 3 mentioned later is mentioned.
[335] For introduction of genes into various host cells, a method of introducing a recombinant vector suitable for various host cells described below can be used.
[336] The activity of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the sugar chain modification in which the first place of fucose is on the 6th of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reduction terminal As a method of selecting a transformant based on the activity of the enzyme to be mentioned, the method as described in (a) of 1 (1) mentioned later is mentioned, for example.
[337] As a method of selecting a transformant based on the sugar chain structure of the sugar protein on a cell membrane, the method as described in (5) of 1 below is mentioned, for example. As a method of selecting a transformant using the sugar chain structure of the produced antibody molecule as an index, for example, the method described in 4 below or 5 below may be mentioned.
[338] (3) Techniques for introducing mutations for enzymes
[339] The host cell used to prepare the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or fucose on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end. The mutation can be produced by introducing a mutation to the gene of the enzyme involved in the sugar chain modification to which the first position of α binds, and selecting a desired cell line that has mutated the enzyme.
[340] Specific enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD, Fx, GFPP, fucokinase and the like. As an enzyme which is involved in the sugar chain modification in which the 1st position of the fucose is on 6 of the N-acetylglucosamine at the N-glycoside-bonded complex sugar chain reduction terminal, specifically, α-1,6-fucosyl Transferase, α-L-fucosidase, and the like.
[341] Methods include: 1) N-glycosidic conjugated sugar chain activity or activity of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose from mutants treated with parental or naturally occurring mutants in mutagenesis. A method of selecting a desired cell line as an indicator of the activity of an enzyme involved in the sugar chain modification in which the 1st position of the fucose is on the 6th position of N-acetylglucosamine at the reducing end; 2) Mutation-treated mutations From a sieve or naturally occurring mutant, a method of selecting a desired cell line as an indicator of the sugar chain structure of the producing antibody molecule, 3) from a mutant or a naturally occurring mutant treated with a parent strain in mutagenesis, The desired cell line is selected based on the sugar chain structure of the sugar protein on the cell membrane of the cell. And a method for such.
[342] As mutagenesis treatment, any treatment can be used as long as point mutations, deletions or frame shift mutations are induced in the DNA of cells of the parent cell.
[343] Specifically, treatment with ethyl nitrosourea, nitrosoguanidine, benzopyrene, acridine pigment, irradiation of radiation, and the like can be given. Various alkylating agents and carcinogens can also be used as mutagenesis agents. As a method of causing a mutagen to act on a cell, for example, the third edition of tissue culture technology (Asakura Bookstore), Japan Society for Tissue Culture (1996), Nature Genet., 24 , 314, (2000) The method described in these etc. can be mentioned.
[344] Examples of naturally occurring mutants include mutants naturally occurring by continuing passage in normal cell culture conditions without performing special mutagenesis treatment.
[345] The activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal As a method of measuring the activity of the enzyme to say, the method as described in (a) of (1) of this paragraph 1 is mentioned, for example. As a method of identifying the sugar chain structure of a production antibody molecule, the method as described in 4 or 5 later is mentioned, for example. As a method of identifying the sugar chain structure of the glycoprotein on a cell membrane, the method as described in 1 (5) of this paragraph is mentioned, for example.
[346] (4) Techniques for inhibiting transcription or translation of enzyme genes
[347] The host cell used to prepare the cells of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or fucose on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end. 1st place of the enzyme targets the genes of enzymes involved in the α-linked sugar chain modification, antisense RNA / DNA technology [Bioscience and Industry, 50 , 322 (1992), Chemistry, 46 , 681 (1991), Biotechnology, 9 , 358 (1992), Trends in Biotechnology, 10 , 87 (1992), Trends in Biotechnology, 10 , 152 (1992), Cell Engineering, 16 , 1463 (1997)], Triple Helix Technology [Trends in Biotechnology, 10 , 132 (1992)] and the like can be produced by inhibiting the transcription or translation of the target gene.
[348] Specific examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD, Fx, GFPP, and fucokinase. As an enzyme which is involved in the sugar chain modification in which the 1st position of the fucose is on 6 of the N-acetylglucosamine at the N-glycoside-bonded complex sugar chain reduction terminal, specifically, α-1,6-fucosyl Transferase, α-L-fucosidase, and the like.
[349] (5) A method of selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain reduction terminal are recognized.
[350] The host cell used for producing the cell of the present invention is a strain that is resistant to a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain reduction terminal are recognized. It can produce by using the method of selecting.
[351] As a method of selecting a strain resistant to a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain-reducing terminal are recognized, for example, a somatic cell And a method using a lectin as described in & Molecular Genetics (Somatic Cell Mol. Genet.), 12 , 51 (1986) and the like.
[352] As the lectin, any lectin can be used as long as the lectin recognizes the sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain reduction terminal are recognized. Soybean lectin LCA (lentil agulutinine derived from Lance cullinaris), pea lectin PSA (pea lectin derived from Pisum sativa), silkworm lectin VFA (agulutinine from B. favia), wild grape lectin AAL (Alleleia) Lectins derived from Aurantia).
[353] Specifically, the cultured cells are passaged for 1 to 2 weeks, preferably 1 to 1 week in a medium containing the above-described lectin at a concentration of 1 µg / ml to 1 mg / mg. By picking up colonies and transferring them to a separate incubator, and continuing the culture in a medium containing lectin again, the 6th position of N-acetylglucosamine at the N-glycosidic linkage sugar chain reducing end of the present invention and the 1st position of fucose A strain resistant to lectins that recognize α-linked sugar chain structures can be selected.
[354] 2. Preparation of transgenic non-human animals or plants of the present invention or their offspring
[355] Transgenic non-human animals or plants or genomes whose genome genes have been modified to control the activity of the enzymes involved in the modification of sugar chains of the antibody molecule of the present invention are enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose or An enzyme involved in a sugar chain modification wherein α-binding of fucose on 6 of N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal 6 targets a gene, and is produced by using 1 above. From embryonic stem cells, fertilized egg cells, and plant callus cells of the invention, for example, it can be produced as follows.
[356] In the case of a transgenic non-human animal, by using the method described in 1. above for a non-human animal of interest, for example, embryonic stem cells such as cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys, rabbits, etc. To sugar chain modifications in which the first position of fucose binds to the 6th position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal, or the activity of an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose. The embryonic stem cells of the present invention in which the activity of the enzyme involved is controlled can be produced.
[357] Specifically, the first position of fucose is α-binding on 6 of N-acetylglucosamine at the N-glycosidic linkage complex sugar chain-reducing terminal or the activity of an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose on the chromosome. Genes encoding enzymes involved in sugar chain modification are inactivated by known homologous recombination techniques (eg, Nature, 326 , 6110, 295 (1987), Cell, 51 , 3, 503 (1987), etc.). Mutated clones that are substituted with a mutated or arbitrary sequence are created. Using the produced embryonic stem cells (e.g., the mutant clones thereof), chimeras consisting of embryonic stem cell clones and normal cells by a method such as injection chimera or aggregate chimera method of animal embryo into blastcyst. The object can be prepared. The cross-linking of chimeric and normal individuals results in the activity of enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose in systemic cells or N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminus. A transgenic non-human animal in which the activity of an enzyme involved in the sugar chain modification to which α-binding fucose is bound can be obtained.
[358] In addition, by using the same method as the method described in the above 1. to fertilized egg cells, such as cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys, rabbits, etc. The activity of enzymes involved in the synthesis of sugar nucleotides GDP-fucose or the sugar chain modification in which the first position of fucose is on the 6th of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reduction terminal The fertilized egg cell of the present invention having reduced activity of the enzyme can be produced.
[359] The fertilized egg cells produced were transplanted and delivered to the fallopian tubes or uterus of females of phantom pregnancy using the embryo transplantation method described in the manifolding mouse embryo 2nd edition and the like. Transgenic with decreased activity of enzyme involved in synthesis or activity of enzyme involved in sugar chain modification at which 1st position of fucose is on 6 of N-acetylglucosamine at N-glycoside-linked complex sugar chain-reducing terminal Nonhuman animals can be produced.
[360] In the case of transgenic plants, N-glycosidic binding or activity of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose by using the same method as described in 1. above, on the desired plant callus or cells. The callus of the present invention can be produced in which the activity of the enzyme involved in the sugar chain modification in which the first position of the fucose is α-bonded on 6 of the N-acetylglucosamine at the complex sugar chain reduction terminal is reduced.
[361] Called callus was known in a known method [tissue culture, 20 (1994); Tissue culture, 21 (1995); According to Trends in Biotechnology, 15 , 45 (1997), the cells were re-differentiated by culturing in a medium containing auxin and cytokinin to be involved in the synthesis of intracellular sugar nucleotide GDP-fucose. A transgenic plant can be produced in which the activity of the enzyme involved in the sugar chain modification in which the first position of the fucose binds to the 6th of N-acetylglucosamine at the N-glycoside-linked complex sugar chain-reducing terminal is α-bonded. Can be.
[362] 3. Manufacturing method of antibody composition
[363] Antibody compositions include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter abbreviated as Antibodies), Monoclonal Antibodies: principles and practice, Third Edition, Acad. Using methods described in Press, 1993 (hereinafter abbreviated to monoclonal antibodies), Antibody Engineering, A Practical Approach, IRL Press at 0xford University Press, 1996 (hereinafter abbreviated as antibody engineering), etc. For example, it can obtain by expressing in a host cell as follows.
[364] The full-length cDNA of the anti-CD2O antibody molecule of this invention is prepared, and the DNA fragment of suitable length containing the part which encodes this antibody molecule is prepared.
[365] The recombinant vector is prepared by inserting the DNA fragment or full-length cDNA downstream of the promoter of a suitable expression vector.
[366] By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant for producing an antibody molecule can be obtained.
[367] Any host cell can be used as long as it can express a gene of interest, such as yeast, animal cells, insect cells, or plant cells.
[368] Enzymes involved in the modification of N-glycosidic linkage sugar chains binding to the Fc region of an antibody molecule, ie enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycoside linkage complex sugar chain reduction ends Select cells in which activity of enzymes involved in sugar chain modification at which 1st place of fucose binds to 6th place of N-acetylglucosamine is reduced or deleted, or hosts cells obtained by various artificial methods shown in 1 above. It can also be used as a cell.
[369] As the expression vector, one containing a promoter at a position capable of autonomous replication or insertion into a chromosome in the host cell and capable of transcribing DNA encoding an antibody molecule of interest is used.
[370] cDNA can be prepared from tissues or cells of human or non-human animals using a probe primer specific for the antibody antibody of interest, according to the method for preparing DNA according to (a) of (1) above. have.
[371] When using yeast as a host cell, as an expression vector, YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), etc. are mentioned, for example.
[372] As the promoter, any one that can be expressed in the yeast strain can be used. For example, the relevant system such as hexkinase is a promoter of the gene, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, gal And 1 promoter, gal 10 promoter, heat shock protein promoter, MFα1 promoter, CUP 1 promoter and the like.
[373] Examples of host cells include microorganisms belonging to the genus Saccharomyces, Schizocarcinomyces, Cleveriberomyces, Tricosphorone, Schwanomyces and the like, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, and the like.
[374] As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into yeast. For example, an electroporation method (Methods. Enzymol., 194 , 182 (1990)) ], Spheroplast method [Proc. Natl. Acad. Sci. USA, 84 , 1929 (1978)], lithium acetate method [J. Bacteriology, 153] , 163 (1983), Proc. Natl. Acad. Sci. USA, 75 , 1929 (1978), and the like.
[375] When animal cells are used as a host, as expression vectors, for example, pcDNAI, pcDM8 (commercially available from Funakoshi Co., Ltd.), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechnology, 3 , 133, (1990)], pAS3-3 [JP-A-2-227075], pCDM8 [Nature, 329 , 840, (1987)], pcDNAI / Amp (Invitrogen G), pREP4 (Invitrogen), pAGE103 (J. Biochemistry, 101 , 1307 (1987)), pAGE210, and the like.
[376] As the promoter, any one that can be expressed in an animal cell can be used. For example, a promoter of an IE (immediate early) gene of cytomegalovirus (CMV), an initial promoter of SV40, a promoter of retrovirus, a metalloti Oneine promoter, heat shock promoter, SR alpha promoter, etc. are mentioned. In addition, the enhancer of the IE gene of human CMV may be used together with a promoter.
[377] As host cells, Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are cells of Chinese hamsters, HBT5637 (Japanese Patent Laid-Open No. 63-299), rat myelomer cells, mice Myelomer cells, Syrian hamster kidney-derived cells, embryonic stem cells, fertilized egg cells and the like.
[378] As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into an animal cell. For example, the electroporation method (Cytotechnology, 3 , 133 (1990)), the calcium phosphate method [ Japanese Patent Laid-Open No. 2-227075], Lipofection Method [Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 84 , 7413 (1987)], Injection Method [Manipulating the Mouse] Emblem of Laboratories], Particle Gun (Gene Gun) [Japanese Patent No. 2606856, Japanese Patent No. 2517813], DEAE-Dextran Method [Bio Manual Series 4-Gene Introduction and Expression Analysis Method ( Yododa Takashi Arai, Shinichi (1994)], the viral vector method [Maniplating Mouse Embryo 2nd Edition], etc. are mentioned.
[379] When using insect cells as hosts, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology, The protein can be expressed by the method described in 6 , 47 (1988) and the like.
[380] In other words, the recombinant transfection vector and the Vercuro virus can be co-introduced into insect cells to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus can be infected with insect cells to express the protein.
[381] As a transduction vector used in this method, pVL1392, pVL1393, pBlueBac III (all are Invitorogen), etc. are mentioned, for example.
[382] As the Bucurero virus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus infected with yadon moths and insects, can be used.
[383] Insect cells include Sf9 and Sf21, ovarian cells of Spodopterafrugiperda [Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992). ], Trichoplusiani ovary cells High 5 (Invitrogen), and the like can be used.
[384] As a method of co-introducing the recombinant gene introduction vector into the insect cell and the Bucure virus for preparing a recombinant virus, for example, the calcium phosphate method (JP-A-2-227075), lipofection method [ Proc. Natl. Acad. Sci. USA, 84 , 7413 (1987), and the like.
[385] When using a plant cell as a host cell, as an expression vector, Ti plasmid, tobacco mosaic virus vector, etc. are mentioned, for example.
[386] As a promoter, any can be used as long as it can be expressed in a plant cell, For example, the 35S promoter of a cauliflower mosaic virus (CaMV), the rice actin 1 promoter, etc. are mentioned.
[387] Examples of the host cells include plant cells such as tobacco, potatoes, tomatoes, carrots, beans, mustards, alfurpers, rice, wheat, and barley.
[388] As a method for introducing a recombinant vector, any method can be used as long as it introduces DNA into plant cells, and for example, a method using Agrobacterium [Japanese Patent Laid-Open No. 59-140885, Japanese Laid-Open Patent Publication] Japanese Patent Application Laid-Open No. 60-70080, WO94 / 00977], Electroporation Method [JP-A-60-251887], Method of using a particle gun (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 2517813], etc. Can be mentioned.
[389] As the gene expression method, in addition to direct expression, secretion production, fusion protein expression of the Fc region with other proteins, and the like can be performed in accordance with the method described in the molecular cloning second edition and the like.
[390] When expressed by bacteria, yeast, animal cells, insect cells or plant cells into which a gene involved in the synthesis of sugar chains is introduced, an antibody molecule to which sugar or sugar chains are added by the introduced gene can be obtained.
[391] The antibody composition can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating antibody molecules in the culture, and extracting from the culture. The method of culturing a transformant in a medium can be performed according to the conventional method used for culturing a host cell.
[392] As a medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryotes such as yeast as a host, the medium contains a carbon source, a nitrogen source, an inorganic salt, etc. which the organism can magnetize and transforms. As long as a medium capable of efficiently culturing the sieve, both natural and synthetic media can be used.
[393] As the carbon source, the organism can be magnetized, and carbohydrates such as glucose, fructose, sucrose, molasses, starch or starch hydrolyzate containing them, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, etc. Can be used.
[394] Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, cornstarch flicker, casein hydrolyzate , Soybean sulphate and soybean sulphate hydrolyzate, various fermented microorganisms and their digestions may be used.
[395] As the inorganic salts, there may be used potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like.
[396] The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. The pH during the culture is maintained at 3.0 to 9.0. Preparation of pH is performed using inorganic or organic acid, alkaline solution, urea, calcium carbonate, ammonia, and the like.
[397] Moreover, you may add antibiotics, such as ampicillin and tetracycline, to a medium as needed during culturing.
[398] When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with a recombinant vector using a lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside, etc., and a recombinant vector using a trp promoter, You may add indole acrylic acid etc. to a medium.
[399] As a medium for culturing transformants obtained using animal cells as a host, RPMI1640 medium (The Journal of the American Medical Association, 199 , 519 (1967)), which is generally used, Eagle MEM medium [Science, 122 , 501 (1952)], Dulbecco's modified MEM medium [Virology, 8 , 396 (1959)], 199 medium [Procedings of the Society for the Biologic Medicine] (Proceeding of the Society for the Biological Medicine), 73 , 1 (1950)], Whitten medium [Developmental Engineering Experiment Manual-Method for Making Transgenic Mice (Kodansha), Katsuki Motoya (1987)] A medium to which fetal serum or the like is added can be used.
[400] The culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and 5% CO ₂ .
[401] Moreover, you may add antibiotic substances, such as kanamycin and penicillin, to a culture medium as needed during the culture.
[402] As a medium for culturing the transformant obtained using insect cells as a host, TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell 400, ExCell 405 (both JRH), which are generally used. Biosciences), Grace's Insect Medium (Nature, 195 , 788 (1962)), and the like.
[403] Cultivation is normally performed for 1 to 5 days under conditions, such as pH6-7 and 25-30 degreeC.
[404] Moreover, you may add antibiotics, such as gentamicin, to a medium as needed during culturing.
[405] A transformant obtained by using a plant cell as a host can be cultured as a cell or by differentiating into cells or organs of a plant. As a medium for culturing the transformant, commonly used Murashige and Skoog (MS) medium, White medium, or auxin and cytokines such as auxin and cytokine are added to these mediums. A medium etc. can be used.
[406] Cultivation is normally performed for 3 to 60 days under conditions of pH 5-9 and 20-40 degreeC.
[407] In addition, antibiotics such as kanamycin and hyglomycin may be added to the medium as necessary during the culture.
[408] As described above, a transformant derived from a microorganism, an animal cell, or a plant cell having a recombinant vector containing a DNA encoding an antibody molecule is cultured according to a conventional culture method to produce and accumulate an antibody composition. The antibody composition can be prepared by extracting the antibody composition from the culture.
[409] As an expression method of an antibody gene, in addition to direct expression, secretion production, fusion protein expression, etc. can be performed according to the method described in molecular cloning 2nd edition.
[410] As the production method of the antibody composition, there is a method of producing in the host cell, a method of secreting other than the host cell, or a method of producing on the host cell outer membrane, and by changing the structure of the host cell to be used and the antibody molecule to be produced. You can choose the method.
[411] When the antibody composition is produced in the host cell or on the host cell outer membrane, Paulson et al. [Journal of Biological Chemistry (J. Biol, Chem.), 264 , 17619 (1989)], Row et al. Of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 86 , 8227 (1989); Gene antibody Develop., 4 , 1288 (1990)], or by applying the method described in Unexamined-Japanese-Patent No. 5-336963, 6-823021, etc., the antibody composition is host | hosted. It can be actively secreted out of the cell.
[412] That is, DNA encoding an antibody molecule and DNA encoding a signal peptide suitable for expression of the antibody molecule are inserted into the expression vector using a method of genetic recombination, and the antibody molecule is expressed after introducing the expression vector into the host cell. By making it possible, the target antibody molecule can be actively secreted out of the host cell.
[413] Moreover, according to the method described in Unexamined-Japanese-Patent No. 2-227075, a production amount can also be raised using the gene amplification system which used the dihydrofolate reductase gene etc.
[414] In addition, by re-differentiating cells of an animal or plant into which a gene is introduced, an animal individual (transgenic non-human animal) or a plant individual (transgenic plant) into which the gene is introduced is prepared, and these individuals are used to prepare an antibody composition. You may.
[415] When the transformant is an animal individual or a plant individual, the antibody composition can be produced by breeding or cultivating according to a conventional method, producing and accumulating the antibody composition, and extracting the antibody composition from the animal individual or plant individual.
[416] Methods for preparing antibody compositions using animal subjects include, for example, known methods [American Journal of Clinical Nutrition, 63 , 639S (1996); Antibodies of interest in animals incorporating genes according to American Journal of Clinical Nutrition, 63 , 627S (1996): Bio / Technology, 9 , 830 (1991)]. The method of producing a composition is mentioned.
[417] In the case of animal individuals, for example, a transgenic non-human animal in which DNA encoding an antibody molecule is introduced is produced, the antibody composition is produced and accumulated in the animal, and the antibody composition is produced by collecting the antibody composition from the animal. can do. As production and accumulation place in the animal, the milk of the animal (Japanese Patent Laid-Open No. 63-309192), eggs, etc. are mentioned, for example. As the promoter used at this time, any one that can be expressed in an animal can be used. For example, α casein promoter, β casein promoter, β lactoglobulin promoter, whey acidic protein promoter and the like which are mammary cell specific promoters It is preferably used.
[418] As a method for producing an antibody composition using a plant individual, for example, a transgenic plant into which DNA encoding an antibody molecule is introduced can be known methods [tissue culture, 20 (1994); Tissue culture, 21 (1995); Trends in Biotechnology, 15 , 45 (1997)], and the method of producing an antibody composition by producing and accumulating the antibody composition in the plant, and extracting the antibody composition in the plant. have.
[419] The antibody composition produced by the transformant which introduce | transduced the gene which codes an antibody molecule is, for example, when an antibody composition is expressed in the lysis state in a cell, after completion | finish of culture, a cell is collect | recovered by centrifugation and water-based After suspension in the buffer solution, the cells are disrupted by an ultrasonic crusher, French press, Mantongaurin homogenizer, dynomil or the like to obtain a cell-free extract. From the supernatant obtained by centrifugation of the cell-free extract, the isolation and purification method of ordinary enzymes, i.e., solvent extraction, salting out with ammonium sulfate, desalting, precipitation with an organic solvent, diethylaminoethyl (DEAE)- Anion exchange chromatography using resin such as Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Co., Ltd.), cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia), butyl sepharose, Antibodies can be used alone or in combination by hydrophobic chromatography using resins such as phenyl sepharose, gel filtration using molecular sieves, affinity chromatography, chromatographic focusing, and electrophoresis such as isoelectric electrophoresis. Purified product of the composition can be obtained.
[420] In addition, when an antibody composition forms and expresses an insoluble substance in a cell, the insoluble substance of an antibody composition is collect | recovered as a precipitation fraction by similarly collect | recovering, crushing and centrifuging a cell. Insoluble material of the recovered antibody composition is solubilized with a protein denaturant. After diluting or dialysis the solubilizing solution, the antibody composition is returned to a normal three-dimensional structure, and the purified product of the antibody composition can be obtained by the above-described isolation and purification method.
[421] When the antibody composition is secreted out of the cell, the antibody composition or its derivative can be recovered to the culture supernatant. That is, a soluble fraction can be obtained by treating the culture by a method such as centrifugation as described above, and purified products of the antibody composition can be obtained from the soluble fraction by using the above-mentioned isolation and purification method.
[422] As an antibody composition obtained in this way, an antibody, a fragment of an antibody, the fusion protein which has the Fc region of an antibody, etc. are mentioned, for example.
[423] Hereinafter, although the manufacturing method of the composition of a humanized antibody is described as a more specific example of acquisition of the antibody composition of this invention, another antibody composition can also be acquired by the method similar to the said method.
[424] (1) Construction of humanized antibody expression vector
[425] Humanized antibody expression vectors are expression vectors for animal cells in which genes encoding the heavy chain (H chain) and light chain (L chain) C regions of a human antibody are inserted, and the H chain and L of the human antibody in the expression vector for animal cells. It can be constructed by cloning each of the genes encoding the chain C region.
[426] The C region of a human antibody may be the H chain and L chain C region of any human antibody, for example, the C region of the IgG1 subclass of the H chain of the human antibody (hereinafter referred to as hCγ1) and human C region of the k-class of the L chain of an antibody (henceforth hCk), etc. are mentioned.
[427] As a gene encoding the H chain and the L chain C region of a human antibody, chromosomal DNA composed of exons and introns can be used, and cDNA can also be used.
[428] As the expression vector for animal cells, any one capable of inserting and expressing a gene encoding the C region of a human antibody can be used. See, eg, pAGE107 (Cytotechnology, 3 , 133 (1990)), pAGE103 [J. Biochem., 101 , 1307 (1987)], pHSG274 [Gene, 27 , 223 (1984)], pKCR [Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78 , 1527 (1981)], pSG1βd2-4 [Cytotechnology, 4 , 173 ( 1990). As promoters and enhancers used in expression vectors for animal cells, early promoters and enhancers of SV40 [Journal Biochemistry (J. Biochem.), 101 , 1307 (1987)], LTRs of the Moroni mouse leukemia virus [Bio Chemical and Biophysical Research Communication (Biochem. Biophys. Res. Commun.), 149 , 960 (1987)], immunoglobulin H chains and promoters [Cell, 41 , 479 (1985)] and Enhancer [Cell ( Cell), 33 , 717 (1983)].
[429] The humanized antibody expression vector may be a type in which the antibody H chain and the L chain are present on different vectors or a type present on the same vector (hereinafter, referred to as a tandem type). Tandem type humanized antibody expression vectors are preferred in view of ease of construction, easy introduction into animal cells, and the balance of expression amounts of antibody H chain and L chain in animal cells. Journal of Immunological Methods (J. Immunol. Methods), 167 , 271 (1994)]. Examples of tandem humanized antibody expression vectors include pKANTEX 93 [Mole. Immunol., 37 , 1035 (2000)], pEE18 [Hybridoma, 17 , 559 (1998)], and the like. Can be.
[430] The constructed humanized antibody expression vector can be used for expression in animal cells of humanized chimeric antibodies and humanized CDR-grafted antibodies.
[431] (2) Acquisition of cDNA encoding V region of antibody of animal other than human
[432] CDNA encoding the H chain and L chain V region of an antibody of an animal other than human, for example, a mouse antibody, can be obtained by the following method.
[433] CDNA is synthesize | combined by extracting mRNA from the hybridoma cell which produces the target mouse antibody. The synthesized cDNA is cloned into a vector such as phage or plasmid to prepare a cDNA library. A recombinant phage having a cDNA encoding an H chain V region or a recombinant plasmid having a cDNA encoding an H chain V region and a cDNA encoding an L chain V region using a C region portion or a V region portion of an existing mouse antibody from the library or Recombinant plasmids are isolated respectively. The entire nucleotide sequence of the H chain and L chain V regions of the desired mouse antibody on the recombinant phage or recombinant plasmid is determined to estimate the entire amino acid sequence of the H chain and L chain V regions from the base sequence.
[434] As animals other than humans, any of hybridoma cells such as mice, rats, hamsters, rabbits, and the like can be used as long as possible.
[435] As a method for preparing total RNA from hybridoma cells, guanidine-trifluoroacetate acetate method (Methods in Enzymol., 154 , 3 (1987)), and total RNA As a method for preparing mRNA in, oligo (dT) immobilized cellulose column method [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989]. Examples of kits for preparing mRNA from hybridoma cells include a Fast Track mRNA Isolation Kit (Invitrogen), a Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.
[436] Synthesis of cDNA and preparation method of cDNA library include conventional methods [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34], or commercially available kits such as Super Script ™ Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by GiBCO BRL) or ZAP -cDNA Synthesis Kit (made by Stratagene), etc. are mentioned.
[437] In the preparation of the cDNA library, any vector that inserts a cDNA synthesized using mRNA extracted from hybridoma cells as a template can be used as long as the vector can insert the cDNA. See, for example, ZAP Express [Strategies, 5 , 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17 , 9494 (1989)], λzap II (manufactured by Stratagene), λgt 10, λgt 11 [DNA Cloning: A Practical Approach, I , 49 (1985)], Lambda BlueMid (manufactured by Clontech), λExCell, pT7T3 18U ( Pharmacia), pcD2 (Molecular and Cellular Biology (Mol. Cell. Biol.), 3 , 280 (1983)) and pUC18 (Gene, 33 , 103 (1985)) and the like can be used.
[438] E. coli, which introduces a cDNA library constructed by phage or plasmid vectors, can be used as long as it can introduce, express, and maintain the cDNA library. For example, XL1-Blue MRF '[Strategies, 5 , 81 (1992)], C600 [Genetics, 39 , 440 (1954)], Y1088, Y1090 [Science, 222 , 778 (1983)], NM522 [Journal of Molecular Biology (J. Mol. Biol.), 166 , 1 (1983)], K802 [Journal of Molecular Biology (J. Mol. Biol.), 16 , 118 (1966) and JM105 (Gene, 38 , 275 (1985)) and the like.
[439] As a selection method of cDNA clones encoding H chain and L chain V regions of antibodies of non-human animals from the cDNA library, colony hybridization or plaque hybridization using an isotope or fluorescently labeled probe (plaque hybridization) [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989]. In addition, primers were prepared, and cDNA or cDNA library synthesized from mRNA was used as a template, and Polymerase Chain Reaction [hereinafter, referred to as PCR method; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34] can also prepare cDNA encoding the H chain and L chain V regions.
[440] The cDNA selected by the above method is cleaved with a suitable restriction enzyme or the like, and then cloned into a plasmid of pBluescript SK (-) (manufactured by Stratagene), and a commonly used sequencing method, for example, Sanger et al. Reactions of the dideoxy method (Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)) and the like, and an automatic sequence analysis device such as ALFDNA The sequence of the DNA can be determined by analyzing using a sequencer (manufactured by Pharmacia).
[441] The total amino acid sequence of the H chain and L chain V region is estimated from the determined base sequence, and the total amino acid sequence of the H chain and L chain V region of a known antibody [Sequences of Proteins of Immunological Interests]. of Imnunological Interest, US Dept. Health and Human Services, 1991], it can be confirmed whether the obtained cDNA codes the complete amino acid sequence of the H chain and L chain V region of the antibody containing the secretion signal sequence.
[442] Moreover, when the amino acid sequence of an antibody variable region or the base sequence of DNA which codes the variable region is already known, it can manufacture using the following method.
[443] If the amino acid sequence is known, the amino acid sequence is determined using the codon usage frequency [Sequences of Proteins of Imnunological Interest, US Dept. Health and Human Services, 1991] is converted into a DNA sequence, and synthesized a plurality of synthetic DNA having a length of about 100 bases based on the designed DNA sequence, and then subjected to PCR method using them to obtain DNA Can be. If the nucleotide sequence is known, a DNA can be obtained by synthesizing a plurality of synthetic DNAs having a length of about 100 bases based on the information, and performing the PCR method using them.
[444] (3) Analysis of amino acid sequence of the V region of an antibody of an animal other than human
[445] For the complete amino acid sequence of the H chain and L chain V regions of an antibody comprising a secretion signal sequence, the entire amino acid sequence of the H chain and L chain V regions of a known antibody [Sequences of Proteins of Immunological Interest ( Sequences of Proteins of Imnunological Interest), US Dept. Health and Human Services, 1991], it is possible to estimate the length of the secretory signal sequence and the N terminal amino acid sequence, as well as to identify the subgroups to which they belong. In addition, the amino acid sequences of the CDRs of the H chain and the L chain V region also include the amino acid sequences of the H chain and the L chain V region of a known antibody [Sequences of Proteins of Imnunological]. Interest), US Dept. Health and Human Services, 1991].
[446] (4) Construction of humanized chimeric antibody expression vector
[447] An H-chain and L-chain V region of an antibody of an animal other than human is encoded upstream of a gene encoding the H chain and L chain C region of the human antibody of the humanized antibody expression vector according to item 3 of the present invention. The cDNA can be cloned to construct a humanized chimeric antibody expression vector. For example, the cDNA encoding the H chain and L chain V regions of an antibody of a non-human animal is obtained from the nucleotide sequence of the 3 'terminal side of the antibody H chain and L chain V region of an animal other than a human, and the H of a human antibody. Humanized antibody expression as described in (1) of this paragraph 3, respectively, linked with the synthetic DNA which consists of the base sequence of the 5 'terminal side of a chain | strand and L chain C region, and has the recognition sequence of a suitable restriction enzyme in both ends, respectively, Humanoid chimeric antibody expression vectors can be constructed by cloning them upstream of the genes encoding the H chain and L chain C regions of the human antibody of the dragon vector so as to be expressed in an appropriate form.
[448] (5) Construction of cDNA encoding V region of humanized CDR grafted antibody
[449] The cDNA encoding the H chain and L chain V regions of the humanized CDR-grafted antibody can be constructed by the following method. First, the amino acid sequence of the framework of the H chain and L chain V region of a human antibody (hereinafter referred to as FR) of the human antibody to which the CDRs of the H chain and L chain V regions of the antibody of a non-human animal of interest are transplanted is selected. do. As the amino acid sequence of FR of the H chain and L chain V region of a human antibody, any one derived from a human antibody can be used. For example, the amino acid sequence of the FR of the H chain and the L chain V region of a human antibody registered in a database such as Protein Data Bank, etc., the common of each subgroup of the FR of the V region of the H chain and the L chain of a human antibody Amino acid sequences [Sequences of Proteins of Imnunological Interest, US Dept. Health and Human Services, 1991], but among others, in order to produce a humanized CDR-grafted antibody having sufficient activity, amino acid sequences of FRs in the H chain and L chain V regions of antibodies of non-human animals of interest. It is preferred to select an amino acid sequence with as high homology as possible (at least 60% or more).
[450] Next, the amino acid sequences of the CDRs of the H chain and the L chain V region of the antibody of a non-human animal are transplanted to the amino acid sequences of the FR of the H chain and the L chain V region of the selected human antibody, and the humanized CDR grafted antibody. Design the amino acid sequences of the H chain and L chain V regions. Frequency of use of codons showing the designed amino acid sequence in the nucleotide sequence of the antibody gene [Sequences of Proteins of Imnunological Interest, US Dept. Health and Human Services, 1991] is designed to convert DNA sequences and to design DNA sequences encoding amino acid sequences of the H chain and L chain V regions of humanized CDR grafted antibodies. Based on the designed DNA sequence, several synthetic DNAs of about 100 bases in length are synthesized, and PCR is performed using these. In this case, it is preferable to design 4 to 6 synthetic DNAs for both the H chain and the L chain from the reaction efficiency in PCR and the length of the synthesizeable DNA.
[451] In addition, by introducing a recognition sequence of a suitable restriction enzyme at the 5 'end of the synthetic DNA located at both ends, it can be easily cloned into the humanized antibody expression vector constructed in (1) of the present invention. After PCR, the amplified product was cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the nucleotide sequence was determined by the method described in (2) of this item 3, and the H of the target human-type CDR grafted antibody. A plasmid having a DNA sequence encoding the amino acid sequence of the chain and L chain V regions is obtained.
[452] (6) Construction of humanized CDR grafted antibody expression vector
[453] The H chain of the human-type CDR grafted antibody constructed in (5) of the present item 3 upstream of the gene encoding the H chain and the L-chain C region of the human antibody of the humanized antibody expression vector of the present item 3 (1). And cDNA encoding the L chain V region can be cloned to construct a humanized CDR grafted antibody expression vector. For example, recognition of a restriction enzyme suitable for the 5 'end of the synthetic DNA located at both ends of the synthetic DNA used when constructing the H chain and the L chain V region of the humanized CDR-grafted antibody in (5) of this item 3 By introducing a sequence, the humanized CDR-grafted antibody was cloned so that they could be expressed in an appropriate form upstream of the genes encoding the H chain and L chain C regions of the human antibody of the humanized antibody expression vector according to (1) of the present invention. Expression vectors can be constructed.
[454] (7) Stable Production of Humanized Antibodies
[455] A transgenic strain stably producing humanized chimeric antibodies and humanized CDR-grafted antibodies (hereinafter collectively referred to as humanized antibodies) by introducing the humanized antibody expression vectors according to (4) and (6) of the present item 3 into appropriate animal cells. You can get it.
[456] As a method of introducing a humanized antibody expression vector into an animal cell, the electroporation method [Unexamined-Japanese-Patent No. 2-257891; Cytotechnology, 3 , 133 (1990)].
[457] As the animal cell into which the humanized antibody expression vector is introduced, any cell can be used as long as it is an animal cell capable of producing a humanized antibody.
[458] Specifically, NS0 cells, SP2 / O cells, Chinese hamster ovary cells CH0 / dhfr- cells, CHO / DG44 cells, rat myelomer YB2 / 0 cells, IR983F cells, and Syrian hamster kidneys, which are mouse myelomer cells, Although BHK cells, the Namalba cell which is a heat myelomer cell, etc. are mentioned, Preferably, CHO / DG44 cells which are Chinese hamster ovary cells, rat myelomer YB2 / 0 cells, etc. are mentioned.
[459] After introduction of the humanized antibody expression vector, the transformant which stably produces the humanized antibody is, according to the method disclosed in Japanese Patent Laid-Open No. 2-257891, G418 sulfate (hereinafter referred to as G418; manufactured by SIGMA Corporation) and the like. It can select with the medium for animal cell culture containing the chemical agent of. As an animal cell culture medium, RPMI1640 medium (made by Nissei Seiyaku Co., Ltd.), GIT medium (manufactured by Nippon Seiyaku Co., Ltd.), EX-CELL302 medium (manufactured by JRH), IMDM medium (manufactured by Gibco Brl), Hybridoma-SFM medium Or a medium to which various additives such as fetal bovine serum (hereinafter referred to as FCS) are added to these mediums. By culturing the obtained transformant in a medium, humanized antibodies can be produced and accumulated in the culture supernatant. Production amount and antigen binding activity of the humanized antibody in the culture supernatant is denoted by the enzyme immune antibody method (hereinafter referred to as ELISA method: Antibody: Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14, 1998, monoclonal antibodies; Monoclonal Antibodies (Principles and Practice), Academic Press Limited, 1996]. The transgenic strain can increase the amount of humanized antibody produced using a DHFR gene amplification system or the like according to the method disclosed in JP-A-2-257891.
[460] Humanized antibodies can be purified using a Protein A column from the transformed supernatant [Antibodies: A Laboratory Manual, Cold spring Harbor Laboratory, Chapter 8, 1988, monoclonal]. Antibodies: Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996]. In addition, the purification method normally used for the purification of a protein can be used. For example, gel filtration, ion exchange chromatography, ultrafiltration and the like can be combined and carried out for purification. The molecular weight of the H chain, L chain, or the whole antibody molecule of the purified humanized antibody was determined by polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE; Nature, 227 , 680 (1970)) or Western blotting [ Antibodies: A Laboratory Manual, Cold spring Harbor Laboratory, Chapter 12, 1988, Monoclonal Antibodies: Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996].
[461] As mentioned above, although the manufacturing method of the antibody composition which used the animal cell as a host was shown, in the yeast, an insect cell, a plant cell, or an animal individual or a plant individual as described above, an antibody composition can be manufactured by the same method as an animal cell. .
[462] If the host cell already has the ability to express the antibody molecule, a cell expressing the antibody molecule is prepared using the method described in 1 below, the cells are oriented, and the desired antibody composition is purified from the culture. The antibody composition of the present invention can be prepared.
[463] 4. Evaluation of Activity of Antibody Compositions
[464] As a method of measuring the protein amount, the binding activity with an antigen, or the effector function of a purified antibody composition, the well-known method described in monoclonal antibodies, antibody engineering, etc. can be used.
[465] As specific examples, when the antibody composition is a humanized antibody, the binding activity with the antigen and the binding activity with respect to the antigen-positive cultured cell line are determined by ELISA method and fluorescent antibody method (Cancer Immunol. Immunother., 36 ). , 373 (1993)]. Cytotoxic activity against antigen positive oriented cell lines can be assessed by measuring CDC activity, ADCC activity and the like (Cancer Immunol. Immunother., 36 , 373 (1993)).
[466] In addition, the safety and therapeutic effect of the antibody composition in humans can be evaluated using an appropriate model of an animal relatively close to humans such as crab-eating macaque.
[467] 5. Analysis of Sugar Chains of Antibody Compositions
[468] The sugar chain structure of the antibody molecule expressed in various cells can be carried out in accordance with the analysis of the sugar chain structure of a conventional glycoprotein. For example, the sugar chains bound to the IgG molecule are composed of neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetylglucosamine, and acidic sugars such as sialic acid. It can carry out using methods, such as sugar chain structure analysis using these etc.
[469] (1) Analysis of neutral sugar and amino sugar composition
[470] Analysis of the composition of sugar chains of an antibody molecule can be carried out by acid hydrolysis of sugar chains with trifluoro acetic acid or the like to liberate neutral sugars or amino sugars and to analyze the composition ratio thereof.
[471] As a specific method, the method of using the composition analysis apparatus per Dionex company make is mentioned. BioLC is a device for analyzing sugar composition by high perfomance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) (J. Liq. Chromatogr., 6 , 1577 (1983)).
[472] The composition ratio can also be analyzed by the fluorescent labeling method with 2-aminopyridine. Specifically, 2-aminopyridyl is obtained by acid hydrolysis of a sample according to a known method (Agric. Biol. Chem., 55 (1) , 283-284 (1991)). The composition ratio can be calculated by fluorescent labeling and HPLC analysis.
[473] (2) sugar chain structure analysis
[474] Structural analysis of sugar chains of antibody molecules is carried out using two-dimensional sugar chain mapping method (Anal. Biochem., 171 , 73 (1988), biochemical method 23-sugar protein sugar chain research method (Academic Press Center) Takahashi Reiko edition (1989)]. The two-dimensional sugar chain mapping method, for example, plots the retention time or elution position of sugar chains by reverse phase chromatography on the X axis, and the retention time or elution position of sugar chains by normal phase chromatography on the Y axis, respectively. By comparing with the result of the, is a method of estimating the sugar chain structure.
[475] Specifically, the hydrazine digestion of the antibody and the release of sugar chains from the antibody resulted in fluorescent labeling of sugar chains by 2-aminopyridine (hereinafter referred to as PA) [Journal of Biochem., 95 , 197 ( 1984)] and the sugar chains are separated by excess PAization reagent and the like by gel filtration and subjected to reverse phase chromatography. Subsequently, normal phase chromatography is performed on each peak of the sugar chains separated and collected. Plotting on a two-dimensional sugar chain map based on these results to determine sugar from spot comparisons with sugar chain standards (manufactured by TaKaRa), Analytical Biochem., 171 , 73 (1988). The chain structure can be estimated.
[476] And the mass estimated by MALDI-TOF-MS etc. of each sugar chain can be confirmed, and the structure estimated by the two-dimensional sugar chain mapping method can be confirmed.
[477] 6. Immunological Quantitative Methods of Identifying Sugar Chain Structures of Antibody Molecules
[478] The antibody composition is composed of antibody molecules having different sugar chain structures that are bound to the Fc region of the antibody. The antibody composition of the present invention has a ratio of 20% or more of sugar chains, which are bound to the Fc region and does not have fucose bonded to N-acetylglucosamine at the sugar chain reduction terminal, of the total N-glycoside-linked complex sugar chains, and has high ADCC. It has the characteristic of showing activity. Such an antibody composition can be identified by using the analysis of the sugar chain structure of the antibody molecule of 5 above. It can also be identified by using an immunological quantitative method using lectins.
[479] Identification of sugar chain structures of antibody molecules using immunological quantitative methods using lectins can be found in Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., ( 1995); enzyme immunoassay, 3rd edition, medical vow (1987); revised edition, enzyme antibody method, interdisciplinary project (1985)], etc., Western immunity, RIA (Radioimmunoassay), VIA (Viroimmunoassay), EIA (Enzymoimmunoassay), FIA According to immunological quantitative methods such as Fluoroimmunoassay and Metalloimmunoassay, MIA can be carried out as follows, for example.
[480] A lectin that recognizes a sugar chain structure of an antibody molecule forming the antibody composition is labeled, and the labeled lectin is reacted with a sample antibody composition. The amount of the complex of labeled lectin and antibody molecule is then measured.
[481] Lectins used to identify sugar chain structures of antibody molecules include, for example, WGA [T. Wheat germ agulutinine from T. vulgaris], ConA [C. Concanavalin A from C. ensiformis], RIC [R. Toxins from R. communis], L-PHA [P. Leukoagglutinin from P. vulgaris], LCA [L. Lentil Agulutinine from L. culinaris], PSA [P. Pea lectins derived from P. sativum], AAL [Aleuria aurantia lectins], ACL [Amaranthus caudatus lectins], BPL [Bauhinia perpurea ( Bauhinia Purpurea Lectin], DSL [Datura stramonium Lectin], DBA [Dolichos biflorus Agulutinine], EBL [Elderberry Balk Lectin], ECL [Eri Erythrina cristagalli lectin], EEL [Euonymus europaeus lectin], GNL [Galanthus nivalis ractine], GSL [Griffonia simplicifolia] ) Lectin], HPA [Helix pomatia agulutinine], HHL [Hippeastrum Hybrid lectin], Jacalin, LTL [Lotus tetragonolobus lectin ], LEL [Lycopersicon esculentum lectins], MAL [Mackia Amu] Maackia amuresis lectin], MPL [Maclura pomifera lectin], NPL [Narcissus pseudonarcissus lectin], PNA [Peanut Agglutinin], E-PHA [P. Phaeseolus vulgaris Erythroagglutinin], PTL [Psophocarpus tetragonolobus lectin], RCA [Ricinus communis Agulutinine] ], STL [Solanum tuberosum lectin], SJA [Sophora japonica agulutinin], SBA [Soybean Agglutinin], UEA [Ulex Europaeus ( Ulex europaeus) agulutinin], VVL [Vicia villosa lectin], WFA [Wisteria floribunda agulutinin].
[482] It is preferable to use a lectin that specifically recognizes a sugar chain structure in which fucose is bound to N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal, and specific examples thereof include lentil lectin LCA (lance). Culinaris-derived lentil agulutinine), pea lectin PSA (pea lectin derived from Pisum sativa), silkworm lectin VFA (Agulutinine derived from Visia fava), wild mushroom lectin ALL (derived from Allurea aurantia) Lectins).
[483] 7. Use of Antibody Molecules of the Invention
[484] Since the antibody composition of the present invention specifically binds to CD20 and has high antibody dependent longitudinal injury activity, it is useful for the prevention and treatment of various CD20 expressing cell-related patients including cancer.
[485] In cancer, ie malignant tumors, cancer cells proliferate, such as in B cell lymphoma, certain B cells multiply. Conventional anticancer agents are characterized by inhibiting the proliferation of cancer cells. However, an antibody having high antibody-dependent cytotoxic activity is more effective as a therapeutic agent than a conventional anticancer agent because cancer can be treated by injuring cancer cells expressing an antigen by a killer cell effect. In particular, the antitumor effect of the antibody medicine alone is insufficient in the therapeutic drug of cancer, and in combination with chemotherapy (Science, 280 , 1197 (1998)), the antibody composition of the present invention alone If stronger antitumor effects are recognized, side effects may be reduced by less dependence on chemotherapy.
[486] A medicament containing the antibody composition of the present invention may be administered alone as a therapeutic agent, but is usually prepared by any method well known in the art of formulation by mixing with one or more pharmaceutically acceptable carriers. It is preferable to provide as a pharmaceutical.
[487] The route of administration is preferably to be used the most effective at the time of treatment, oral administration or parenteral administration such as oral, airway, rectal, subcutaneous, intramuscular and intravenous, preferably in the case of antibody preparations Intravenous administration is mentioned.
[488] Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
[489] Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
[490] Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, and anti-corrosive agents such as p-hydroxybenzoic acid esters. Perfumes, such as strawberry flavor and peppermint, can be manufactured as an additive.
[491] Capsules, tablets, powders, granules, etc. include excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxypropyl cellulose, gelatin It can manufacture using binders, surfactants, such as fatty acid ester, plasticizers, such as glycerin, as an additive.
[492] Suitable formulations for parenteral administration include injections, suppositories, and sprays.
[493] Injectables are prepared using a carrier consisting of a salt solution, a glucose solution or a mixture of both. Alternatively, the powder composition may be prepared by lyophilizing the antibody composition according to a conventional method and adding sodium chloride thereto.
[494] Suppositories are formulated using carriers such as cacao oil, hydrogenated fats or carboxylic acids.
[495] In addition, the nebulizer is prepared by using the antibody composition itself, or a carrier which does not irritate the oral cavity and airway mucosa of the recipient, and also easily disperses the antibody composition as fine particles to easily absorb it.
[496] Specific examples of the carrier include lactose, glycerin, and the like. Preparations such as aerosols and dry powders are possible depending on the nature of the antibody composition and the carrier used. Moreover, these parenteral agents can also add the component illustrated as an additive in an oral preparation.
[497] The dosage or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, weight, and the like, but is usually 10 µg / kg to 20 mg / kg per adult as the amount of the active ingredient.
[498] Moreover, the method of examining the antitumor effect on various tumor cells of an antibody composition can mention CDC activity measurement method, ADCC activity measurement method, etc. in an in vitro experiment, Tumor in experimental animals, such as a mouse, in an in vivo experiment. And antitumor experiments using the system.
[499] CDC activity, ADCC activity, and anti-tumor experiments were performed according to the methods described in Cancer Immunology Immunotherapy, 36 , 373 (1993); Cancer Research, 54 , 1511 (1994). It can be carried out.
[500] The present invention will be described in more detail with reference to the following examples, which are merely illustrative of the present invention and do not limit the scope of the present invention.
[528] Example 1. Preparation of anti-CD20 humanoid chimeric antibody
[529] 1. Construction of anti-CD20 humanoid chimeric antibody expression vector
[530] (1) Construction of cDNA encoding L chain V region of anti-CD20 mouse monoclonal antibody
[531] The cDNA (described in SEQ ID NO: 11) encoding the amino acid sequence of the L chain V region (hereinafter referred to as VL) of the anti-CD20 mouse monoclonal antibody 2B8 described in WO94 / 11026 using PCR was described below. It was built in the same way.
[532] First, binding base sequences (including restriction enzyme recognition sequences for cloning to humanized antibody expression vectors) of amplification primers for PCR reaction are added to the 5 'and 3' ends of the base sequence of VL described in WO94 / 11026. It was. The designed nucleotide sequence is divided into 6 nucleotide sequences of about 100 bases from the 5 'end side (adjacent nucleotide sequences have an overlapping sequence of about 20 bases at the ends thereof), and these are replaced by a sense chain and an antisense chain. In fact, six synthetic DNAs of SEQ ID NOs: 15, 16, 17, 18, 19, and 20 were produced (consigned to GENSET Corporation).
[533] 50 μl of reaction solution [KOD DNA Polymerase-attached PCR Buffer # 1 (manufactured by Toyo Spin Co., Ltd.), 0.2 mM dNTPs, 1 mM magnesium chloride, 0.5 μM M13 primer M4 (produced by Takarazu Corporation) so that the final concentration of each oligonucleotide was 0.1 μM. ), 0.5 μM M13 primer RV (manufactured by Takara Co., Ltd.), and heated at 94 ° C. for 3 minutes using a DNA thermal cycler GeneAmp PCR System 9600 (manufactured by Perkin Elmer), followed by 2.5 units of KOD DNA. Polymerase (made by Toyo Spin Co., Ltd.) was added, followed by 25 cycles of 1 minute at 74 ° C for 30 seconds, 55 ° C for 30 seconds, and further reaction at 72 ° C for 10 minutes. After 25 µl of the reaction solution was subjected to agarose gel electrophoresis, a PCR product of about 0.44 kb was recovered using a QIAquick Gel Extraction Kit (produced by QIAGEN).
[534] Next, 7.5 µl of DNA 0.1 g obtained from the plasmid pBluescript II SK (-) (manufactured by Stratagene) and the restriction enzyme Sma I (manufactured by Takara Co., Ltd.) and about 0.1 g of the PCR product obtained above were added to sterile water and 7.5 µl. 7.5 µl of solution I (manufactured by Takarazu Corporation) and 0.3 µl of restriction enzyme Sma I (manufactured by Takarazu Corporation) of TAKARA ligation kit ver.2 were added thereto and reacted at 22 ° C for 2 hours. E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) was transformed using the recombinant plasmid DNA solution thus obtained. Each plasmid DNA was prepared from a clone of the transgenic strain, reacted according to the attached instructions using the BigDye Teminator Cycle Sequencing Ready Reaction Kit v2.0 (manufactured by Applied Biosystems), and then subjected to base by the DNA sequencer ABI PRISM 377 of the company. The sequence was interpreted. This gave the plasmid pBS-2B8L shown in FIG. 1 with the desired base sequence.
[535] (2) Construction of cDNA encoding H chain V region of anti-CD20 mouse monoclonal antibody
[536] CDNA (described in SEQ ID NO: 13) encoding the amino acid sequence of the H chain V region (hereinafter referred to as VH) of the anti-CD20 mouse monoclonal antibody 2B8 described in WO94 / 11026 using PCR method as follows. Built.
[537] First, binding base sequences (including restriction enzyme recognition sequences for cloning to a humanized antibody expression vector) of amplification primers for PCR reaction are added to the 5 'and 3' ends of the base sequence of VH described in WO94 / 11026. It was. The designed base sequence is divided into 6 base sequences of about 100 bases from the 5 'end side (adjacent base sequences have a duplicate sequence of about 20 bases at the ends thereof), and the sense and antisense chains are alternated. In fact, six synthetic DNAs of SEQ ID NOs: 25, 26, 27, 28, 29 and 30 were produced (consigned to GENSET).
[538] 50 μl of reaction solution [KOD DNA Polymerase-attached PCR Buffer # 1 (manufactured by Toyo Spin Co.), 0.2 mM dNTPs, 1 mM magnesium chloride, 0.5 μM M13 primer M4 ), 0.5 μM M13 primer RV (manufactured by Takara Co., Ltd.), and heated at 94 ° C. for 3 minutes using a DNA thermal cycler GeneAmp PCR System 9600 (manufactured by Perkin Elmer), followed by 2.5 units of KOD DNA Polymerase. (Toyo Spun Co., Ltd.) was added, and 25 cycles of 1 minute was performed at 74 degreeC for 30 second, 55 degreeC for 30 second, and 74 degreeC, and it was made to react at 72 degreeC for 10 minutes. After 25 µl of the reaction solution was subjected to agarose gel electrophoresis, a PCR product of about 0.49 kb was recovered using a QIAquick Gel Extraction Kit (produced by QIAGEN).
[539] Next, 0.1 µg of DNA obtained by plasmid pBluescript II SK (-) (manufactured by Stratagene) and the restriction enzyme SmaI (manufactured by Takara Corporation) and about 0.1 µg of the PCR product obtained above were added to sterile water to 7.5 µl. Then, 7.5 µl of solution I (manufactured by Takarazu Corporation) and 0.3 µl of restriction enzyme Sma I (manufactured by Takarazu Corporation) of TAKARA ligation kit ver.2 were added and reacted overnight at 22 ° C.
[540] E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) was transformed using the recombinant plasmid DNA solution thus obtained. Each plasmid DNA was prepared from clones of the transgenic strain, reacted according to the attached instructions using the BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (manufactured by Applied Biosystems), and then subjected to base by the DNA sequencer ABI PRISM 377. The sequence was interpreted. This gave the plasmid pBS-2B8H shown in FIG. 2 with the desired base sequence.
[541] Next, the synthetic DNA shown in SEQ ID NO: 31 was designed to replace the 14th amino acid residue from Ala to Pro, and was then analyzed by PCR using LA PCR in vitro Mutagenesis Primer Set for pBluescript II (manufactured by Takarazu Corporation). The base was substituted. 50 μl of reaction solution containing 1 ng of the plasmid pBS-2B8H [LA PCR Buffer II (manufactured by Takara Co., Ltd.), 2.5 units of TaKaRa LA Taq, 0.4 mM dNTPs, 2.5 mM magnesium chloride, 50 nM T3 BcaBEST sequencing primer ( Takanara Co., Ltd.), 50 nM of the above-mentioned mutation introduction primer (SEQ ID NO: 31, manufactured by GENSET)] was prepared, using a DNA thermal cycler GeneAmp PCR System 9600 (manufactured by Perkin Elmer) for 55 seconds at 94 ° C. 25 cycles of 1 minute 30 seconds were performed at 72 degreeC for 2 minutes. After 30 µl of the reaction solution was subjected to agarose gel electrophoresis, about 0.44 kb of PCR product was recovered using a QIAquick Gel Extraction Kit (produced by QIAGEN) to obtain an aqueous solution of 30 µl. Similarly, 50 µl of the reaction solution containing 1 ng of the plasmid pBS-2B8H [LA PCR Buffer II (manufactured by Takara Co., Ltd.), 2.5 units of TaKaRa LA Taq, 0.4 mM dNTPs, 2.5 mM magnesium chloride, 50 nM T7 BcaBEST sequence Assay primers (manufactured by Takarazu Corporation) and 50nM MUT B1 primer (manufactured by Takarazu Corporation) were subjected to PCR reaction. After 30 µl of the reaction solution was subjected to agarose gel electrophoresis, about 0.63 kb of PCR product was recovered using a QIAquick Gel Extraction Kit (produced by QIAGEN) to obtain an aqueous solution of 30 µl. Subsequently, 0.45 kb of the PCR product and 0.63 kb of the PCR product were added to 47.5 µl of the reaction solution [LA PCR Buffer II (manufactured by Takaraz Corporation), 0.4 mM dNTPs, 2.5 mM magnesium chloride). After addition, the mixture was heated at 90 ° C for 10 minutes using a DNA thermal cycler GeneAmp PCR System 9600 (manufactured by Perkin Elmer), cooled to 37 ° C over 60 minutes, and then annealed by holding at 37 ° C for 15 minutes. I was. After adding 2.5 units of TaKaRa LA Taq (manufactured by Takara Co., Ltd.) and reacting at 72 ° C. for 3 minutes, T3BcaBEST sequencing primers (manufactured by Takara Co., Ltd.) and T7 BcaBEST sequencing primers (manufactured by Takara Co., Ltd.) at 10 pmol each were prepared. ) Was added to make 50 µl of the reaction solution, and 10 cycles were performed at 94 ° C for 30 seconds, at 55 ° C for 2 minutes, and at 72 ° C for 1 minute 30 seconds. After 25 µl of the reaction solution was purified by a QIA quick PCR purification kit (produced by QIAGEN), 1/2 of the amount was determined by 10 units of restriction enzyme Kpn I (manufactured by Takara Co., Ltd.) and 10 units of restriction enzyme Sac I (Takara State Irradiation). Reaction) at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis to recover a Kpn I- Sac I fragment of about 0.59 kb .
[542] Next, 1 μg of pBluescriptII SK (-) (manufactured by Stratagene) was reacted at 37 ° C. for 1 hour using 10 units of restriction enzyme Kpn I (manufactured by Takarazon Corporation) and 10 units of Sac I (manufactured by Takarazon Corporation). The reaction solution was then fractionated by agarose gel electrophoresis to recover a Kpn I- Sac I fragment of about 2.9 kb .
[543] The PCR fragment of Kpn Ⅰ- Sac Ⅰ products derived and obtained from the plasmid pBluescriptⅡ SK (-) attached to the guide Kpn Ⅰ- Sac Ⅰ fragment derived using the Solution Ⅰ of DNA Ligation Kit Ver.2 (manufactured by Takara Shuzo Co., Ltd.) Connected according to. E. coli DH5α strain (manufactured by Toyoji Co., Ltd.) was transformed using the recombinant plasmid DNA solution thus obtained, each plasmid DNA was prepared from clones of the transformant strain, and BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (manufactured by Applied Biosystems, Inc.). ) Was reacted according to the attached instructions, and then the base sequence was analyzed by the DNA sequencer ABI PRISM 377.
[544] This gave the plasmid pBS-2B8Hm shown in FIG. 2 with the desired base sequence.
[545] (3) Construction of anti-CD20 humanoid chimeric antibody expression vector
[546] Anti-CD20 humanoid chimeric antibody using vector pKANTEX93 (Mol. Immunol., 37 , 1035, 2000) for expression of humanized antibody and plasmids pBS-2B8L and pBS-2B8Hm obtained in paragraphs (1) and (2) of Example 1 An expression vector pKANTEX2B8P (hereinafter referred to as an anti-CD20 chimeric antibody) was constructed as follows.
[547] 2 μg of the plasmid pBS-2B8L obtained in Example 1 (1) of Example 1 was reacted at 55 ° C. for 1 hour using 10 units of restriction enzyme Bsi WI (manufactured by New England Biolabs), followed by another 10 units of restriction. It was made to react at 37 degreeC for 1 hour using the enzyme Eco RI (manufactured by Takarazu Corporation). The reaction solution was fractionated by agarose gel electrophoresis to recover a Bsi WI- Eco RI fragment of about 0.14 kb .
[548] Next, 2 µg of the humanized antibody expression vector pKANTEX93 was reacted at 55 ° C for 1 hour using 10 units of restriction enzyme Bsi WI (manufactured by New England Biolabs), and then 10 units of restriction enzyme Eco RI Irradiated at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis to recover a Bsi WI- Eco RI fragment of about 12.75 kb .
[549] Next, the plasmid pBS-2B8L-derived Bsi WI- Eco RI fragment and the plasmid pKANTEX93-derived Bsi WI- Eco RI fragment were attached using Solution I of DNA Ligation Kit Ver.2 (manufactured by Takara Co., Ltd.). Connected according to. E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) was transformed using the recombinant plasmid DNA solution thus obtained to obtain plasmid pKANTEX2B8-L shown in FIG. 3.
[550] Next, 2 µg of the plasmid pBS-2B8Hm obtained in paragraph 1 (2) of Example 1 was reacted at 37 ° C for 1 hour using 10 units of restriction enzyme Apa I (manufactured by Takarazon Corporation), and then 10 units again. The reaction was carried out at 37 ° C. for 1 hour using the restriction enzyme Not I (manufactured by Takarazu Corporation). The reaction solution was fractionated by agarose gel electrophoresis to recover about 0.45 kb of Apa I- Not I fragment.
[551] Next, 3 μg of the plasmid pKANTEX2B8-L obtained above was reacted at 37 ° C. for 1 hour using 10 units of restriction enzyme Apa I (manufactured by Takarazon Corporation), and then 10 units of restriction enzyme Not I (Takara) Irradiated at 37 ° C for 1 hour. The reaction solution was fractionated by agarose gel electrophoresis to recover an Apa I- Not I fragment of about 13.16 kb.
[552] Next, the plasmid pBS-derived 2B8Hm of Apa Ⅰ- Not Ⅰ fragment with Apa Ⅰ- Not Ⅰ pKANTEX2B8 fragment of the plasmid-derived L obtained in the use of Solution Ⅰ of DNA Ligation Kit Ver.2 (manufactured by Takara Shuzo Co., Ltd.) Connections were made according to the attached instructions. E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) was transformed using the recombinant plasmid DNA solution thus obtained, and each plasmid DNA was prepared from clones of the transformant strain.
[553] Using the obtained plasmid, the DNA sequence of BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (manufactured by Applied Biosystems) was analyzed using the DNA sequencer 377 of the company. As a result, the target DNA was cloned. It was confirmed that the plasmid pKANTEX2B8P was obtained.
[554] 2. Stable expression using animal cells of anti-CD20 chimeric antibody
[555] (1) Production of production cells using rat myeloma B / O cells
[556] The anti-CD20 chimeric antibody expression vector pKANTEX2B8P obtained in item 1 (3) of Example 1 was used to express the anti-CD20 chimeric antibody in animal cells as follows.
[557] 10 μg of the plasmid pKANTEX2B8P was introduced into the rat myelomer cell line YB2 / O cells (ATCC CRL1662) of 4 × 10 ⁶ cells by the electroporation method (Cytotechnology, 3 , 133 (l990)) and then 40 200 ml / well was dispensed in 96-well microtiter plates (manufactured by Sumitomo Bakelite Co., Ltd.) in suspension in ml of H-SFM (manufactured by GIBCO-BRL) medium (5% fetal bovine serum (FCS) was added). It was. After incubation at 37 ° C. for 24 hours in a 5% CO ₂ incubator, G418 was added to 1 mg / ml and incubated for 1-2 weeks. A colony of transgenic strains showing G418 resistance appeared, and the culture supernatant was recovered from the confluent well, and the ELISA method shown in the paragraph 2 (2) of Example 1 in the production amount of human IgG antibody in the culture supernatant. Was measured.
[558] For transformed strains of wells in which human IgG antibody expression was recognized in the culture supernatant, 1 mg / ml of G418 and dihydrofolic acid of the dhfr gene product were used for the purpose of increasing the amount of antibody expression using the dhfr gene amplification system. Suspended to 1 to 2 x 10 ⁵ cells / ml in H-SFM medium containing 50 nM of methotrexate (hereinafter referred to as MTX: manufactured by SIGMA), an inhibitor of a reductase (hereinafter referred to as DHFR), 24 1 ml was dispensed in the well plate (the Greiner company make). Incubated for 1-2 weeks at 37 ° C. in a 5% CO ₂ incubator to induce transformants exhibiting 50 nM MTX resistance. When the transformant became a confluent in the well, the production amount of the human IgG antibody in the culture supernatant was measured by the ELISA method shown in 2 (2) of Example 1. For transformed strains of wells in which human IgG antibody expression was recognized in the culture supernatant, MTX concentrations were sequentially raised to 100 nM and 200 nM by the same method as described above. Finally, G418 was 1 mg / ml and MTX was 20 OnM. Transformants that were proliferated with H-SFM medium containing the concentration and highly expressed anti-CD20 chimeric antibody were obtained. The resulting transformants were cloned (cloned) by limiting dilution to obtain clone KM3065 expressing anti-CD20 chimeric antibody. In addition, by using the quantification method of the gene transcript of α-1,6-fucoosyltransferase shown in Example 8 of WO00 / 61739, a strain having a relatively low amount of the transcript was selected and used as a superior strain.
[559] The transgenic clone KM3065, which produced the anti-CD20 chimeric antibody thus obtained, was assigned to the Patent Biological Deposit Center of the Industrial Technology Research Institute of Japan (Dec. 6, Ibaraki, Higashi 1-chome, 1-Chuo Chuo, Ibaraki, Japan) on December 21, 2001. Deposited as FERM BP-7834.
[560] (2) Measurement of human IgG antibody concentration in culture supernatant (ELISA method)
[561] A goat anti-human IgG (H & L) antibody (manufactured by American Qualex) was diluted with phosphate buffered saline (hereinafter referred to as PBS) to 1 µg / ml, and was placed on a 96-well ELISA plate (manufactured by Grainer). Aliquots to μl / well and left overnight at 4 ° C. to adsorb. After washing with PBS, PBS (hereinafter referred to as 1% BSA-PBS) containing 1% bovine serum albumin (hereinafter referred to as BSA; manufactured by APC Co., Ltd.) was added to 100 µl / well and allowed to react at room temperature for 1 hour. Remaining active groups were blocked. 1% BSA-PBS was discarded, and various diluted solutions of the culture supernatant of the transformed strain and purified humanized chimeric antibody were added at 50 µl / well, and reacted at room temperature for 2 hours. After the reaction, each well was washed with PBS containing 0.15% Tween20 (hereinafter referred to as Tween-PBS), and then peroxidase-labeled goat anti-human IgG (1 ml) diluted 3000-fold with 1% BSA-PBS. An antibody solution (manufactured by American Qualex) was added as a secondary antibody solution at 50 µl / well and allowed to react at room temperature for 1 hour. After the reaction, washing with Tween-PBS, and dissolving 0.15 g of ABTS substrate solution [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium in 1 L of 0.1 M citric acid buffer (pH 4.2) Then, a solution obtained by adding 1 μl / ml of hydrogen peroxide immediately before use was added at 50 μl / well and developed, and the absorbance at 415 nm (hereinafter referred to as OD415) was measured.
[562] 3. Purification from the Culture Supernatant of Anti-CD20 Chimeric Antibodies
[563] H-SFM (GIBCO-) containing a transgenic cell clone KM3065 expressing the anti-CD20 chimeric antibody obtained in Example 1 (2) (200 nM) and Daigo's GF21 (manufactured by Wako Pure Chemical) at a concentration of 5% It was suspended so as to be 1 × 10 ⁵ cells / ml by BRL Co., Ltd. and 50 ml was dispensed into a 182 cm 2 flask (manufactured by Grainer). Culture supernatants were recovered at the point of confluent by incubating at 37 ° C. for 7 days in a 5% CO ₂ incubator. Anti-CD20 chimeric antibody KM3065 was purified from the culture supernatant using the Prosep-A (Millipore) column according to the attached instructions. About 3 µg of the obtained anti-CD20 chimeric antibody KM3065 was electrophoresed according to a known method (Nature, 227 , 680 (1970)) to investigate molecular weight and purity. As a result, the purified anti-CD20 chimeric antibody KM3065 was about 150 kilodaltons (hereinafter referred to as Kd) under non-reducing conditions, and two bands of about 50 Kd and about 25 Kd were recognized under reducing conditions. The size of these proteins is that the IgG-type antibody has a molecular weight of about 150 Kd under non-reducing conditions, and disulfide bonds (hereinafter referred to as SS bonds) in the molecule are cleaved under reducing conditions, and thus an H chain having a molecular weight of about 50 Kd. And an L chain having a molecular weight of about 25 Kd [Antibodies: A Laboratory Manual, Cold spring Harbor Laboratory, Chapter 14, 1988, Monoclonal Antibodies: Principal] In line with Monoclonal Antibodies (Principles and Practice), Academic Press Limited, 1996, and in close agreement with the Rituxan ^TM electrophoresis pattern, the anti-CD20 chimeric antibody KM3065 was expressed as an antibody molecule of the correct structure. Confirmed.
[564] Example 2. Assessment of Activity of Anti-CD20 Chimeric Antibodies
[565] 1. Binding Activity of Anti-CD20 Chimeric Antibody to CD20 Expressing Cells (Fluorescent Antibody Method)
[566] The binding activity of the purified CD2O chimeric antibody obtained in Example 3 of Example 1 was evaluated by the fluorescent antibody method using flow cytometry. Human lymphoma cell line Raji cells (JCRB9012), which are CD20 positive cells, were divided into 96-well U-shaped plates (manufactured by Falcon) in 2 × 10 ⁵ cells. Antibody CD20 chimeric antibody diluted with FACS buffer (1% BSA-PBS, 0.02% EDTA, 0.05% NaN ₃ ) was added as 50 μl / well of antibody solution (concentration 0.039-40 μg / ml) for 30 minutes in ice. Reacted. After washing twice with 200 µl / well in a buffer for FACS, 50 µl / well was added to a 100-fold diluted PE-labeled anti-human IgG antibody (manufactured by Coulter) using a buffer for FACS. After shading and reacting for 30 minutes in ice, the mixture was washed three times with 200 µl / well and finally suspended in 500 µl, and the fluorescence intensity was measured with a flow cytometer. The results are shown in FIG. It was confirmed that KM3065 and Rituxan ^™ showed almost equivalent binding activity, with an increase in fluorescence intensity recognized in dependence of antibody concentration. In addition, binding activity to human CCRF-CEM cells (ATCC CCL119), which are CD20 negative cells, was examined by the same method with an antibody concentration of 40 µg / ml. The results are shown in FIG. Both KM3065 and Rituxan ^™ did not bind, suggesting that KM3065 binds specifically to CD20.
[567] 2. In vitro Cytotoxic Activity (ADCC Activity) of Anti-CD20 Chimeric Antibody
[568] In order to evaluate the in vitro cytotoxic activity of the purified anti-CD20 chimeric antibody obtained in Example 3 above, ADCC activity was measured according to the following method.
[569] (1) Preparation of Target Cell Solution
[570] Human B lymphocyte cultured cell line WIL2-S cells (ATCC CRL8885) or Ramos cells (ATCC CRL1596), Raji cells (cultured in RPMI1640-FCS (10) medium (RPMI1640 medium containing 10% FCS (manufactured by GIBCO BRL)) JCRB9012) was washed with RPMI1640-FCS (5) medium (RPM1640 medium containing 5% FCS (manufactured by GIBCO BRL)) by centrifugation and suspension, followed by 2 × 10 with RPMI1640-FCS (5) medium. Prepared at ⁵ cells / ml to give a target cell solution.
[571] (2) Preparation of Effector Cell Solution
[572] 50 ml of healthy venous blood was collected, and 0.5 ml of heparin sodium (manufactured by Shimizu Pharmaceutical Co., Ltd.) was added and mixed slowly. This was centrifuged (800 g, 20 minutes) according to the instructions using Lymphoprep (manufactured by AXIS SHIELD) to separate the monocyte layer. After centrifugation three times with RPMI1640-FCS (5) medium to wash, the medium was resuspended at a concentration of 4 × 10 ⁶ cells / ml using the same medium to prepare an effector cell solution.
[573] (3) Determination of ADCC Activity
[574] To each well of a 96-well U-bottom plate (manufactured by Falcon), 50 μl (1 × 10 ⁴ cells / well) of the target cell solution prepared in the above (1) was dispensed. Next, 50 mu l of the effector cell solution prepared in (2) was added (2x10 ⁵ cells / well, the ratio of effector cells to target cells was 20: 1). In addition, various anti-CD20 chimeric antibodies were added so as to have a final concentration of 0.3 to 3000 ng / ml, the total amount was set to 150 µl, and the reaction was carried out at 37 ° C for 4 hours. After the reaction, the plates were centrifuged and the lactic acid dehydrogenase (LDH) activity in the supernatant was measured by using the CytoTox96Non-Radioactive Cytotoxicity Assay (promega) to obtain absorbance data according to the attached instructions. The absorbance data of the target cell natural glass is obtained by using only the medium instead of the effector cell solution and the antibody solution, and the absorbance data of the effector cell natural glass is obtained by performing the above operation using only the medium instead of the target cell solution and the antibody solution. It was. Absorbance data of the entire target cell glass was measured by adding 15 μl of 9% Triton X-100 solution 45 minutes before the end of the reaction using the medium instead of the antibody solution and the effector cell solution to measure the LDH activity of the supernatant. Obtained by ADCC activity was calculated by the following equation.
[575] Cytotoxic activity (%) = {[absorbance of the sample]-[absorbance of the effector cell natural glass]-[absorbance of the target cell natural glass]} / {[absorbance of the target cell whole glass]-[absorbance of the target cell natural glass] ]} × 100
[576] 6 shows the results of targeting three types of cell lines. FIG. 6A shows the results of targeting Raji cells (JCRB9012), FIG. 6B targeting Ramos cells (ATCC CRL1596), and FIG. 6C targeting WIL2-S cells (ATCC CRL8885). As shown in FIG. 6, KM3065 showed higher ADCC activity than Rituxan ^™ at any antibody concentration, and had the highest cytotoxic activity.
[577] Example 3 Sugar Chain Analysis of Anti-CD20 Chimeric Antibodies
[578] The sugar chain analysis of the anti-CD20 chimeric antibody purified in Section 3 of Example 1 was performed. Hydrazine digestion of KM3065 and Rituxan ^™ resulted in cleavage of sugar chains from proteins (Method in Enzymology, 83 , 263 (1982)). After removing hydrazine by distillation under reduced pressure, an aqueous ammonium acetate solution and acetic anhydride were added, followed by N-acetylation. After freeze drying, fluorescent labeling with 2-aminopyridine was performed (Journal of Biochemistry, 95 , 197, 1984). Fluorescently labeled sugar chain group (hereinafter referred to as PA-ized sugar chain group) was separated from the excess reagent using Surperdex Peptide HR10 / 30 column (manufactured by Pharmacia). The sugar chain fractions were dried with a centrifugal concentrator to obtain purified PA-ized sugar chain groups. Next, reverse phase HPLC analysis of the purified PA-ized sugar chain group was performed using a CLC-ODS column (manufactured by Shimadzu Co., Ltd.).
[579] Figure 7 shows the elution degree obtained by analyzing the reversed-phase HPLC of PA-sugar chains prepared from anti-CD20 chimeric antibody. The elution degree of KM3065 in FIG. 7A and Rituxan ^™ is shown in FIG. 7B, respectively. The relative fluorescence intensity is shown on the vertical axis and the elution time on the horizontal axis, respectively. 10 mM sodium phosphate buffer (pH 3.8) as buffer A and 10 mM sodium phosphate buffer (pH 3.8) + 0.5% -1-butanol as buffer B were analyzed with the following gradients.
[580] Minutes0809090.1120 Buffer B (%)0606000
[581] The peak of (1)-(b) shown in FIG. 7 shows the structure of following (1)-(10).
[582]
[583]
[584] GlcNAc represents N-acetylglucosamine, Gal represents galactose, Man represents mannose, Fuc represents fucose, and PA represents pyridylamino group. In Fig. 7, the 6th position of the fucose to the 1st position of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reduction terminal has a sugar chain group in which α-, 6,6-fucose is not α-linked. Sugar chain group or α-1,6-fucose unlinked sugar chain group) of the sum of the areas occupied by the peaks ① to ④, ⑨ and ⑩ It calculated from the sum total of area. In addition, the sugar chain group in which the 6th position of fucose is α-linked to the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal (hereinafter, α-1,6-fucose-bonded sugar chain group) Ratio) was calculated from the sum of the areas occupied by the peaks ⑤ to ⑧ of the sum of the areas occupied by the respective peaks 1 to VII.
[585] As a result, the sugar chain content of α-1,6-fucose to which Rituxan ^™ was not bound was 6%, and the sugar content to α-1,6-fucose bound 94%. The sugar chain content of α-1,6-fucose to which KM3065 was not bound was 96%, and the sugar chain content of α-1,6-fucose was 4%. From the above results, it was found that KM3065 had a high ratio of sugar chain content to which α-1,6-fucose was not bound.
[586] Example 4 Acquisition of CHO Cell α-1,6-Fucoosyltransferase (FUT8) Gene
[587] (1) Acquisition of CHO cell α-1,6-fucoosyltransferase (FUT8) cDNA sequence
[588] Chinese hamster FUT8 cDNA was obtained from the single chain cDNA prepared from Chinese hamster ovary-derived CHO / DG44 cells on the second day of culture in Example 8 (1) of WO00 / 61739 in the following order (FIG. 8).
[589] First, a forward primer specific to the 5 'side untranslated region (shown in SEQ ID NO: 21) and a reverse primer specific to the 3' side untranslated region (shown in SEQ ID NO: 22) from the cDNA sequence of mouse FUT8 (GenBank, AB025198) Was designed.
[590] Next, a 25 μl reaction solution containing 1 μl of CDNA derived from CHO / DG44 cells described above using DNA polymerase ExTaq (manufactured by Takara Co., Ltd.) [ExTaq buffer (manufactured by Takara Co., Ltd.), 0.2 mmol / L dNTPs , 4% DMSO, 0.5 μmol / L The specific primers (SEQ ID NO: 21 and SEQ ID NO: 22) were prepared and PCR was performed. PCR was carried out under the condition of heating at 94 ° C for 1 minute, 30 cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 cycles for 2 minutes at 1 cycle, followed by heating at 72 ° C for 10 minutes. It was.
[591] After PCR, the reaction solution was subjected to 0.8% agarose gel electrophoresis to purify about 2 Kb of the specific amplified fragment. 4 µl of this DNA fragment was inserted into plasmid pCR2.1 according to the instructions of the TOPO TA cloning kit (manufactured by Invitrogen), and the E. coli DH5α strain was transformed using the reaction solution. Plasmid DNA was isolated from the 8 clones in which cDNA was inserted among the obtained kanamycin resistant colonies according to a known method.
[592] The base sequence of the cDNA inserted into each plasmid was determined using DNA sequencer 377 (manufactured by Applied Biosystems) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by Applied Biosystems), and the method was in accordance with the attached manual. By this method, it was confirmed that all inserted cDNAs encode a sequence including the ORF full length of CHO cell FUT8. Of these, plasmid DNA was selected in which the misreading of the base accompanying PCR was not included in the sequence. Hereinafter, this plasmid is called CHfFUT8-pCR2.1. The base sequence of the determined CHO cell FUT8 cDNA is shown in SEQ ID NO: 1. The translation part (open reading frame: ORF) of SEQ ID NO: 1 is SEQ ID NO: 10-1827, and shows the amino acid sequence corresponding to SEQ ID NO: 100-1824 from which the stop codon is removed.
[593] (2) Acquisition of CHO cell α-1,6-fucoosyltransferase (FUT8) genomic sequence
[594] Using the CHO cell FUT8 ORF full-length cDNA fragment obtained in this section (1) as a probe, molecular cloning second edition, current protocols in molecular biool from CHO-K1 cell-derived λ-phage genome library (manufactured by STATEGENE) Lodge, A Laboratory Manual, 2nd Ed. CHO cell FUT8 genomic clones were obtained according to known genomic screening methods described in (1989) and the like. Next, after digesting the obtained genomic clones with various restriction enzymes, the AfaI-Sau3AI fragment (about 280bp) containing the start codon of the CHO cell FUT8 cDNA was Southern hybridized as a probe, and among the restriction enzyme fragments showing positive results. Xba I- Xba I fragments (about 2.5Kb) and Sac I- Sac I fragments (about 6.5Kb) were selected and inserted into pBluescriptII KS (+) (manufactured by Strategene), respectively.
[595] The base sequence of each obtained genomic fragment was determined using DNA sequencer 377 (manufactured by Applied Biosystems) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by Applied Biosystems). The method was in accordance with the attached manual. The sequence of the Xba Ⅰ- Xba Ⅰ fragment upstream intron containing exon 2 of the CHO cell FUT8 about 2.5Kb by the present method, Sac Ⅰ- Sac Ⅰ fragment downstream intron of about 6.5Kb containing the exon 2 of the CHO cell FUT8 It was confirmed that the sequence of each was encoded. Hereinafter, Xba Ⅰ- Xba plasmids the plasmids containing the fragment containing Ⅰ pFUT8fgE2-2, Sac Ⅰ- Sac Ⅰ fragment is referred to as pFUT8fgE2-4. The base sequence (about 9.0 Kb) of the genomic region containing exon 2 of the determined CHO cell FUT8 is shown in SEQ ID NO: 3.
[596] Example 5 Preparation of CHO Cells Degrading α-1,6-fucose Transfer Enzyme Gene
[597] CHO Cells CHO cells lacking a genomic region containing α-1,6-fucoosyltransferase (FUT8) gene exon 2 were prepared and evaluated for ADCC activity of the antibodies produced by the cells.
[598] 1. Construction of Chinese hamster α-1,6-fucoosyltransferase (FUT8) gene exon 2 targeting vector plasmid pKOFUT8Puro
[599] (1) Construction of the plasmid ploxPPuro
[600] The plasmid ploxPPuro was constructed in the following order (FIG. 9).
[601] 1.0 µg of plasmid pKOSelectPuro (manufactured by Lexicon) was dissolved in 35 µl of NEBuffer 4 (manufactured by New England Biolabs), and 20 units of restriction enzyme Asc I (manufactured by New England Biolabs) were added to perform digestion at 37 ° C for 2 hours. . After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify a DNA fragment of about 1.5 Kb containing the puromycin resistance gene expression unit.
[602] On the other hand, 1.0 µg of the plasmid ploxP described in Japanese Patent Application Laid-Open No. 11-314512 was dissolved in 35 µl of NEBuffer 4 (manufactured by New England Biolabs), and 20 units of restriction enzyme Asc I (manufactured by New England Biolabs) was added to 37 ° C. Digestion reaction was carried out for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 2.0 Kb.
[603] The plasmid obtained in the above pKOSelectPuro Asc Ⅰ- Asc Ⅰ fragment derived from (approx 1.5Kb) 4.5㎕, plasmid ploxP Asc Ⅰ- Asc Ⅰ fragment derived from (approx 2.0Kb) 0.5㎕, Ligation High (Toyobo yarn production) mixing 5.0㎕ The reaction was carried out by reacting at 16 ° C. for 30 minutes. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as ploxPPuro.
[604] (2) Construction of the plasmid pKOFUT8gE2-1
[605] Plasmid pKOFUT8gE2-1 was constructed in the following order using the plasmid pFUT8fgE2-2 having the genomic region containing exon 2 of Chinese hamster FUT8 obtained in Example 4 (2) (FIG. 10).
[606] 2.0 µg of plasmid pFUT8fgE2-2 was dissolved in 35 µl of NEBuffer 1 (manufactured by New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and 20 units of restriction enzyme Sac I (manufactured by New England Biolabs) The addition was carried out for 2 hours at 37 ℃ digestion reaction. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then dissolved in 35 µl of NEBuffer 2 (manufactured by New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and 20 units of restriction enzyme Eco RV (New England Biolabs, Inc.) was added, and the digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was added to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 1.5 Kb.
[607] Meanwhile, 1.0 µg of plasmid LITMUS28 (manufactured by New England Biolabs) was dissolved in 35 µl of NEBuffer 1 (manufactured by New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and the restriction enzyme Sac I (New). 20 units of England Biolabs) were added and digested for 2 hours at 37 ° C. After recovering the DNA fragment by ethanol precipitation from the reaction solution 100㎍ / ㎖ BSA (New England Biolabs Co.) dissolved in NEBuffer 2 (New England Biolabs Co.) 35㎕, and 20 units of restriction enzyme containing Eco RV (New England Biolabs, Inc.) was added, and the digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was added to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 2.8 Kb.
[608] Eco RV- Sac Ⅰ fragment of the plasmid pFUT8fgE2-2 origin obtained in the above (approximately 1.5Kb) 4.5㎕, plasmid LITMUS28 derived Eco RV- Sac Ⅰ fragment (approximately 2.8Kb) 0.5㎕, Ligation High (Toyobo yarn production) 5.0㎕ The reaction was carried out by mixing and reacting at 16 ° C. for 30 minutes. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOFUT8gE2-1.
[609] (3) Construction of the plasmid pKOFUT8gE2-2
[610] The plasmid pKOFUT8gE2-2 was constructed using the plasmid pKOFUT8gE2-1 obtained in this section (2) in the following order (FIG. 11).
[611] 2.0 µg of plasmid pKOFUT8gE2-1 was dissolved in 30 µl of NEBuffer 2 (manufactured by New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and 20 units of the restriction enzyme Eco RV (manufactured by New England Biolabs) Was added and the digestion reaction was carried out at 37 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then dissolved in 30 µl of NEBuffer 1 (New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and 20 units of restriction enzyme. Kpn I (manufactured by New England Biolabs) was added, and a digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 1.5 Kb.
[612] Meanwhile, 1.0 µg of the plasmid ploxPPuro was dissolved in 30 µl of NEBuffer 4 (manufactured by New England Biolabs), and 20 units of the restriction enzyme Hpa I (manufactured by New England Biolabs) were added to perform a digestion reaction at 37 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then dissolved in 30 µl of NEBuffer 1 (New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and 20 units of restriction enzyme. Kpn I (manufactured by New England Biolabs) was added, and a digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 3.5 Kb.
[613] Eco RV- Kpn plasmid pKOFUT8gE2-1 Ⅰ fragment derived from the obtained in the above (approximately 1.5Kb) 4.0㎕, plasmid ploxPPuro Hpa Ⅰ- Kpn Ⅰ fragment derived from (approx 3.5Kb) 1.0㎕, Ligation High (Toyobo yarn production) 5.0㎕ The reaction was carried out by mixing and reacting at 16 ° C. for 30 minutes. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOFUT8gE2-2.
[614] (4) Construction of the plasmid pscFUT8gE2-3
[615] Plasmid pscFUT8gE2-3 was constructed in the following order using the plasmid pFUT8fgE2-4 having a genomic region containing exon 2 of Chinese hamster FUT8 obtained in Example 4 (2) (FIG. 12).
[616] 2.0 µg of the plasmid pFUT8fgE2-4 was dissolved in 35 µl of NEBuffer 1 (manufactured by New England Biolabs), and 20 units of restriction enzyme Hpa II (manufactured by New England Biolabs) were added to perform a digestion reaction at 37 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then blunted using the Blunting High (manufactured by Toyo Spin Co., Ltd.) according to the attached instructions. After recovering the DNA fragments by phenol / chloroform extraction and ethanol precipitation, it was dissolved in 35 µl of NEBuffer 2 (New England Biolabs), and added to 20 units of restriction enzyme Hin dIII (New England Biolabs). Digestion reaction was carried out for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 3.5 Kb.
[617] Meanwhile, 1.0 µg of plasmid LITMUS39 (manufactured by New England Biolabs) was dissolved in 35 µl of NEBuffer 2 (manufactured by New England Biolabs), and 20 units of restriction enzyme Eco RV (NewEngland Biolabs) and 20 units of restriction enzyme Hin dIII (New England Biolabs Co., Ltd.) was added, and a digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 2.8 Kb.
[618] 4.0 μl of Hpa II- Hin dIII fragment (approximately 3.5 Kb) derived from the plasmid pFUT8fgE2-4 obtained above, 1.0 μl of Eco RV- Hin dIII fragment (approximately 2.8 Kb) derived from plasmid LITMUS39, 5.0 μl of Ligation High The reaction was carried out by mixing and reacting at 16 ° C. for 30 minutes. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pscFUT8gE2-3.
[619] (5) Construction of plasmid pK0FUT8gE2-3
[620] Plasmid pKOFUT8gE2-3 was constructed in the following order using the plasmid pFUT8fgE2-4 having a genomic region containing exon 2 of Chinese hamster FUT8 obtained in Example 4 (2) (FIG. 13).
[621] 2.0 µg of the plasmid pFUT8fgE2-4 was dissolved in 35 µl of NEBuffer for Eco RI (manufactured by New England Biolabs), and 20 units of restriction enzymes Eco RI (manufactured by New England Biolabs) and 20 units of restriction enzyme Hin dIII (New England Biolabs) Co., Ltd.) was added, and a digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 1.8 Kb.
[622] Meanwhile, 1.0 µg of plasmid pBluescript II KS (+) (manufactured by Strategene) was dissolved in 35 µl of NEBuffer for Eco RI (manufactured by New England Biolabs), and 20 units of restriction enzyme Eco RI (manufactured by New England Biolabs) and 20 units The restriction enzyme Hin dIII (manufactured by New England Biolabs) was added thereto, and the digestion reaction was performed at 37 ° C for 2 hours. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 3.0 Kb.
[623] Hin dⅢ- Eco RI fragment of the plasmid pFUT8fgE2-4 origin obtained in the above (approximately 1.8Kb) 4.0㎕, plasmid pBluescriptⅡ KS (+) derived from the Eco RI Hin dⅢ- fragment (approximately 3.0Kb) 1.0㎕, Ligation High (Toyobo yarns Preparation) 5.0 μl of the mixture was mixed and reacted at 16 ° C. for 30 minutes to conduct a coupling reaction. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOFUT8gE2-3.
[624] (6) Construction of the plasmid pKOFUT8gE2-4
[625] Plasmids pKOFUT8gE2-4 were constructed in the following order using the plasmids pscFUT8gE2-3 and pKOFUT8gE2-3 obtained in this section (4) and (5) (FIG. 14).
[626] 1.0 µg of plasmid pscFUT8gE2-3 was dissolved in 35 µl of NEBuffer for Sal I (manufactured by New England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and the restriction enzyme Sal I (manufactured by New England Biolabs) 20 units were added and the digestion reaction was carried out at 37 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then dissolved in 30 µl of NEBuffer 2 (manufactured by New England Biolabs), and 20 units of restriction enzyme Hin dIII (manufactured by New England Biolabs) were added thereto at 37 ° C. Time digestion reaction was performed. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 3.6 Kb.
[627] Meanwhile, 1.0 µg of plasmid pKOFUT8gE2-3 was dissolved in 35 µl of NEBuffer for Sal I (manufactured by Ncw England Biolabs) containing 100 µg / ml BSA (manufactured by New England Biolabs), and the restriction enzyme Sal I (New England Biolabs company) 20 units were added, and a digestion reaction was performed at 37 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, dissolved in 35 µl of NEBuffer 2 (manufactured by New England Biolabs), and 20 units of restriction enzyme Hin dIII (manufactured by New England Biolabs) were added thereto at 37 ° C. Time digestion reaction was performed. After the digestion reaction, 35 µl of 1 mol / L Tri-HCl buffer at pH 8.0 and 3.5 µl of Alkaline Phosphatase derived from Escherichia coli C15 strain were added, followed by reaction at 65 ° C for 30 minutes for dephosphorylation of DNA ends. After dephosphorylation, phenol / chloroform extraction and ethanol precipitation were performed, and the recovered DNA fragment was dissolved in 10 µl of sterile water.
[628] Sal Ⅰ- Hin dⅢ fragment of plasmid pscFUT8gE2-3 origin obtained in the above (approximately 3.1Kb) 4.0㎕, plasmid pKOFUT8gE2-3 Sal Ⅰ- Hin dⅢ fragment derived from (approx 4.8Kb) 1.0㎕, Ligation High (Toyobo yarn production) The coupling reaction was performed by mixing 5.0 microliters and reacting at 16 degreeC for 30 minutes. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOFUT8gE2-4.
[629] (7) Construction of plasmid pKOFUT8gE2-5
[630] Plasmids pKOFUT8gE2-5 were constructed in the following order using the plasmids pKOFUT8gE2-2 and pKOFUT8gE2-4 obtained in this section (3) and (6) (FIG. 15).
[631] 1.0 µg of the plasmid pKOFUT8gE2-2 was dissolved in 30 µl of NEBuffer 4 (manufactured by New England Biolabs), and 20 units of the restriction enzyme Sma I (manufactured by New England Biolabs) were added to perform a digestion reaction at 25 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then dissolved in 30 µl of NEBuffer2 (manufactured by New England Biolabs), and 20 hours of restriction enzyme Bam HI (manufactured by New England Biolabs) was added thereto at 37 ° C for 2 hours. Digestion reaction was performed. After the digestion reaction, 30 µl of 1 mol / L Tri-HCl buffer at pH 8.0 and 3.0 µl of Alkaline Phosphatase derived from Escherichia coli C15 strain were added, followed by reaction at 65 ° C for 1 hour to dephosphorylation of DNA ends. After dephosphorylation, phenol / chloroform extraction and ethanol precipitation were performed, and the recovered DNA fragment was dissolved in 10 µl of sterile water.
[632] On the other hand, 1.0 µg of plasmid pKOFUT8gE2-4 was dissolved in 30 µl of NEBuffer 4 (manufactured by New England Biolabs), and 20 units of the restriction enzyme Sma I (manufactured by New England Biolabs) were added to carry out a digestion reaction at 25 ° C for 2 hours. The DNA fragment was recovered from the reaction solution by ethanol precipitation, and then dissolved in 30 µl of NEBuffer 2 (manufactured by New England Biolabs), and 20 units of restriction enzyme Bam HI (manufactured by New England Biolabs) were added thereto at 37 ° C. Time digestion reaction was performed. After digestion, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify DNA fragments of about 5.2 Kb.
[633] Bam HI fragment of plasmid pKOFUT8gE2-2 Sma Ⅰ- the origin obtained in the above (approximately 5.0Kb) 0.5㎕, plasmid pKOFUT8gE2-4 Sma Ⅰ- Bam HI fragment derived from (approx 5.2Kb) 4.5㎕, Ligation High (Toyobo yarn production) 5.0 µl was mixed and the reaction was carried out by reacting at 16 ° C for 15 hours. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOFUT8gE2-5.
[634] (8) Construction of the plasmid pKOFUT8Puro
[635] Plasmid pKOFUT8Puro was constructed in the following order using the plasmid pKOFUT8gE2-5 obtained in this section (7) (FIG. 16).
[636] 1.0 µg of the plasmid pKOSelectDT (manufactured by Lexicon) was dissolved in 50 µl of NEBuffer 4 (manufactured by New England Biolabs), and 16 units of the restriction enzyme Rsr II (manufactured by New England Biolabs) were added to carry out a digestion reaction at 37 ° C for 2 hours. After the digestion reaction, the solution was subjected to 0.8% (w / v) agarose gel electrophoresis to purify a DNA fragment of about 1.2 Kb containing diphtheria toxin expression unit.
[637] On the other hand, 1.0 µg of plasmid pKOFUT8gE2-5 was dissolved in 50 µl of NEBuffer 4 (manufactured by New England Biolabs), and 16 units of the restriction enzyme Rsr II (manufactured by New England Biolabs) were added to perform digestion reaction at 37 ° C for 2 hours. After the digestion reaction, 30 µl of 1 mol / L Tris-HCl buffer at pH 8.0 and 3.0 µl of Alkaline Phosphatase derived from Escherichia coli C15 strain were added, followed by reaction at 65 ° C for 1 hour to perform dephosphorylation of DNA ends. . After dephosphorylation, phenol / chloroform extraction and ethanol precipitation were performed, and the recovered DNA fragment was dissolved in 10 µl of sterile water.
[638] 1.0 µl of the Rsr II- Rsr II fragment (about 1.2 Kb) derived from the plasmid pKOSelectDT obtained above, 1.0 µl of the Rsr II- Rsr II fragment (about 10.4 Kb) derived from the plasmid pKOFUT8gE2-5, 3.0 µl of sterile water, Ligation High 5.0 microliters of spinning yarns were mixed, and the reaction was carried out by reacting at 16 캜 for 30 minutes. E. coli DH5α strains were transformed using the reaction solution, and plasmid DNA was isolated from known ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOFUT8Puro. The plasmid is used as a targeting vector for producing FUT8 gene knockout cells of CHO cells.
[639] Example 6 Preparation of Lectin Resistant CHO / DG44 Cells and Production of Antibodies Using the Cells
[640] 1. Acquisition of lectin-resistant CHO / DG44 strains
[641] CHO / DG44 cells were adhered to IMDM-FBS (10) medium (IMDM medium containing 10% fetal bovine serum (FBS) and 1-fold concentration of HT supplement (manufactured by GIBCO BRL)). Cultured in Liner, Inc.) and propagated until immediately before confluent. After washing the cells with 5 ml of Dulbecco PBS (Invitrogen), 1.5 ml of 0.05% trypsin (Invitrogen) diluted with Dulbeco PBS was added and left at 37 ° C for 5 minutes to incubate the cells. It peeled from the bottom face. The detached cells were collected by centrifugal manipulation carried out in a normal cell culture, and added with IMDM-FBS (10) medium so as to have a density of 1 × 10 ⁵ cells / ml, and then suspended or unadded or 0.1 µg / ml. An alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidin (hereinafter referred to as MNNG, manufactured by Sigma) was added. After standing at 37 ° C. for 3 days in a CO ₂ incubator (TABAI), the culture supernatant is removed, and the cells are washed, peeled, and recovered by the same operation as described above, suspended in IMDM-FBS (10) medium, and then adhered. The cells were seeded at a density of 1000 cells / well in a 96-well plate (manufactured by Iwakigaras Corporation). Each well contains 1 mg / mL lentil agglomerate (Lens culinaris agglutinin; hereinafter referred to as LCA, manufactured by Vector) or 1 mg / mL wild mushroom agglomerate (Aleuria aurantia Lectin; hereinafter referred to as AAL). (Manufactured by Vector, Inc.) or 1 mg / ml kidney bean coagulant (Phaseolus vulgaris Leucoagglutinin; hereafter referred to as L-PHA, manufactured by Vector). After two weeks of incubation at 37 ° C. in a CO ₂ incubator, the colonies that appeared were obtained as lectin resistant CHO / DG44 strains. For each of the acquired lectin resistant CHO / DG44 strains, the LCA resistant strains were named CHO-LCA strains, the AAL resistant strains were CHO-AAL strains, and the L-PHA resistant strains were named CHO-PHA strains. Examination of resistance to various lectins obtained from these strains revealed that CHO-LCA strains were resistant to AAL and that CHO-AAL strains were also resistant to LCA. Also, CHO-LCA and CHO-AAL strains are lectins that recognize the same sugar chain structure as the sugar chain structure recognized by LCA or AAL, that is, the 6th position of N-acetylglucosamine residue at the N-glycosidic linkage sugar chain reducing end and Fuco The first place in the os showed resistance to lectins that recognized sugar chain structures added by α bonds. Specifically, it was found that CHO-LCA strains and CHO-AAL strains survived even in a medium to which a pea aggregate (Pisum sativum Agglutinin; hereinafter referred to as PSA, manufactured by Vector) was added at a final concentration of 1 mg / ml. In addition, even in the absence of the alkylating agent MNNG, lectin-resistant strains were obtained by increasing the number of cells subjected to the aforementioned treatment.
[642] 2. Construction of Anti-CD20 Humanoid Chimeric Antibody Producing Cells
[643] 4 µg of the anti-CD20 humanoid chimeric antibody expression vector pKANTEX2B8P to LCA lectin-resistant strains obtained in 1 above was electroporated to CHO / DG44 cells of 1.6 × 10 ⁶ cells [Cytotechnology, 3 , 133 (1990)]. IMDM medium containing 10 ml of IMDM-dFBS (10) -HT (1) [dFBS (manufactured by Invitrogen) and 1-fold concentration of HT supplement (manufactured by Invitrogen) after introduction by Invitrogen, Inc.), and 100 μl / well was dispensed into 96 well culture plates (manufactured by Iwakigaras Corporation). After incubation at 37 ° C. for 24 hours in a 5% CO ₂ incubator, the cells were cultured in IMDM-dFBS 10 (IMDM medium containing 10% dialysis FBS) for 1 to 2 weeks. Since a colony of transformant strains showing HT independent growth appeared, antibody production was increased using a DHFR gene amplification system for well-transformed wells. Specifically, the mixture was suspended in IMDM-dFBS (10) medium containing 50 nM of MTX so as to be 1-2 x 10 ⁵ cells / mL, and 0.5 mL was dispensed into 24 well plates (manufactured by Iwaki Glass Co., Ltd.). Transformants exhibiting 50 nM MTX resistance were induced by incubating for 1-2 weeks at 37 ° C. in a 5% CO ₂ incubator. Regarding well-transfected strains, the MTX concentration can be raised to 200 nM by the above-described method, and finally, the MTX can be grown to IMDM-dFBS (10) medium containing MTX at a concentration of 200 nM. Transformants were produced which produced high anti-CD20 humanoid chimeric antibody.
[644] 3. Culture of antibody expressing cell line and purification of antibody
[645] LCA lectin-resistant CHO / DG44 transformed cells producing high anti-CD20 chimeric antibody obtained in step 2. were named R92-3-1 strain. As of March 26, 2002, R92-3-1 has been deposited as FERM BP-7976 at the Institute of Patented Biological Deposits of the Industrial Technology Research Institute (1st Chuo 6th Higashi, Tsukubashi, Ibaraki, Japan).
[646] R92-3-1 strain was incubated with IMDM-dFBS (10) at a concentration of 200 nM MTX, washed with Dulbecco PBS (manufactured by Invitrogen), and then EX-CELL301 (manufactured by JRH). Medium was exchanged. The culture supernatant was recovered by incubating at 37 ° C. for 7 days in a 5% CO ₂ incubator. Anti-CD20 chimeric antibody was purified from the culture supernatant using a Prosep-A (Millipore) column. The obtained antibody was named R92-3-1 antibody.
[647] Example 7 Purification and Activity Evaluation of Anti-CD20 Chimeric Antibodies Produced by Lectin Resistant CHO / DG44 Cells
[648] 1. Evaluation of binding activity of lectin resistant CHO / DG44 cell-derived antibody (fluorescent antibody method)
[649] The binding activity of the R92-3-1 antibody obtained in paragraph 3 of Example 6 to the CD20 expressing cell line Raji cells was examined according to the fluorescent antibody method shown in paragraph 1 of Example 2, and a commercial antibody derived from ordinary CHO cells. It was compared with the activity of Rituxan ^™ . As shown in FIG. 17, it was confirmed that the R92-3-1 antibody and Rituxan ^™ showed an increase in fluorescence intensity depending on antibody concentration, and showed almost the same binding activity.
[650] 2. Evaluation of in vitro cytotoxic activity of lectin resistant CHO / DG44 cell-derived antibodies (ADCC activity)
[651] In order to evaluate the in vitro ADCC activity of the R92-3-1 antibody obtained in Section 3 of Example 6, ADCC activity was measured according to the method shown in Section 2 of Example 2. The ratio of effector cells to Raji cells as target cells was 25: 1 and the final antibody concentration was 0.001 to 10 µg / ml. The results are shown in FIG.
[652] R92-3-1 antibody derived from LCA lectin resistant CHO / DG44 cells showed higher ADCC activity than Rituxan ^™ .
[653] 3. Sugar Chain Analysis of Lectin-resistant CHO / DG44 Cell-derived Antibodies
[654] The sugar chain analysis of the R92-3-1 antibody obtained in Section 3 of Example 6 was carried out according to the method shown in Example 3. The result is shown in FIG. The sugar chain structures of the peaks 1 to 8 shown in Fig. 19 are the same as the sugar chain structures of the peaks 1 to 8 shown in Fig. 7.
[655] In Fig. 19, the ratio of the sugar chain group without α-1,6-fucose is calculated from the sum of the areas occupied by the peaks 1, 4, 9 and 9 of the sum of the areas occupied by each of the peaks 1-⑩. It was. In addition, the ratio of the sugar chain group which the (alpha) -1, 6- fucose couple | bonded was computed from the sum total of the area which the peak of (5)-(8) occupies among the sum total of the area which each peak of (1)-(b) occupies.
[656] As a result, the α-1,6-fucose-linked sugar chain content of the R92-3-1 antibody was 33%, and the α-1,6-fucose-linked sugar chain content was 67%. Compared to the sugar chain assay Rituxan ^™ , antibodies produced with LCA lectin resistant CHO / DG44 cells had higher sugar chain content without α-1,6-fucose binding.
[657] Example 8. Acquisition of GMHO Gene Derived from CHO Cells
[658] 1. Determination of GMHO cDNA sequence derived from CHO cell
[659] (1) cDNA acquisition of GMHO gene derived from CHO cell (partial cDNA acquisition except 5 'and 3' terminal sequences)
[660] The rodent-derived GMD cDNA was searched using a public database (BLAST) using a human GMD cDNA sequence registered in GenBank (GenBank Accession No. AF042377) as a query to obtain three kinds of mouse EST sequences. (GenBank Accesssion No. BE986856, BF158988, BE284785). By linking these EST sequences, putative mouse GMD cDNA sequences were determined.
[661] From this mouse GMD cDNA sequence, a primer of 28mer having a nucleotide sequence represented by SEQ ID NO: 32, a primer of 27mer having a nucleotide sequence represented by SEQ ID NO: 33, a primer of 25mer having a nucleotide sequence represented by SEQ ID NO: 34, and a sequence A primer of 24mer having the nucleotide sequence shown in No. 35 and a primer of 25mer having the nucleotide sequence shown in SEQ ID NO: 36 were prepared.
[662] CHO / DG44 cells were then incubated for 4 days after passage in a 37 ° C. 5% CO ₂ incubator. After cultivation, each RNA was prepared using Rneasy Protect Mini kit (manufactured by Kiagen) and RT-PCR (manufactured by Gibco BRL) according to the attached instructions from each 1 × 10 ⁷ cell, according to the attached instructions. One chain cDNA was synthesize | combined from 20 micrograms of reaction liquid from 5 micrograms.
[663] In order to amplify this CHO cell-derived cDNA, PCR was performed by the following method. 20 μl of reaction solution containing 0.5 μl of single chain cDNA derived from CHO cells as a template [1 × EX Taq Buffer (manufactured by Takarazu Corporation), 0.2 mM dNTP's, 0.5 unit of EX Taq polymerase (manufactured by Takarazu Corporation), 0.5 [micro] m synthetic DNA primers]. As the synthetic DNA primer, a combination of SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 33, SEQ ID NO: 32 and SEQ ID NO: 35, SEQ ID NO: 32, and SEQ ID NO: 36 was used. The reaction solution was heated at 94 ° C for 5 minutes using DNA Thermal Cycura 480 (manufactured by Parkin Elmar Co., Ltd.), and then cycled for 1 minute at 94 ° C and 2 minutes at 68 ° C for 30 cycles.
[664] The PCR reaction solution was fractionated by agarose electrophoresis. As a result, the PCR product using the synthetic DNA primers of SEQ ID NO: 32 and SEQ ID NO: 33 was about 1.2 kbp, and the PCR product using the synthetic DNA primers of SEQ ID NO: 33 and SEQ ID NO: 34 was obtained. About 1.1 bp, a DNA fragment of about 350 bp was amplified in a PCR product using synthetic DNA primers of SEQ ID NO: 32 and SEQ ID NO: 35, and a synthetic DNA primer of SEQ ID NO: 32 and SEQ ID NO: 36. These DNA fragments were recovered using a Gene Clean II kit (manufactured by BIO101) according to the attached manual. The recovered DNA fragment was linked to the pT7Blue (R) vector (manufactured by Novagen) using a DNA Ligation kit (manufactured by Takara Co., Ltd.), and transformed into E. coli DH5 strain (manufactured by Toyoro Spin Co.) using the obtained recombinant plasmid DNA. Plasmid 22-8 (having about 1.2 kbp of DNA fragments amplified from the synthetic DNA primers of SEQ ID NO: 32 and SEQ ID NO: 33), 23-3 (about 1.1 amplified from the synthetic DNA primers of SEQ ID NO: 34 and SEQ ID NO: 33) kbp DNA fragment), 31-5 (having about 350 bp DNA fragment amplified from the synthetic DNA primers of SEQ ID NO: 32 and SEQ ID NO: 35), 34-2 (synthetic DNA primer of SEQ ID NO: 32 and SEQ ID NO: 36) Having a DNA fragment of about 1 kbp amplified therefrom). GMD cDNA sequences derived from CHO cells contained in these plasmids were determined according to a conventional method using a DNA sequencer ABI PRISM 377 (manufactured by Parkin Elma Co., Ltd.) (a sequence of 28 bases downstream from the starting methionine at the 5 'end side). And a sequence of 27 bases upstream from the end codon at the 3 'end side is a mouse GMD cDNA sequence since it is derived from a synthetic oligo DNA sequence.
[665] In addition, the following steps were carried out to produce a plasmid obtained by combining GMHO cDNA derived from CHO cells contained in plasmids 22-8 and 34-2. 1 µg of plasmid 22-8 was reacted with a restriction enzyme Eco RI (manufactured by Takara Co., Ltd.) for 16 hours at 37 ° C, followed by agarose electrophoresis, and a DNA fragment of about 4 kbp was prepared by Gene Clean II kit (BIO101). It was collected in accordance with the attached manual. 2 μg of plasmid 34-2 was reacted with a restriction enzyme Eco RI at 37 ° C. for 16 hours, followed by agarose electrophoresis, and the DNA fragment of about 150bp was prepared using a Gene Clean II kit (manufactured by BIO101) according to the attached manual. Recovered. The recovered DNA fragments were dephosphorylated at their ends with Calf Intestine Alkaline Phosphatase (manufactured by Takara Co., Ltd.), and then linked using a DNA Ligation kit (manufactured by Takara Co., Ltd.). (Toyo Spin Co., Ltd. product) was transformed to obtain plasmid CHO-GMD (FIG. 20).
[666] (2) Determination of 5 'terminal sequence of GMHO cDNA derived from CHO cell
[667] A primer of 24mer having the nucleotide sequence represented by SEQ ID NO: 37 from the base sequence of the 5 'terminal non-coding region of the GMHO cDNA derived from CHO cell, and the base represented by SEQ ID NO: 38 from the CHO derived GMD cDNA sequence A 32mer primer having a sequence was prepared, and PCR was performed in the following manner to amplify the cDNA. 20 µl of reaction solution containing 0.5 µl of one-chain cDNA derived from CHO cells as a template [1 × EX Taq Buffer (manufactured by Takarazu Corporation), 0.2 mM dNTP's, 0.5 unit of EX Taq polymerase (manufactured by Takarazu Corporation) , 0.5 µm SEQ ID NO: 37 and SEQ ID NO: 38 Synthetic DNA primer] were prepared, and heated at 94 ° C. for 5 minutes using DNA Thermal Cycla 480 (manufactured by Parkin Elma), and then 1 minute at 94 ° C. After 20 cycles of 1 minute at 55 ° C and 2 minutes at 72 ° C, 18 cycles of 1 minute at 94 ° C and 2 minutes at 68 ° C were performed. After the PCR reaction solution was fractionated by agarose electrophoresis, a DNA fragment of about 300 bp was recovered using a Gene Clean II kit (manufactured by BIO101) according to the attached instructions. The recovered DNA fragment was linked to a pT7Blue (R) vector (manufactured by Novagen) using a DNA Ligation kit (manufactured by Takara Co., Ltd.), and transformed into E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) using the obtained recombinant plasmid DNA. Plasmid 5'GMD was obtained. The DNA sequencer 377 (manufactured by Parkin Elmar Co., Ltd.) was used to determine the sequence of 28 bases downstream from the starting methionine of the CHO-derived GMD cDNA contained in this plasmid.
[668] (3) Determination of 3 'terminal sequence of GMHO cDNA derived from CHO cell
[669] In order to obtain the 3 'terminal cDNA sequence of GMHO derived from CHO cells, the RACE method was performed by the following method. From CHO cell-derived RNA, the production of single chain cDNA for 3'RACE using SMART ^TM RACE cDNA Amplification kit (CLONTECH Inc.) was performed according to the attached manual. However, PowerScript ^TM Reverse Transcriptase (manufactured by CLONTECH) was used for the reverse transcriptase. One-chain cDNA after preparation was diluted 10-fold with Tricin-EDTA buffer with kit as a template for PCR.
[670] Subsequently, 20 µl of the reaction solution containing 1 µl of the 1-chain cDNA for 3 'RACE as a template [1 × EX Taq Buffer (manufactured by Takara Co., Ltd.), 0.2 mM dNTP's, and 0.5 units of EX Taq polymerase (manufactured by Takara Corporation) ), 24mer synthetic DNA primer (SEQ ID NO: 39) prepared with CHO cell-derived GMD cDNA sequence determined in this section (1), 1-fold Universal Primer Mix (SMART ^TM RACE cDNA Amplification kit) ; CLONTECH Co., Ltd.] was prepared, and heated at 94 ° C. for 5 minutes using DNA Thermal Cycura 480 (manufactured by Parkin Elmar Co., Ltd.), followed by 30 cycles of 1 minute at 94 ° C. and 2 minutes at 68 ° C. It was done.
[671] After completion of the reaction, 1 µl was taken from the PCR reaction solution and 20 µl of the reaction solution containing 1 µl of an aqueous solution diluted 20-fold with Tricin-EDTA buffer (manufactured by CLONTECH) as a template [1 × EX Taq Buffer (Takuraju) Irradiation)), 0.2mM dNTP's, 0.5 unit EX Taq polymerase (manufactured by Takarazu Corporation), 25mer of synthetic DNA primer represented by SEQ ID NO: 40 of 0.5 µm [CHO cell-derived GMD cDNA sequence determined in this section (1) [Preparation], 0.5 µm Nested Universal Primer (supplied with SMART ^TM RACE cDNA Amplification kit; manufactured by CLONTECH)] and heated at 94 ° C. for 5 minutes using DNA Thermal Cycura 480 (manufactured by Parkin Elmar). Then, 30 cycles of 1 minute at 94 degreeC and 2 minutes at 68 degreeC were performed.
[672] After completion of the reaction, the PCR reaction solution was fractionated by agarose electrophoresis, and the DNA fragment of about 700 bp was recovered using a Gene Clean II kit (manufactured by BIO101) according to the attached manual. The recovered DNA was linked to a pT7Blue (R) vector (manufactured by Novagen) using a DNA Ligation kit (manufactured by Takara Co., Ltd.), and transformed into E. coli DH5α strain (manufactured by Toyoro Cotton Co., Ltd.) using the resulting recombinant plasmid DNA. 3'GMD was obtained. Using DNA sequencer 377 (manufactured by Parkin Elma Co., Ltd.), the sequence of upstream 27 bases and the base sequence of the non-coding region 415bp on the 3 'side were determined from the stop codon of the CHO-derived GMD cDNA contained in this plasmid.
[673] As described above, the full length cDNA sequence of the CHO-derived GMD gene determined from the items (1), (2), and (3) is shown in SEQ ID NO: 41, and the amino acid sequence corresponding thereto is shown in SEQ ID NO: 61.
[674] 2. Determination of Genomic Sequences Containing GMD Genes of CHO / DG44 Cells
[675] From the mouse GMD cDNA sequence determined in paragraph 1 of Example 8, a 25mer primer having the nucleotide sequence represented by SEQ ID NO: 56 was prepared. Subsequently, genomic DNA derived from CHO cells was obtained by the following method. CHO / DG44 cells were placed in IMDM-dFBS (10) -HT (1) medium [IMDM-dFBS (10) medium containing HT supplement (Invitrogen) at 1-fold concentration] 3 × 10 ⁵ cells / ml The cells were suspended in 2 ml / well in flat bottom 6 well plates (manufactured by Greiner) for adhesion cells. After incubation in a 5% CO ₂ incubator at 37 ° C. until confluent, the plate was prepared from known methods (Nucleic Acids Research, 3 , 2303 (1976)). Genomic DNA was thus prepared and dissolved in 150 μl of TE-RNase buffer (pH8.0) (10 mmol / L Tri-HCl, 1 mmol / L EDTA, 200 μg / mL RNase A) overnight.
[676] 100 ng, 20 μl of the CHO / DG44 cell-derived genomic DNA obtained above was reacted with 1 × EX Taq Buffer (manufactured by Takarazon Corporation), 0.2 mM dNTP's, 0.5 unit of EX Taq polymerase (manufactured by Takarazon Corporation), 0.5 μm. Synthetic DNA primers of SEQ ID NO: 35 and SEQ ID NO: 56] were prepared, and were heated at 94 ° C for 5 minutes using DNA thermal cyclura 480 (manufactured by Parkin Elma Co., Ltd.), followed by 1 minute at 94 ° C, and 2 at 68 ° C. 30 minutes of cycles were performed. After completion of the reaction, the reaction solution was fractionated by agarose electrophoresis, and about 100 bp of DNA fragment was recovered using the Gene Clean II kit (manufactured by BIO101) according to the attached manual. The recovered DNA fragment was linked to a pT7Blue (R) vector (manufactured by Novagen) using a DNA Ligation kit (manufactured by Takara Co., Ltd.), and transformed into E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) using the obtained recombinant plasmid DNA. Plasmid ex3 was obtained. DNA sequencer 377 (manufactured by Parkin Elmar Co., Ltd.) was used to determine the nucleotide sequence of the CHO cell-derived genomic DNA contained in this plasmid. The determined nucleotide sequence is shown in SEQ ID NO: 57.
[677] Next, a 25mer primer having a nucleotide sequence represented by SEQ ID NO: 58 and a 25mer primer having a nucleotide sequence represented by SEQ ID NO: 59 were prepared from the CHO cell-derived GMD cDNA sequence determined in Example 1 of Example 8. Subsequently, 100 ng of CHO / DG44-derived genomic DNA was added to 20 μl of a reaction solution [1 × EX Taq Buffer (manufactured by Takara Co., Ltd.), 0.2 mM dNTP's, 0.5 unit of EX Taq polymerase (manufactured by Takarazon Corporation), sequence number of 0.5 μm. 58 and the synthetic DNA primer of SEQ ID NO: 59], were heated at 94 ° C for 5 minutes using DNA Thermal Cycura 480 (manufactured by Parkin Elma Co.), and then cycled at 94 ° C for 1 minute and at 68 ° C for 2 minutes. 30 cycles were carried out.
[678] After completion of the reaction, the reaction solution was fractionated by agarose electrophoresis, and a DNA fragment of about 200 bp was recovered using a Gene Clean II kit (manufactured by BIO101) according to the attached manual. The recovered DNA fragment was linked to the pT7Blue (R) vector (manufactured by Novagen) using a DNA Ligation kit (manufactured by Takara Co., Ltd.), and transformed into E. coli DH5α strain (manufactured by Toyo Spin Co., Ltd.) using the obtained recombinant plasmid DNA. Plasmid ex4 was obtained. DNA sequencer 377 (manufactured by Parkin Elmar Co., Ltd.) was used to determine the nucleotide sequence of the CHO cell-derived genomic DNA contained in this plasmid. The determined nucleotide sequence is shown in SEQ ID NO: 60.
[679] Example 9 Acquisition of various enzyme genes related to sugar chain synthesis derived from CHO cells
[680] 1. Determination of Fx cDNA Sequence of CHO Cells
[681] (1) Extraction of Total RNA from CHO / DG44 Cells
[682] CHO / DG44 cells were suspended in IMDM medium (manufactured by Life Technologies) with 10% fetal bovine serum (manufactured by Life Technologies) and HT supplement (manufactured by Life Technologies) at a concentration of 2 × 10 ⁵ / 15 ml were sown into T75 flasks for adherent cell culture (manufactured by Greiner) at a density of ml. After culturing in a 5% CO ₂ incubator at 37 ° C., 1 × 10 ⁷ cells were recovered on the second day of culture, and total RNA was extracted by RNAeasy (produced by QIAGEN) according to the attached instructions.
[683] (2) Preparation of one-chain cDNA derived from CHO / DG44 cells
[684] The total RNA prepared in the above (1) was dissolved in 45 µl of sterile water, 1 µl of RQ1 RNase-Free DNase (promega), 5 µl of the attached 10 × DNase buffer, 0.5 µl of RNasin Ridonuclease inhibitor (manufactured by Promega) Were added to each of them and reacted at 37 ° C for 30 minutes to decompose genomic DNA incorporated into the sample. After the reaction, total RNA was purified by RNAeasy (produced by QIAGEN) and dissolved in 50 µl of sterile water.
[685] 3 μl of the total RNA obtained was subjected to reverse transcription in a 20 μl system based on oligo (dT) according to the attached instructions using SUPERSCRIPT ^™ Preamplification System for First Strand cDNA Synthesis (manufactured by Life Technologies). Dog chain cDNA was synthesized. A 50-fold diluted aqueous solution of this reaction solution was used for the cloning of GFPP and Fx. Store at -80 ° C until use.
[686] (3) Acquisition of cDNA partial fragment of Chinese hamster Fx
[687] The cDNA partial fragment of Chinese hamster Fx was obtained by the following procedures. First primers specific for the nucleotide sequence common to the cDNA of human Fx (Genebank Accession No. U58766) and mouse cDNA (Genebank Accession No. M30127) registered in the public database (indicated by SEQ ID NO: 42 and SEQ ID NO: 43) Was designed.
[688] Next, a 25 µl reaction solution containing 1 µl of the CHO / DG44-derived one-chain cDNA prepared in this section (2) using DNA polymerase ExTaq (manufactured by Takara Co., Ltd.) [ExTaq buffer Production), 0.2 mM dNTPs, 0.5 µmol / L of the above gene-specific primers (SEQ ID NO: 42 and SEQ ID NO: 43)] were prepared, and a polymerase chain reaction (PCR) was performed. PCR was carried out under the condition of heating at 94 ° C. for 5 minutes, heating at 94 ° C. for 1 minute, 58 ° C. for 2 minutes, and 72 ° C. for 3 minutes as one cycle, 30 cycles later, and then heating at 72 ° C. for 10 minutes. It was.
[689] After PCR, the reaction solution was subjected to 2% agarose gel electrophoresis, and the specific amplified fragment 301 bp was purified by QiaexII Gel Extraction kit (Kiagen) and eluted with 20 µl of sterile water (hereinafter, DNA from agarose gel). Use this method for purification of fragments). 4 μl of the amplified fragment was inserted into the plasmid pCR2.1 according to the instructions of the TOPO TA cloning kit (manufactured by Invitrogen), and E. coli DH5α was coenized using the reaction solution [Procedings of the National Academy of Sciences ( Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)] (hereinafter, this method is used for transformation of Escherichia coli).
[690] Isolation of plasmid DNA from a plurality of kanamycin resistant colonies obtained according to a known method (Nucleic Acids Research, 7 , 1513 (1979)) (hereafter, this method is used for the isolation method of plasmid). Then, 2 clones into which the Fx cDNA partial fragment was inserted were obtained. Each is referred to as pCRFX clone 8, pCRFX clone 12.
[691] The base sequence of the cDNA inserted into Fx clone 8 and Fx clone 12 was determined using DNA sequencer 377 (manufactured by Applied Biosystems) and Big Dye Terminator Cycle Sequencing FS Raedy Reaction Kit (manufactured by Applied Biosystems). The method was followed in the attached manual. It was confirmed that the inserted cDNA sequenced by this method codes the open reading frame (ORF) partial sequence of Fx of Chinese hamster.
[692] (4) Synthesis of single chain cDNA for RACE
[693] The production of single-chain cDNA for 5 'and 3' RACE from CHO / DG44 total RNA extracted in this item (1) was performed using SMART ^TM RACE cDNA Amplification Kit (manufactured by CLONTECH). The method was according to the attached manual. However, PowerScript ^™ Reverse Transcriptase (manufactured by CLONTECH) was used as a reverse transcriptase. The one-chain cDNA after preparation was diluted 10-fold with each kit's Tricin-EDTA buffer, and was used as a template for PCR. (5) Determination of Chinese hamster Fx full-length cDNA by RACE method
[694] Primer FXGSP1-1 (SEQ ID NO: 44) and FXGSP1-2 (SEQ ID NO: 45) and Chinese Hamster for 5 'RACE specific to Chinese Hamster Fx based on the partial sequence of Chinese Hamster Fx determined in (3) above Primers FXGSP2-1 (SEQ ID NO 46) and FXGSP2-2 (SEQ ID NO 47) for the Fx specific 3 ′ RACE were designed.
[695] Next, using a Advantage2 PCR Kit (manufactured by CLONTECH Co., Ltd.), a 50 µl reaction solution containing 1 µL of the CHO / DG44-derived RACE single chain cDNA prepared in this section (4) [Advantage 2 PCR buffer (CLONTECH Corporation) Production) 0.2 mM dNTPs, 0.2 μmol / L chinis for Hamster Fx specific RACE primer, and a common primer of 1-fold concentration (manufactured by CLONTECH)] were prepared, and polymerase chain reaction (PCR) was performed.
[696] PCR was carried out under the condition of repeating 20 cycles of 1 cycle of reaction consisting of 5 seconds at 94 ° C, 10 seconds at 68 ° C, and 2 minutes at 72 ° C.
[697] After completion | finish of reaction, 1 microliter was taken from the reaction liquid, and 1 microliter of the aqueous solution diluted 50-fold with Tricin-EDTA buffer was used as a template, the reaction liquid was prepared again, and PCR was performed on the same conditions. Table 2 shows the lengths of DNA fragments that are recombined and amplified by the primers used in the first and second PCRs.
[698] Combination of Primers Used in Chinese Hamster FxcDNA RACE PCR and Length of PCR Product 5 'RACEFX specific primerCommon primerSize of PCR Amplification Products 1st timeFXGSP1-1Univarsal primer mix (UPM)2nd timeFXGSP1-2Nested Univarsal primer (NUP)300 bp 3 'RACEFX specific primerCommon primerSize of PCR Amplification Products 1st timeFXGSP2-1Univarsal primer mix (UPM)2nd timeFXGSP2-2Nested Univarsal primer (NUP)1100bp
[699] After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The specific amplified fragment of interest was purified by QiaexII Gel Extraction kit (manufactured by Kiagen) and eluted with 20 µl of sterile water. 4 µl of the amplified fragment was inserted into the plasmid pCR2.1 according to the instructions of the TOPO TA cloning kit (manufactured by Invitrogen), and transformed into E. coli DH5α using the reaction solution.
[700] Plasmid DNA was isolated from the obtained kanamycin resistant colonies, and 6 clones of cDNA containing the 5 'region of Chinese hamster Fx were obtained. Each is called Fx5 'clone 25, Fx5' clone 26, Fx5 'clone 27, Fx5' colon 28, Fx5 'clone 31, Fx5' clone 32.
[701] Similarly, 5 clones of cDNA containing the 3 'region of Chinese hamster Fx were obtained. Fx3 'is referred to as Fx3' clone 1, Fx3 'clone 3, Fx3' clone 6, Fx3 'clone 8, and Fx3' clone 9.
[702] The base sequence of the cDNA portion of each clone obtained by the 5 'and 3' RACE was determined using DNA sequencer 377 (manufactured by Applied Biosystems). The method was in accordance with the attached manual. The base sequence of each cDNA determined by this method was compared, and the misreading of the base accompanying PCR was eliminated, and the base sequence of the full length of Chinese hamster FxcDNA was determined. The determined nucleotide sequence is shown in SEQ ID NO: 48. ORF of sequence number 48 is base numbers 95-1060, and shows the amino acid sequence corresponding to base numbers 95-1057 except a terminal codon by sequence number 62.
[703] 2. Determination of GFPP cDNA Sequences in CHO Cells
[704] (1) Acquisition of cDNA partial fragment of Chinese hamster GFPP
[705] The cDNA partial fragment of Chinese hamster GFPP was obtained by the following procedures. First, cDNA (Genebank Accession No. AF017445) of human GFPP registered in the public database, mouse EST sequence (Genebank Accession No. AI467195, AA422658, BE304325, AI466474) having high homology with this sequence, and Rat EST sequence (Genebank Accession No. BF546372) , AI058400, AW144783) were compared, and primers GFPP FW9 and GFPP RV9 (SEQ ID NO: 49 and SEQ ID NO: 50) specific for rat GFPP were designed in regions of high conservativeness among the three species.
[706] Next, a 25 µl reaction solution containing 1 µl of the CHO / DG44-derived single-chain cDNA prepared in item 1 (2) using DNA polymerase ExTaq (manufactured by Takara Co., Ltd.) [ExTaQ buffer (Takaraju) Irradiation production), 0.2 mM dNTPs, 0.5 µmol / L The above GFPP specific primers GFPP FW9 and GFPP RV9 (SEQ ID NO: 49 and SEQ ID NO: 50)] were prepared, and a polymerase chain reaction (PCR) was performed. PCR was carried out under the condition of heating at 94 ° C. for 5 minutes, 1 cycle at 94 ° C., 2 minutes at 58 ° C., and 3 minutes at 72 ° C. for 30 cycles, and then heating at 72 ° C. for 10 minutes. It was.
[707] After PCR, the reaction solution was subjected to 2% agarose gel electrophoresis, 1.4kbp of the specific amplified fragment was purified by QiaexII Gel Extraction kit (Kiagen), and eluted with 20 µl of sterile water. 4 µl of the amplified fragment was inserted into the plasmid pCR2.1 according to the instructions of the TOPO TA cloning kit (manufactured by Invitrogen), and transformed into E. coli DH5α using the reaction solution.
[708] Plasmid DNA was isolated from the plurality of kanamycin resistant colonies obtained to obtain 3 clones into which the GFPPcDNA partial fragment was inserted. GFPP clone 8, GFPP clone 11, and GFPP clone 12, respectively.
[709] The base sequence of the cDNA inserted into GFPP clone 8, GFPP clone 11, and GFPP clone 12 was determined using DNA sequencer 377 (manufactured by Applied Biosystems) and Big Dye Terminator Cycle Sequencing FS Raedy Reaction Kit (manufactured by Applied Biosystems). The method was followed in the attached manual. It was confirmed that the inserted cDNA sequenced by this method codes the partial sequence of the open reading frame (ORF) of GFPP of Chinese hamster.
[710] (2) Determination of Chinese hamster GFPP full-length cDNA by RACE method
[711] Primer for 5 'RACE GFPP GSP-1 (SEQ ID NO: 52) and GFPPGSP1-2 (SEQ ID NO: 53), based on the partial sequence of Chinese Hamster Fx determined in paragraph 2 (1) Primer GFPP GSP2-1 (SEQ ID NO: 54) and GFPP GSP2-2 (SEQ ID NO: 55) for Nice Hamster GFPP specific 3 'RACE were designed.
[712] Next, using a Advantage2 PCR Kit (manufactured by CLONTECH), 50 µl of reaction solution containing 1 µl of one-chain cDNA for CACE / DG44-derived RACE prepared in this section (4) [Advantage2 PCR buffer (manufactured by CLONTECH) , 0.2 mM dNTPs, 0.2 μmol / L Chinese Hamster GFPP-specific RACE primer, 1-fold concentration common primer (manufactured by CLONTECH)] was prepared, and polymerase chain reaction (PCR) was performed.
[713] PCR was carried out under the condition that 20 cycles of reactions were performed with 1 cycle of reaction consisting of 5 seconds at 94 ° C, 10 seconds at 68 ° C, and 2 minutes at 72 ° C.
[714] After the completion of the reaction, 1 µl was taken from the reaction solution, 1 µl of an aqueous solution diluted 50-fold with Tricin-EDTA buffer was used as a template to prepare a reaction solution, and PCR was carried out under the same conditions. The combination of the primers used in the first and second PCRs and the length of the DNA fragment to be amplified are shown in Table 3,
[715] Combination of primers used in Chinese hamster GFPP cDNA RACE PCR and length of PCR product 5 'RACEGFPP specific primerCommon primerSize of PCR Amplification Products 1st timeGFPPGSP1-1Univarsal primer mix (UPM)2nd timeGFPPGSP1-2Nested Univarsal primer (NUP)1100bp 3 'RACEGFPP specific primerCommon primerSize of PCR Amplification Products 1st timeGFPPGSP2-1Univarsal primer mix (UPM)2nd timeGFPPGSP2-2Nested Univarsal primer (NUP)1400 bp
[716] After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis, and the target specific amplified fragment was purified by a QiaxII Gel Extraction kit (manufactured by Kiagen) and eluted with 20 µl of sterile water. 4 μl of the amplified fragment was inserted into the plasmid pCR2.1 according to the instructions of the TOPO TA cloning kit (manufactured by Invitrogen), and transformed into E. coli DH5α using the reaction solution.
[717] Plasmid DNA was isolated from the obtained kanamycin resistant colonies, and 4 clones were obtained of cDNA containing the 5 'region of Chinese hamster CFPP. Each is called GFPP5 'clone 1, GFPP5' clone 2, GFPP5 'clone 3, GFPP5' clone 4.
[718] Similarly, 3 clones of cDNA comprising the 3 'region of Chinese hamster GFPP were obtained. Each is referred to as GFPP3 'clone 10, GFPP3' clone 16, and GFPP3 'clone 20.
[719] The base sequence of the cDNA portion of each clone obtained by the 5 'and 3' RACE was determined using DNA sequencer 377 (manufactured by Applied Biosystems). The method was followed in the attached manual. After the base sequence was determined, the base sequences of the respective cDNAs were compared to eliminate misreading of the bases involved in PCR, and the base sequence of the full length of the Chinese hamster GFPP cDNA was determined. The determined nucleotide sequence is shown in SEQ ID NO: 51. The ORF of SEQ ID NO: 51 is SEQ ID NO: 27 to 1799, and the amino acid sequence corresponding to SEQ ID NO: 27 to 1796 except for the stop codon is represented by SEQ ID NO: 63.
[720] Example 10 Activity Evaluation of Anti-CD20 Chimeric Antibodies with Different Proportions of Antibody-linked Sugar Molecules Without α-1,6-fucose
[721] 1.Preparation of anti-CD20 chimeric antibody having different ratios of antibody molecules bound to sugar chains without α-1,6-fucose
[722] KM3065 purified in item 3 of Example 1 and Rituxan ^™ derived from CHO cells were used and mixed in a ratio of KM3065: Rituxan ^™ = 24: 66, 34:56, 44:46. These samples were subjected to sugar chain analysis according to the method of Example 3. The proportion of antibody molecules bound to sugar chains without α-1,6-fucose was 26%, 35% and 44%, respectively. These samples are hereinafter referred to as anti-CD20 chimeric antibodies (26%), anti-CD20 chimeric antibodies (35%) and anti-CD20 chimeric antibodies (44%). Fig. 21 shows the results of sugar chain analysis of each sample.
[723] 2. Evaluation of binding activity against CD20 expressing cell line (fluorescent antibody method)
[724] KM3065 which was subjected to sugar chain analysis in Example 3 to an anti-CD20 chimeric antibody having a different ratio of antibody molecules bound to sugar chains not having three kinds of α-1,6-fucose prepared in Example 1 of Example 10, and The binding activity of five kinds of antibodies to which Rituxan ^TM (represented as anti-CD20 chimeric antibody (96%) and anti-CD20 chimeric antibody (6%), respectively) was measured according to the fluorescence antibody method shown in Section 1 of Example 2. It was. As shown in FIG. 22, at the antibody concentration of 0.016 to 2 µg / ml, all the antibodies showed approximately equivalent binding activity to CD20-curing Raji cells (JCRB9012), and sugar chains having no α-1,6-fucose bound thereto. It was found that the ratio of the antibody molecule did not affect the antigen binding activity of the antibody.
[725] 3. Evaluation of Cytotoxic Activity on CD20 Expressing Cell Line ( ⁵¹ Cr Release Method)
[726] ADCC activity against CD20 positive human B lymphocyte cell line WIL2-S (ATCC CRL8885) was measured as follows using effector cells harvested from healthy donor A.
[727] (1) Preparation of Target Cell Solution
[728] 2 x 10 ⁶ cells of WIL2-S cells were prepared, and 3.7 MBq equivalent of Na ₂ ⁵¹ CrO ₄ , a radioactive substance, was added and reacted at 37 ° C. for 1 hour to radiolabel the cells. After the reaction, the mixture was washed three times by suspension and centrifugation using RPMI1640-FCS (10) medium, resuspended in the medium, and left in ice at 4 ° C. for 30 minutes to dissociate radioactive material naturally. After centrifugation, 10 ml of medium was added and prepared at 2 x 10 ⁵ cells / ml to obtain a target cell solution.
[729] (2) Preparation of Human Effector Cell Solution
[730] 50 ml of healthy peripheral blood was collected, 0.5 ml of heparin sodium (manufactured by Shimizu Pharmaceutical Co., Ltd.) was added and mixed slowly. The mononuclear cell layer was separated by centrifugation (800 g, 20 minutes) using Lymphoprep (manufactured by AXIS SHIELD) according to the instructions. After centrifugation (1400 rpm, 5 minutes) in the medium and washing, the medium was resuspended at a concentration of 2 x 10 ⁶ cells / ml using the medium to prepare a human effector cell solution.
[731] (3) Determination of ADCC Activity
[732] 50 μl (1 × 10 ⁴ cells / well) of the target cell solution prepared in (1) was dispensed into each well of a 96 well U-bottomed plate (manufactured by Falcon). Subsequently, 100 μl (2 × 10 ⁵ cells / well, ratio of human effector cells to target cells became 20: 1) of the human effector cell solution prepared in (2). Further, anti-CD20 chimeric antibodies having different ratios of sugar molecules bound to various sugar chains without various α-1,6-fucose were added so as to have a final concentration of 0.001 to 1 µg / ml, and the reaction was carried out at 37 ° C for 4 hours. I was. After the reaction, the plate was centrifuged and the amount of ⁵¹ Cr in the supernatant was measured by γ-counter. The amount of spontaneous dissociation of ⁵¹ Cr was determined by measuring the amount of ¹⁵ Cr in the supernatant by the same procedure as described above using only the medium of human effector cell solution and antibody solution. The total amount of ⁵¹ Cr of dissociation was determined by adding 1 mol / L hydrochloric acid solution instead of the antibody solution and human effector cell solution, and measuring ⁵¹ Cr in the supernatant in the same manner as above. Cytotoxic activity (%) was calculated | required by the following formula.
[733] Cytotoxic activity (%) = {( ⁵¹ Cr amount-natural dissociation ⁵¹ Cr amount in sample supernatant) / (total dissociation ⁵¹ Cr amount-natural dissociation ⁵¹ Cr amount)} × 100
[734] 23 shows ADCC activity at various concentrations (0.001 to 1 µg / ml) of anti-CD20 chimeric antibodies having different ratios of sugar molecules bound to sugar chains without α-1,6-fucose. The result measured by the said method using an effector cell was shown. As shown in FIG. 23, the ADCC activity of the anti-CD20 chimeric antibody showed a tendency to increase when the proportion of antibody molecules bound to sugar chains without α-1,6-fucose was increased at any antibody concentration. Lower antibody concentrations lower ADCC activity. At an antibody concentration of 0.01 μg / mL, 26%, 35%, 44% and 96% of the antibody chains with no α-1,6-fucose bound showed approximately the same high ADCC activity, but α-1 The ADCC activity was low in the antibody having 6% sugar chain to which the, 6-fucose was not bound.
[735] 4. Evaluation of ADCC Activity on CD20 Expressing Cell Line (LDH Method)
[736] ADCC activity against Raji cells was evaluated using effector cells harvested from healthy donor B by the LDH (lactic acid dehydrogenase) activity assay shown in Section 2 of Example 2. The ratio of effector cells to target cells was 20: 1, and the final antibody concentration was 0.0001 to 1 µg / ml, and the total amount was 200 µl, followed by 4 hours of reaction at 37 ° C. It was. Fig. 24 shows ADCC activity at various concentrations (0.0001 to 1 µg / ml) of anti-CD20 chimeric antibodies having different ratios of sugar molecules bound to sugar chains without α-1,6-fucose. The results measured using effector cells are shown. As shown in FIG. 24, ADCC activity of the anti-CD20 chimeric antibody showed a tendency to increase when the proportion of the antibody molecule to which sugar chains without α-1,6-fucose bound was increased at any antibody concentration. Lower antibody concentrations lower ADCC activity. At an antibody concentration of 0.01 μg / ml, 26%, 35%, 44% and 96% of the sugar chains without α-1,6-fucose bound showed high ADCC activity, but α-1,6 -ADCC activity was low in 6% antibody without sugar chains.
[737] The results in FIGS. 23 and 24 show that ADCC activity is increased according to the ratio of the antibody molecules to which sugar chains without α-1,6-fucose are bound, and sugars without α-1,6-fucose. The antibody composition with the ratio of the chain | strand-bonded antibody molecule about 20% or more shows that it has sufficiently high ADCC activity, and the same result was obtained even if the donor or target cell of human effector cells differs.
[738] Example 11 Activity Evaluation of Anti-CD20 Chimeric Antibodies with Different Ratio of Sugar Molecules Conjugated to Sugar Chains Having Bisector GlcNAc
[739] (1) Isolation of Anti-CD20 Chimeric Antibody by Lectin Chromatography
[740] The anti-CD20 chimeric antibody KM3065 purified in Section 3 of Example 1 was isolated using a column in which a lectin having affinity for sugar chains having a bisecting GlcNAc was immobilized.
[741] The solution containing the purified anti CD20 chimeric antibody KM3065 was subjected to a lectin column (LA-PHA-E ₄ , 4.6 × 150 mm, manufactured by Honen Corporation). As the HPLC system, lectin chromatography was performed at a flow rate of 0.5 ml / min and the column temperature at room temperature using LC-6A manufactured by Shimadzu. Equilibrate the column with 50 mM Tris-Sulfate Buffer (pH8.0) and inject the solution containing purified KM3065 and then add potassium tetraborate (K ₂ from 0M to 58 mM in 50 mM Tris-Sulfate buffer (pH8.0) Elution was carried out with linear gradient (35 minutes) by B ₄ O ₇ , manufactured by Nakara-Ritex Co., Ltd.). After that, the potassium tetraborate concentration was maintained at 100 mM for 5 minutes, and then 50mM tris-sulfate buffer (pH8.0) was passed through for 20 minutes, thereby preventing the anti-CD20 chimeric antibody KM3065 for 9-14 minutes, 14-17 minutes, and 17 minutes. Four fractions eluted at ˜22 min and 22-34 min (1 to ④ of fraction) were separated (FIG. 25).
[742] (2) sugar chain analysis
[743] The sugar chain analysis of the four fractions (fractions 1-4) isolate | separated in the previous paragraph and the anti CD20 chimeric antibody KM3065 before isolation | separation was performed by the method shown in Example 3. PA sugar chain groups were eluted in the range of 15 to 45 minutes. The ratio of sugar chains having a bisecting GlcNAc to the sum of the peak areas of each PA-sugar sugar chain was calculated. As a result, the ratio of the sugar chains of the anti-CD20 chimeric antibody KM3065 before separation was 20%. 0%, fraction ②: 8%, fraction ③: 33%, fraction ④: 45% (FIG. 26). The ratio of antibody molecules bound to sugar chains without α-1,6-fucose was the anti-CD20 chimeric antibody KM3065: 96%, fraction ①: 93%, fraction ②: 94%, fraction ③: 92% before separation, respectively. , Fraction ④: 90%. From the above results, using a column in which a lectin having affinity for sugar chains having a bisecting GlcNAc is immobilized, the ratio of antibody molecules to which sugar chains without α-1,6-fucose are bound is approximately uniform. It was confirmed that anti-CD20 chimeric antibodies having different ratios of antibody molecules to which sugar chains having bisecting GlcNAc are bound were prepared.
[744] (3) Determination of in vitro cytotoxic activity (ADCC activity)
[745] In vitro cytotoxic activity (ADCC activity) measurement of four fractions (fractions 1 to 4) separated by lectin chromatography and anti-CD20 chimeric antibody KM3065 before separation was performed by the method shown in Section 2 of Example 2. (Figure 27). As a result, the four fractions separated by lectin chromatography showed ADCC activity of approximately the same size as the anti-CD20 chimeric antibody KM3065 before separation. The proportion of antibody molecules to which sugar chains without α-1,6-fucose are bound is approximately uniform from 90% to 96% from the result of the preceding paragraph, and sugar chains without α-1,6-fucose are bound. The effect of the antibody molecules on the ADCC activity was considered to be approximately the same. The addition of bisecting GlcNAc to the antibody having a high proportion of antibody molecules bound to sugar chains having no α-1,6-fucose and high ADCC activity did not enhance the magnitude of ADCC activity. In other words, the antibody having a high ratio of sugar chains having no α-1,6-fucose is bound to a high ratio of sugar chains having α-1,6-fucose regardless of the presence or absence of bisecting GlcNAc. It can be seen that the ADCC activity is higher than that.
[746] According to the present invention, a composition comprising an antibody molecule which specifically binds to CD20 and has an N-glycoside-binding complex sugar chain in an Fc region, a cell or a transgenic non-human animal or plant producing the antibody composition, and the antibody composition There is provided a method for producing a medicament containing the antibody composition.
<110> KYOWA HAKKO KOGYO CO., LTD.<120> ANTI-CD20 ANTIBODY COMPOSITION<130> 11440WO1<150> JP2001-392753<151> 2001-12-25<150> JP2002-106948<151> 2002-04-09<150> JP2002-319975<151> 2002-11-01<160> 63<170> Patent In Ver. 2.1<210> 1<211> 2008<212> DNA<213> Cricetulus griseus<400> 1aacagaaact tattttcctg tgtggctaac tagaaccaga gtacaatgtt tccaattctt 60tgagctccga gaagacagaa gggagttgaa actctgaaaa tgcgggcatg gactggttcc 120tggcgttgga ttatgctcat tctttttgcc tgggggacct tattgtttta tataggtggt 180catttggttc gagataatga ccaccctgac cattctagca gagaactctc caagattctt 240gcaaagctgg agcgcttaaa acaacaaaat gaagacttga ggagaatggc tgagtctctc 300cgaataccag aaggccctat tgatcagggg acagctacag gaagagtccg tgttttagaa 360gaacagcttg ttaaggccaa agaacagatt gaaaattaca agaaacaagc taggaatgat 420ctgggaaagg atcatgaaat cttaaggagg aggattgaaa atggagctaa agagctctgg 480ttttttctac aaagtgaatt gaagaaatta aagaaattag aaggaaacga actccaaaga 540catgcagatg aaattctttt ggatttagga catcatgaaa ggtctatcat gacagatcta 600tactacctca gtcaaacaga tggagcaggt gagtggcggg aaaaagaagc caaagatctg 660acagagctgg tccagcggag aataacatat ctgcagaatc ccaaggactg cagcaaagcc 720agaaagctgg tatgtaatat caacaaaggc tgtggctatg gatgtcaact ccatcatgtg 780gtttactgct tcatgattgc ttatggcacc cagcgaacac tcatcttgga atctcagaat 840tggcgctatg ctactggagg atgggagact gtgtttagac ctgtaagtga gacatgcaca 900gacaggtctg gcctctccac tggacactgg tcaggtgaag tgaaggacaa aaatgttcaa 960gtggtcgagc tccccattgt agacagcctc catcctcgtc ctccttactt acccttggct 1020gtaccagaag accttgcaga tcgactcctg agagtccatg gtgatcctgc agtgtggtgg 1080gtatcccagt ttgtcaaata cttgatccgt ccacaacctt ggctggaaag ggaaatagaa 1140gaaaccacca agaagcttgg cttcaaacat ccagttattg gagtccatgt cagacgcact 1200gacaaagtgg gaacagaagc agccttccat cccattgagg aatacatggt acacgttgaa 1260gaacattttc agcttctcga acgcagaatg aaagtggata aaaaaagagt gtatctggcc 1320actgatgacc cttctttgtt aaaggaggca aagacaaagt actccaatta tgaatttatt 1380agtgataact ctatttcttg gtcagctgga ctacacaacc gatacacaga aaattcactt 1440cggggcgtga tcctggatat acactttctc tcccaggctg acttccttgt gtgtactttt 1500tcatcccagg tctgtagggt tgcttatgaa atcatgcaaa cactgcatcc tgatgcctct 1560gcaaacttcc attctttaga tgacatctac tattttggag gccaaaatgc ccacaaccag 1620attgcagttt atcctcacca acctcgaact aaagaggaaa tccccatgga acctggagat 1680atcattggtg tggctggaaa ccattggaat ggttactcta aaggtgtcaa cagaaaacta 1740ggaaaaacag gcctgtaccc ttcctacaaa gtccgagaga agatagaaac agtcaaatac 1800cctacatatc ctgaagctga aaaatagaga tggagtgtaa gagattaaca acagaattta 1860gttcagacca tctcagccaa gcagaagacc cagactaaca tatggttcat tgacagacat 1920gctccgcacc aagagcaagt gggaaccctc agatgctgca ctggtggaac gcctctttgt 1980gaagggctgc tgtgccctca agcccatg 2008<210> 2<211> 1728<212> DNA<213> Mus musculus<400> 2atgcgggcat ggactggttc ctggcgttgg attatgctca ttctttttgc ctgggggacc 60ttgttatttt atataggtgg tcatttggtt cgagataatg accaccctga tcactccagc 120agagaactct ccaagattct tgcaaagctt gaacgcttaa aacagcaaaa tgaagacttg 180aggcgaatgg ctgagtctct ccgaatacca gaaggcccca ttgaccaggg gacagctaca 240ggaagagtcc gtgttttaga agaacagctt gttaaggcca aagaacagat tgaaaattac 300aagaaacaag ctagaaatgg tctggggaag gatcatgaaa tcttaagaag gaggattgaa 360aatggagcta aagagctctg gttttttcta caaagcgaac tgaagaaatt aaagcattta 420gaaggaaatg aactccaaag acatgcagat gaaattcttt tggatttagg acaccatgaa 480aggtctatca tgacagatct atactacctc agtcaaacag atggagcagg ggattggcgt 540gaaaaagagg ccaaagatct gacagagctg gtccagcgga gaataacata tctccagaat 600cctaaggact gcagcaaagc caggaagctg gtgtgtaaca tcaataaagg ctgtggctat 660ggttgtcaac tccatcacgt ggtctactgt ttcatgattg cttatggcac ccagcgaaca 720ctcatcttgg aatctcagaa ttggcgctat gctactggtg gatgggagac tgtgtttaga 780cctgtaagtg agacatgtac agacagatct ggcctctcca ctggacactg gtcaggtgaa 840gtaaatgaca aaaacattca agtggtcgag ctccccattg tagacagcct ccatcctcgg 900cctccttact taccactggc tgttccagaa gaccttgcag accgactcct aagagtccat 960ggtgaccctg cagtgtggtg ggtgtcccag tttgtcaaat acttgattcg tccacaacct 1020tggctggaaa aggaaataga agaagccacc aagaagcttg gcttcaaaca tccagttatt 1080ggagtccatg tcagacgcac agacaaagtg ggaacagaag cagccttcca ccccatcgag 1140gagtacatgg 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atgacagcga gatggagtga tgagaatttg tagaaatgaa 1980ttcacttata ctgagaactt gttttgcttt tagataatga acatattagc ctgaagtaca 2040tagccgaatt gattaattat tcaaagatat aatcttttaa tccctataaa agaggtatta 2100cacaacaatt caagaaagat agaattagac ttccagtatt ggagtgaacc atttgttatc 2160aggtagaacc ctaacgtgtg tggttgactt aaagtgttta ctttttacct gatactgggt 2220agctaattgt ctttcagcct cctggccaaa gataccatga aagtcaactt acgttgtatt 2280ctatatctca aacaactcag ggtgtttctt actctttcca cagcatgtag agcccaggaa 2340gcacaggaca agaaagctgc ctccttgtat caccaggaag atctttttgt aagagtcatc 2400acagtatacc agagagacta attttgtctg aagcatcatg tgttgaaaca acagaaactt 2460attttcctgt gtggctaact agaaccagag tacaatgttt ccaattcttt gagctccgag 2520aagacagaag ggagttgaaa ctctgaaaat gcgggcatgg actggttcct ggcgttggat 2580tatgctcatt ctttttgcct gggggacctt attgttttat ataggtggtc atttggttcg 2640agataatgac caccctgacc attctagcag agaactctcc aagattcttg caaagctgga 2700gcgcttaaaa caacaaaatg aagacttgag gagaatggct gagtctctcc ggtaggtttg 2760aaatactcaa ggatttgatg aaatactgtg cttgaccttt aggtataggg tctcagtctg 2820ctgttgaaaa atataatttc tacaaaccgt ctttgtaaaa ttttaagtat tgtagcagac 2880tttttaaaag tcagtgatac atctatatag tcaatatagg tttacatagt tgcaatctta 2940ttttgcatat gaatcagtat atagaagcag tggcatttat atgcttatgt tgcatttaca 3000attatgttta gacgaacaca aactttatgt gatttggatt agtgctcatt aaattttttt 3060attctatgga ctacaacaga gacataaatt ttgaaaggct tagttactct taaattctta 3120tgatgaaaag caaaaattca ttgttaaata gaacagtgca tccggaatgt gggtaattat 3180tgccatattt ctagtctact aaaaattgtg gcataactgt tcaaagtcat cagttgtttg 3240gaaagccaaa gtctgattta aatggaaaac ataaacaatg atatctattt ctagatacct 3300ttaacttgca gttactgagt ttacaagttg tctgacaact ttggattctc ttacttcata 3360tctaagaatg atcatgtgta cagtgcttac tgtcacttta aaaaactgca gggctagaca 3420tgcagatatg aagactttga cattagatgt ggtaattggc actaccagca agtggtatta 3480agatacagct gaatatatta ctttttgagg aacataattc atgaatggaa agtggagcat 3540tagagaggat gccttctggc tctcccacac cactgtttgc atccattgca tttcacactg 3600cttttagaac tcagatgttt catatggtat attgtgtaac tcaccatcag ttttatcttt 3660aaatgtctat ggatgataat gttgtatgtt aacactttta caaaaacaaa tgaagccata 3720tcctcggtgt gagttgtgat ggtggtaatt gtcacaatag gattattcag caaggaacta 3780agtcagggac aagaagtggg cgatactttg ttggattaaa tcattttact ggaagttcat 3840cagggagggt tatgaaagtt gtggtctttg aactgaaatt atatgtgatt cattattctt 3900gatttaggcc ttgctaatag taactatcat ttattgggaa tttgtcatat gtgccaattt 3960gtcatgggcc agacagcgtg ttttactgaa tttctagata tctttatgag attctagtac 4020tgttttcagc cattttacag atgaagaatc ttaaaaaatg ttaaataatt tagtttgccc 4080aagattatac gttaacaaat ggtagaacct tctttgaatt ctggcagtat ggctacacag 4140tccgaactct tatcttccta agctgaaaac agaaaaagca atgacccaga aaattttatt 4200taaaagtctc aggagagact tcccatcctg agaagatctc ttttcccttt tataatttag 4260gctcctgaat aatcactgaa ttttctccat gttccatcta tagtactgtt atttctgttt 4320tccttttttc ttaccacaaa gtatcttgtt tttgctgtat gaaagaaaat gtgttattgt 4380aatgtgaaat tctctgtccc tgcagggtcc cacatccgcc tcaatcccaa ataaacacac 4440agaggctgta ttaattatga aactgttggt cagttggcta gggcttctta ttggctagct 4500ctgtcttaat tattaaacca taactactat tgtaagtatt tccatgtggt cttatcttac 4560caaggaaagg gtccagggac ctcttactcc tctggcgtgt tggcagtgaa gaggagagag 4620cgatttccta tttgtctctg cttattttct gattctgctc agctatgtca cttcctgcct 4680ggccaatcag ccaatcagtg ttttattcat tagccaataa aagaaacatt tacacagaag 4740gacttccccc atcatgttat ttgtatgagt tcttcagaaa atcatagtat cttttaatac 4800taatttttat aaaaaattaa ttgtattgaa aattatgtgt atatgtgtct gtgtgtcgat 4860ttgtgctcat aagtagcatg gagtgcagaa gagggaatca gatctttttt taagggacaa 4920agagtttatt cagattacat tttaaggtga taatgtatga ttgcaaggtt atcaacatgg 4980cagaaatgtg aagaagctgg tcacattaca tccagagtca agagtagaga gcaatgaatt 5040gatgcatgca ttcctgtgct cagctcactt ttcctggagc tgagctgatt gtaagccatc 5100tgatgtcttt gctgggaact aactcaaagg caagttcaaa acctgttctt aagtataagc 5160catctctcca gtccctcata tggtctctta agacactttc tttatattct tgtacataga 5220aattgaattc ctaacaactg cattcaaatt acaaaatagt ttttaaaagc tgatataata 5280aatgtaaata caatctagaa catttttata aataagcata ttaactcagt aaaaataaat 5340gcatggttat tttccttcat tagggaagta tgtctcccca ggctgttctc tagattctac 5400tagtaatgct gtttgtacac catccacagg ggttttattt taaagctaag acatgaatga 5460tggacatgct tgttagcatt tagacttttt tccttactat aattgagcta gtatttttgt 5520gctcagtttg atatctgtta attcagataa atgtaatagt aggtaatttc tttgtgataa 5580aggcatataa attgaagttg gaaaacaaaa gcctgaaatg acagttttta agattcagaa 5640caataatttt caaaagcagt tacccaactt tccaaataca atctgcagtt ttcttgatat 5700gtgataaatt tagacaaaga aatagcacat tttaaaatag ctatttactc ttgatttttt 5760tttcaaattt aggctagttc actagttgtg tgtaaggtta tggctgcaaa catctttgac 5820tcttggttag ggaatccagg atgatttacg tgtttggcca aaatcttgtt ccattctggg 5880tttcttctct atctaggtag ctagcacaag ttaaaggtgt ggtagtattg gaaggctctc 5940aggtatatat ttctatattc tgtatttttt tcctctgtca tatatttgct ttctgtttta 6000ttgatttcta ctgttagttt gatacttact ttcttacact ttctttggga tttattttgc 6060tgttctaaga tttcttagca agttcatatc actgatttta acagttgctt cttttgtaat 6120atagactgaa tgccccttat ttgaaatgct tgggatcaga aactcagatt tgaacttttc 6180ttttttaata tttccatcaa gtttaccagc tgaatgtcct gatccaagaa tatgaaatct 6240gaaatgcttt gaaatctgaa acttttagag tgataaagct tccctttaaa ttaatttgtg 6300ttctatattt tttgacaatg tcaacctttc attgttatcc aatgagtgaa catattttca 6360atttttttgt ttgatctgtt atattttgat ctgaccatat ttataaaatt ttatttaatt 6420tgaatgttgt gctgttactt atctttatta ttatttttgc ttattttcta gccaaatgaa 6480attatattct gtattatttt agtttgaatt ttactttgtg gcttagtaac tgccttttgt 6540tggtgaatgc ttaagaaaaa cgtgtggtct actgatattg gttctaatct tatatagcat 6600gttgtttgtt aggtagttga ttatgctggt cagattgtct tgagtttatg caaatgtaaa 6660atatttagat gcttgttttg ttgtctaaga acaaagtatg cttgctgtct cctatcggtt 6720ctggtttttc cattcatctc ttcaagctgt tttgtgtgtt gaatactaac tccgtactat 6780cttgttttct gtgaattaac cccttttcaa aggtttcttt tctttttttt tttaagggac 6840aacaagttta ttcagattac attttaagct gataatgtat gattgcaagg ttatcaacat 6900ggcagaaatg tgaagaagct aggcacatta catccacatg gagtcaagag cagagagcag 6960tgaattaatg catgcattcc tgtggtcagc tcacttttcc tattcttaga tagtctagga 7020tcataaacct ggggaatagt gctaccacaa tgggcatatc cacttacttc agttcatgca 7080atcaaccaag gcacatccac aggaaaaact gatttagaca acctctcatt gagactcttc 7140ccagatgatt agactgtgtc aagttgacaa ttaaaactat cacacctgaa gccatcacta 7200gtaaatataa tgaaaatgtt gattatcacc ataattcatc tgtatccctt tgttattgta 7260gattttgtga agttcctatt caagtccctg ttccttcctt aaaaacctgt tttttagtta 7320aataggtttt ttagtgttcc tgtctgtaaa tactttttta aagttagata ttattttcaa 7380gtatgttctc ccagtctttg gcttgtattt tcatcccttc aatacatata tttttgtaat 7440ttattttttt tatttaaatt agaaacaaag ctgcttttac atgtcagtct cagttccctc 7500tccctcccct cctcccctgc tccccaccta agccccaatt ccaactcctt tcttctcccc 7560aggaagggtg aggccctcca tgggggaaat cttcaatgtc tgtcatatca tttggagcag 7620ggcctagacc ctccccagtg tgtctaggct gagagagtat ccctctatgt ggagagggct 7680cccaaagttc atttgtgtac taggggtaaa tactgatcca ctatcagtgg ccccatagat 7740tgtccggacc tccaaactga cttcctcctt cagggagtct ggaacagttc tatgctggtt 7800tcccagatat cagtctgggg tccatgagca accccttgtt caggtcagtt gtttctgtag 7860gtttccccag cccggtcttg acccctttgc tcatcacttc tccctctctg caactggatt 7920ccagagttca gctcagtgtt tagctgtggg tgtctgcatc tgcttccatc agctactgga 7980tgagggctct aggatggcat ataaggtagt catcagtctc attatcagag aagggctttt 8040aaggtagcct cttgattatt gcttagattg ttagttgggg tcaaccttgt aggtctctgg 8100acagtgacag aattctcttt aaacctataa tggctccctc tgtggtggta tcccttttct 8160tgctctcatc cgttcctccc ctgactagat cttcctgctc cctcatgtcc tcctctcccc 8220tccccttctc cccttctctt tcttctaact ccctctcccc tccacccacg atccccatta 8280gcttatgaga tcttgtcctt attttagcaa aacctttttg gctataaaat taattaattt 8340aatatgctta tatcaggttt attttggcta gtatttgtat gtgtttggtt agtgttttta 8400accttaattg acatgtatcc ttatatttag acacagattt aaatatttga agtttttttt 8460tttttttttt ttaaagattt atttattttt tatgtcttct gcctgcatgc cagaagaggg 8520caccagatct cattcaaggt ggttgtgagc caccatgtgg ttgctgggaa ttgaactcag 8580gacctctgga agaacagtca gtgctcttaa ccgctgagcc atctctccag cccctgaagt 8640gtttctttta aagaggatag cagtgcatca tttttccctt tgaccaatga ctcctacctt 8700actgaattgt tttagccatt tatatgtaat gctgttacca ggtttacatt ttcttttatc 8760ttgctaaatt tcttccctgt ttgtctcatc tcttattttt gtctgttgga ttatataggc 8820ttttattttt ctgtttttac agtaagttat atcaaattaa aattatttta tggaatgggt 8880gtgttgacta catgtatgtc tgtgcaccat gtgctgacct ggtcttggcc agaagaaggt 8940gtcatattct ctgaaactgg tattgtggat gttacgaact gccatagggt gctaggaatc 9000aaaccccagc tcctctggaa aagcagccac tgctctgagc cactgagtcc tctcttcaag 9060caggtgatgc caacttttaa tggttaccag tggataagag tgcttgtatc tctagcaccc 9120atgaaaattt atgcattgct atatgggctt gtcacttcag cattgtgtga cagagacagg 9180aggatcccaa gagctc 9196<210> 4<211> 297<212> PRT<213> Homo sapiens<400> 4Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro  1 5 10 15Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg             20 25 30Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu         35 40 45Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile     50 55 60Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile 65 70 75 80Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile                 85 90 95Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu            100 105 110Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile        115 120 125Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser    130 135 140His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro145 150 155 160Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn                165 170 175Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly            180 185 190Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile        195 200 205Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys    210 215 220Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile225 230 235 240Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro                245 250 255Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu            260 265 270Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser        275 280 285Ser Pro Ile Glu Asn Asp Ser Ser Pro    290 295<210> 5<211> 10<212> PRT<213> Mus musculus<400> 5Arg Ala Ser Ser Ser Val Ser Tyr Ile His  1 5 10<210> 6<211> 7<212> PRT<213> Mus musculus<400> 6Ala Thr Ser Asn Leu Ala Ser  1 5<210> 7<211> 9<212> PRT<213> Mus musculus<400> 7Gln Gln Trp Thr Ser Asn Pro Pro Thr  1 5<210> 8<211> 5<212> PRT<213> Mus musculus<400> 8Ser Tyr Asn Met His  1 5<210> 9<211> 17<212> PRT<213> Mus musculus<400> 9Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys  1 5 10 15Gly   <210> 10<211> 12<212> PRT<213> Mus musculus<400> 10Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val  1 5 10<210> 11<211> 384<212> DNA<213> Mus musculus<220><221> CDS(222) (1) .. (384)<400> 11atg gat ttt cag gtg cag att atc agc ttc ctg cta atc agt gct tca 48Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser  1 5 10 15gtc ata atg tcc aga gga caa att gtt ctc tcc cag tct cca gca atc 96Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile             20 25 30ctg tct gca tct cca ggg gag aag gtc aca atg act tgc agg gcc agc 144Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser         35 40 45tca agt gta agt tac atc cac tgg ttc cag cag aag cca gga tcc tcc 192Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser     50 55 60ccc aaa ccc tgg att tat gcc aca tcc aac ctg gct tct gga gtc cct 240Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80gtt cgc ttc agt ggc agt ggg tct ggg act tct tac tct ctc acc atc 288Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile                 85 90 95agc aga gtg gag gct gaa gat gct gcc act tat tac tgc cag cag tgg 336Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp            100 105 110act agt aac cca ccc acg ttc gga ggg ggg acc aag ctg gaa atc aaa 384Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys        115 120 125<210> 12<211> 128<212> PRT<213> Mus musculus<400> 12Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser  1 5 10 15Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile             20 25 30Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser         35 40 45Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser     50 55 60Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile                 85 90 95Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp            100 105 110Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys        115 120 125<210> 13<211> 420<212> DNA<213> Mus musculus<220><221> CDS(222) (1) .. (420)<400> 13atg ggt tgg agc ctc atc ttg ctc ttc ctt gtc gct gtt gct acg cgt 48Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg  1 5 10 15gtc ctg tcc cag gta caa ctg cag cag cct ggg gct gag ctg gtg aag 96Val Leu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys             20 25 30cct ggg gcc tca gtg aag atg tcc tgc aag gct tct ggc tac aca ttt 144Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe         35 40 45acc agt tac aat atg cac tgg gta aaa cag aca cct ggt cgg ggc ctg 192Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu     50 55 60gaa tgg att gga gct att tat ccc gga aat ggt gat act tcc tac aat 240Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn 65 70 75 80cag aag ttc aaa ggc aag gcc aca ttg act gca gac aaa tcc tcc agc 288Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                 85 90 95aca gcc tac atg cag ctc agc agc ctg aca tct gag gac tct gcg gtc 336Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val            100 105 110tat tac tgt gca aga tcg act tac tac ggc ggt gac tgg tac ttc aat 384Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn        115 120 125gtc tgg ggc gca ggg acc acg gtc acc gtc tct gca 420Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala    130 135 140<210> 14<211> 140<212> PRT<213> Mus musculus<400> 14Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg  1 5 10 15Val Leu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys             20 25 30Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe         35 40 45Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu     50 55 60Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn 65 70 75 80Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                 85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val            100 105 110Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn        115 120 125Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala    130 135 140<210> 15<211> 91<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 15caggaaacag ctatgacgaa ttcgcctcct caaaatggat tttcaggtgc agattatcag 60cttcctgcta atcagtgctt cagtcataat g 91<210> 16<211> 91<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 16gtgaccttct cccctggaga tgcagacagg attgctggag actgggagag aacaatttgt 60cctctggaca ttatgactga agcactgatt a 91<210> 17<211> 90<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 17ctccagggga gaaggtcaca atgacttgca gggccagctc aagtgtaagt tacatccact 60ggttccagca gaagccagga tcctccccca 90<210> 18<211> 89<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 18ccagacccac tgccactgaa gcgaacaggg actccagaag ccaggttgga tgtggcataa 60atccagggtt tgggggagga tcctggctt 89<210> 19<211> 91<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 19tcagtggcag tgggtctggg acttcttact ctctcaccat cagcagagtg gaggctgaag 60atgctgccac ttattactgc cagcagtgga c 91<210> 20<211> 90<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 20gttttcccag tcacgaccgt acgtttgatt tccagcttgg tcccccctcc gaacgtgggt 60gggttactag tccactgctg gcagtaataa 90<210> 21<211> 24<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 21gtctgaagca ttatgtgttg aagc 24<210> 22<211> 23<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 22gtgagtacat tcattgtact gtg 23<210> 23<211> 575<212> PRT<213> Cricetulus griseus<400> 23Met Arg Ala Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe  1 5 10 15Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp             20 25 30Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala         35 40 45Lys Leu Glu Arg Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met Ala     50 55 60Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Thr Ala Thr 65 70 75 80Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln                 85 90 95Ile Glu Asn Tyr Lys Lys Gln Ala Arg Asn Asp Leu Gly Lys Asp His            100 105 110Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe        115 120 125Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Lys Leu Glu Gly Asn Glu    130 135 140Leu Gln Arg His Ala Asp Glu Ile Leu Leu Asp Leu Gly His His Glu145 150 155 160Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala                165 170 175Gly Glu Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln            180 185 190Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Arg        195 200 205Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu    210 215 220His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr225 230 235 240Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu                245 250 255Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Leu            260 265 270Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val        275 280 285Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro Tyr Leu    290 295 300Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Leu Arg Val His305 310 315 320Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile                325 330 335Arg Pro Gln Pro Trp Leu Glu Arg Glu Ile Glu Glu Thr Thr Lys Lys            340 345 350Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp        355 360 365Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val    370 375 380His Val Glu Glu His Phe Gln Leu Leu Glu Arg Arg Met Lys Val Asp385 390 395 400Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Ser Leu Leu Lys Glu                405 410 415Ala Lys Thr Lys Tyr Ser Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile            420 425 430Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg        435 440 445Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val    450 455 460Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln465 470 475 480Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile                485 490 495Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Val Tyr Pro            500 505 510His Gln Pro Arg Thr Lys Glu Glu Ile Pro Met Glu Pro Gly Asp Ile        515 520 525Ile Gly Val Ala Gly Asn His Trp Asn Gly Tyr Ser Lys Gly Val Asn    530 535 540Arg Lys Leu Gly Lys Thr Gly Leu Tyr Pro Ser Tyr Lys Val Arg Glu545 550 555 560Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Glu Lys                565 570 575<210> 24<211> 575<212> PRT<213> Mus musculus<400> 24Met Arg Ala Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe  1 5 10 15Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp             20 25 30Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala         35 40 45Lys Leu Glu Arg Leu Lys Gln Gln Asn Glu Asp Leu Arg Arg Met Ala     50 55 60Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Thr Ala Thr 65 70 75 80Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln                 85 90 95Ile Glu Asn Tyr Lys Lys Gln Ala Arg Asn Gly Leu Gly Lys Asp His            100 105 110Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe        115 120 125Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys His Leu Glu Gly Asn Glu    130 135 140Leu Gln Arg His Ala Asp Glu Ile Leu Leu Asp Leu Gly His His Glu145 150 155 160Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala                165 170 175Gly Asp Trp Arg Glu Lys Glu Ala Lys Asp Leu Thr Glu Leu Val Gln            180 185 190Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Arg        195 200 205Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu    210 215 220His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr225 230 235 240Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu                245 250 255Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Leu            260 265 270Ser Thr Gly His Trp Ser Gly Glu Val Asn Asp Lys Asn Ile Gln Val        275 280 285Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro Tyr Leu    290 295 300Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Leu Arg Val His305 310 315 320Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile                325 330 335Arg Pro Gln Pro Trp Leu Glu Lys Glu Ile Glu Glu Ala Thr Lys Lys            340 345 350Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp        355 360 365Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val    370 375 380His Val Glu Glu His Phe Gln Leu Leu Ala Arg Arg Met Gln Val Asp385 390 395 400Lys Lys Arg Val Tyr Leu Ala Thr Asp Asp Pro Thr Leu Leu Lys Glu                405 410 415Ala Lys Thr Lys Tyr Ser Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile            420 425 430Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg        435 440 445Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val    450 455 460Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln465 470 475 480Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile                485 490 495Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Val Tyr Pro            500 505 510His Lys Pro Arg Thr Glu Glu Glu Ile Pro Met Glu Pro Gly Asp Ile        515 520 525Ile Gly Val Ala Gly Asn His Trp Asp Gly Tyr Ser Lys Gly Ile Asn    530 535 540Arg Lys Leu Gly Lys Thr Gly Leu Tyr Pro Ser Tyr Lys Val Arg Glu545 550 555 560Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Glu Lys                565 570 575<210> 25<211> 99<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 25caggaaacag ctatgacgcg gccgcgaccc ctcaccatgg gttggagcct catcttgctc 60ttccttgtcg ctgttgctac gcgtgtcctg tcccaggta 99<210> 26<211> 98<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 26atgtgtagcc agaagccttg caggacatct tcactgaggc cccagccttc accagctcag 60ccccaggctg ctgcagttgt acctgggaca ggacacgc 98<210> 27<211> 97<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 27caaggcttct ggctacacat ttaccagtta caatatgcac tgggtaaaac agacacctgg 60tcggggcctg gaatggattg gagctattta tcccgga 97<210> 28<211> 99<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 28gtaggctgtg ctggaggatt tgtctgcagt caatgtggcc ttgcctttga acttctgatt 60gtaggaagta tcaccatttc cgggataaat agctccaat 99<210> 29<211> 99<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 29aatcctccag cacagcctac atgcagctca gcagcctgac atctgaggac tctgcggtct 60attactgtgc aagatcgact tactacggcg gtgactggt 99<210> 30<211> 98<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 30gttttcccag tcacgacggg cccttggtgg aggctgcaga gacggtgacc gtggtccctg 60cgccccagac attgaagtac cagtcaccgc cgtagtaa 98<210> 31<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequense: Synthetic DNA<400> 31gagctggtga agcctggggc ctcag 25<210> 32<211> 28<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 32atggctcaag ctcccgctaa gtgcccga 28<210> 33<211> 27<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 33tcaagcgttt gggttggtcc tcatgag 27<210> 34<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 34tccggggatg gcgagatggg caagc 25<210> 35<211> 24<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 35cttgacatgg ctctgggctc caag 24<210> 36<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 36ccacttcagt cggtcggtag tattt 25<210> 37<211> 24<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 37cgctcacccg cctgaggcga catg 24<210> 38<211> 32<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 38ggcaggtgct gtcggtgagg tcaccatagt gc 32<210> 39<211> 24<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 39ggggccatgc caaggactat gtcg 24<210> 40<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 40atgtggctga tgttacaaaa tgatg 25<210> 41<211> 1504<212> DNA<213> Cricetulus griseus<220><221> CDS(222) (1) .. (1116)<400> 41atg gct cac gct ccc gct agc tgc ccg agc tcc agg aac tct ggg gac 48Met Ala His Ala Pro Ala Ser Cys Pro Ser Ser Arg Asn Ser Gly Asp  1 5 10 15ggc gat aag ggc aag ccc agg aag gtg gcg ctc atc acg ggc atc acc 96Gly Asp Lys Gly Lys Pro Arg Lys Val Ala Leu Ile Thr Gly Ile Thr             20 25 30ggc cag gat ggc tca tac ttg gca gaa ttc ctg ctg gag aaa gga tac 144Gly Gln Asp Gly Ser Tyr Leu Ala Glu Phe Leu Leu Glu Lys Gly Tyr         35 40 45gag gtt cat gga att gta cgg cga tcc agt tca ttt aat aca ggt cga 192Glu Val His Gly Ile Val Arg Arg Ser Ser Ser Phe Asn Thr Gly Arg     50 55 60att gaa cat tta tat aag aat cca cag gct cat att gaa gga aac atg 240Ile Glu His Leu Tyr Lys Asn Pro Gln Ala His Ile Glu Gly Asn Met 65 70 75 80aag ttg cac tat ggt gac ctc acc gac agc acc tgc cta gta aaa atc 288Lys Leu His Tyr Gly Asp Leu Thr Asp Ser Thr Cys Leu Val Lys Ile                 85 90 95atc aat gaa gtc aaa cct aca gag atc tac aat ctt ggt gcc cag agc 336Ile Asn Glu Val Lys Pro Thr Glu Ile Tyr Asn Leu Gly Ala Gln Ser            100 105 110cat gtc aag att tcc ttt gac tta gca gag tac act gca gat gtt gat 384His Val Lys Ile Ser Phe Asp Leu Ala Glu Tyr Thr Ala Asp Val Asp        115 120 125gga gtt ggc acc ttg cgg ctt ctg gat gca att aag act tgt ggc ctt 432Gly Val Gly Thr Leu Arg Leu Leu Asp Ala Ile Lys Thr Cys Gly Leu    130 135 140ata aat tct gtg aag ttc tac cag gcc tca act agt gaa ctg tat gga 480Ile Asn Ser Val Lys Phe Tyr Gln Ala Ser Thr Ser Glu Leu Tyr Gly145 150 155 160aaa gtg caa gaa ata ccc cag aaa gag acc acc cct ttc tat cca agg 528Lys Val Gln Glu Ile Pro Gln Lys Glu Thr Thr Pro Phe Tyr Pro Arg                165 170 175tcg ccc tat gga gca gcc aaa ctt tat gcc tat tgg att gta gtg aac 576Ser Pro Tyr Gly Ala Ala Lys Leu Tyr Ala Tyr Trp Ile Val Val Asn            180 185 190ttt cga gag gct tat aat ctc ttt gcg gtg aac ggc att ctc ttc aat 624Phe Arg Glu Ala Tyr Asn Leu Phe Ala Val Asn Gly Ile Leu Phe Asn        195 200 205cat gag agt cct aga aga gga gct aat ttt gtt act cga aaa att agc 672His Glu Ser Pro Arg Arg Gly Ala Asn Phe Val Thr Arg Lys Ile Ser    210 215 220cgg tca gta gct aag att tac ctt gga caa ctg gaa tgt ttc agt ttg 720Arg Ser Val Ala Lys Ile Tyr Leu Gly Gln Leu Glu Cys Phe Ser Leu225 230 235 240gga aat ctg gac gcc aaa cga gac tgg ggc cat gcc aag gac tat gtc 768Gly Asn Leu Asp Ala Lys Arg Asp Trp Gly His Ala Lys Asp Tyr Val                245 250 255gag gct atg tgg ctg atg tta caa aat gat gaa cca gag gac ttt gtc 816Glu Ala Met Trp Leu Met Leu Gln Asn Asp Glu Pro Glu Asp Phe Val            260 265 270ata gct act ggg gaa gtt cat agt gtc cgt gaa ttt gtt gag aaa tca 864Ile Ala Thr Gly Glu Val His Ser Val Arg Glu Phe Val Glu Lys Ser        275 280 285ttc atg cac att gga aag acc att gtg tgg gaa gga aag aat gaa aat 912Phe Met His Ile Gly Lys Thr Ile Val Trp Glu Gly Lys Asn Glu Asn    290 295 300gaa gtg ggc aga tgt aaa gag acc ggc aaa att cat gtg act gtg gat 960Glu Val Gly Arg Cys Lys Glu Thr Gly Lys Ile His Val Thr Val Asp305 310 315 320ctg aaa tac tac cga cca act gaa gtg gac ttc ctg cag gga gac tgc 1008Leu Lys Tyr Tyr Arg Pro Thr Glu Val Asp Phe Leu Gln Gly Asp Cys                325 330 335tcc aag gcg cag cag aaa ctg aac tgg aag ccc cgc gtt gcc ttt gac 1056Ser Lys Ala Gln Gln Lys Leu Asn Trp Lys Pro Arg Val Ala Phe Asp            340 345 350gag ctg gtg agg gag atg gtg caa gcc gat gtg gag ctc atg aga acc 1104Glu Leu Val Arg Glu Met Val Gln Ala Asp Val Glu Leu Met Arg Thr        355 360 365aac ccc aac gcc tgag cacctctaca aaaaaattcg cgagacatgg actatggtgc 1160Asn Pro Asn Ala    370agagccagcc aaccagagtc cagccactcc tgagaccatc gaccataaac cctcgactgc 1220ctgtgtcgtc cccacagcta agagctgggc cacaggtttg tgggcaccag gacggggaca 1280ctccagagct aaggccactt cgcttttgtc aaaggctcct ctcaatgatt ttgggaaatc 1340aagaagttta aaatcacata ctcattttac ttgaaattat gtcactagac aacttaaatt 1400tttgagtctt gagattgttt ttctcttttc ttattaaatg atctttctat gacccagcaa 1460aaaaaaaaaa aaaaagggat ataaaaaaaa aaaaaaaaaa aaaa 1504<210> 42<211> 17<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 42gccatccaga aggtggt 17<210> 43<211> 17<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 43gtcttgtcag ggaagat 17<210> 44<211> 28<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 44ggcaggagac caccttgcga gtgcccac 28<210> 45<211> 28<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 45gggtgggctg taccttctgg aacagggc 28<210> 46<211> 28<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 46ggcgctggct tacccggaga ggaatggg 28<210> 47<211> 30<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 47ggaatgggtg tttgtctcct ccaaagatgc 30<210> 48<211> 1316<212> DNA<213> Cricetulus griseus<400> 48gccccgcccc ctccacctgg accgagagta gctggagaat tgtgcaccgg aagtagctct 60tggactggtg gaaccctgcg caggtgcagc aacaatgggt gagccccagg gatccaggag 120gatcctagtg acagggggct ctggactggt gggcagagct atccagaagg tggtcgcaga 180tggcgctggc ttacccggag aggaatgggt gtttgtctcc tccaaagatg cagatctgac 240ggatgcagca caaacccaag ccctgttcca gaaggtacag cccacccatg tcatccatct 300tgctgcaatg gtaggaggcc ttttccggaa tatcaaatac aacttggatt tctggaggaa 360gaatgtgcac atcaatgaca acgtcctgca ctcagctttc gaggtgggca ctcgcaaggt 420ggtctcctgc ctgtccacct gtatcttccc tgacaagacc acctatccta ttgatgaaac 480aatgatccac aatggtccac cccacagcag caattttggg tactcgtatg ccaagaggat 540gattgacgtg cagaacaggg cctacttcca gcagcatggc tgcaccttca ctgctgtcat 600ccctaccaat gtctttggac ctcatgacaa cttcaacatt gaagatggcc atgtgctgcc 660tggcctcatc cataaggtgc atctggccaa gagtaatggt tcagccttga ctgtttgggg 720tacagggaaa ccacggaggc agttcatcta ctcactggac ctagcccggc tcttcatctg 780ggtcctgcgg gagtacaatg aagttgagcc catcatcctc tcagtgggcg aggaagatga 840agtctccatt aaggaggcag ctgaggctgt agtggaggcc atggacttct gtggggaagt 900cacttttgat tcaacaaagt cagatgggca gtataagaag acagccagca atggcaagct 960tcgggcctac ttgcctgatt tccgtttcac acccttcaag caggctgtga aggagacctg 1020tgcctggttc accgacaact atgagcaggc ccggaagtga agcatgggac aagcgggtgc 1080tcagctggca atgcccagtc agtaggctgc agtctcatca tttgcttgtc aagaactgag 1140gacagtatcc agcaacctga gccacatgct ggtctctctg ccagggggct tcatgcagcc 1200atccagtagg gcccatgttt gtccatcctc gggggaaggc cagaccaaca ccttgtttgt 1260ctgcttctgc cccaacctca gtgcatccat gctggtcctg ctgtcccttg tctaga 1316<210> 49<211> 23<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 49gatcctgctg ggaccaaaat tgg 23<210> 50<211> 22<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 50cttaacatcc caagggatgc tg 22<210> 51<211> 1965<212> DNA<213> Cricetulus griseus<400> 51acggggggct cccggaagcg gggaccatgg cgtctctgcg cgaagcgagc ctgcggaagc 60tgcggcgctt ttccgagatg agaggcaaac ctgtggcaac tgggaaattc tgggatgtag 120ttgtaataac agcagctgac gaaaagcagg agcttgctta caagcaacag ttgtcggaga 180agctgaagag aaaggaattg ccccttggag ttaactacca tgttttcact gatcctcctg 240gaaccaaaat tggaaatgga ggatcaacac tttgttctct tcagtgcctg gaaagcctct 300atggagacaa gtggaattcc ttcacagtcc tgttaattca ctctggtggc tacagtcaac 360gacttcccaa tgcaagcgct ttaggaaaaa tcttcacggc tttaccactt ggtgagccca 420tttatcagat gttggactta aaactagcca tgtacatgga tttcccctca cgcatgaagc 480ctggagtttt ggtcacctgt gcagatgata ttgaactata cagcattggg gactctgagt 540ccattgcatt tgagcagcct ggctttactg ccctagccca tccatctagt ctggctgtag 600gcaccacaca tggagtattt gtattggact ctgccggttc tttgcaacat ggtgacctag 660agtacaggca atgccaccgt ttcctccata agcccagcat tgaaaacatg caccacttta 720atgccgtgca tagactagga agctttggtc aacaggactt gagtgggggt gacaccacct 780gtcatccatt gcactctgag tatgtctaca cagatagcct attttacatg gatcataaat 840cagccaaaaa gctacttgat ttctatgaaa gtgtaggccc actgaactgt gaaatagatg 900cctatggtga ctttctgcag gcactgggac ctggagcaac tgcagagtac accaagaaca 960cctcacacgt cactaaagag gaatcacact tgttggacat gaggcagaaa atattccacc 1020tcctcaaggg aacacccctg aatgttgttg tccttaataa ctccaggttt tatcacattg 1080gaacaacgga ggagtatctg ctacatttca cttccaatgg ttcgttacag gcagagctgg 1140gcttgcaatc catagctttc agtgtctttc caaatgtgcc tgaagactcc catgagaaac 1200cctgtgtcat tcacagcatc ctgaattcag gatgctgtgt ggcccctggc tcagtggtag 1260aatattccag attaggacct gaggtgtcca tctcggaaaa ctgcattatc agcggttctg 1320tcatagaaaa agctgttctg cccccatgtt ctttcgtgtg ctctttaagt gtggagataa 1380atggacactt agaatattca actatggtgt ttggcatgga agacaacttg aagaacagtg 1440ttaaaaccat atcagatata aagatgcttc agttctttgg agtctgtttc ctgacttgtt 1500tagatatttg gaaccttaaa gctatggaag aactattttc aggaagtaag acgcagctga 1560gcctgtggac tgctcgaatt ttccctgtct gttcttctct gagtgagtcg gttgcagcat 1620cccttgggat gttaaatgcc attcgaaacc attcgccatt cagcctgagc aacttcaagc 1680tgctgtccat ccaggaaatg cttctctgca aagatgtagg agacatgctt gcttacaggg 1740agcaactctt tctagaaatc agttcaaaga gaaaacagtc tgattcggag aaatcttaaa 1800tacaatggat tttgcctgga aacaggattg caaatgcagg catattctat agatctctgg 1860gttcttcttt ctttctcccc tctctccttt cctttccctt tgatgtaatg acaaaggtaa 1920aaatggccac ttctgatgga aaaaaaaaaa aaaaaaaaaa aaaaa 1965<210> 52<211> 27<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 52caggggtgtt cccttgagga ggtggaa 27<210> 53<211> 27<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 53cactgagcca ggggccacac agcatcc 27<210> 54<211> 23<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 54cccctcacgc atgaagcctg gag 23<210> 55<211> 27<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 55tgccaccgtt tcctccataa gcccagc 27<210> 56<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 56atgaagttgc actatggtga cctca 25<210> 57<211> 59<212> DNA<213> Cricetulus griseus<400> 57ccgacagcac ctgcctagta aaaatcatca atgaagtcaa acctacagag atctacaat 59<210> 58<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 58gacttagcag agtacactgc agatg 25<210> 59<211> 25<212> DNA<213> Artificial Sequence<220><223> Description of Artificial Sequence: Synthetic DNA<400> 59accttggata gaaaggggtg gtctc 25<210> 60<211> 125<212> DNA<213> Cricetulus griseus<400> 60ttgatggagt tggcaccttg cggcttctgg atgcaattaa gacttgtggc cttataaatt 60ctgtgaagtt ctaccaggcc tcaactagtg aactgtatgg aaaagtgcaa gaaatacccc 120agaaa 125<210> 61<211> 372<212> PRT<213> Cricetulus griseus<400> 61Met Ala His Ala Pro Ala Ser Cys Pro Ser Ser Arg Asn Ser Gly Asp  1 5 10 15Gly Asp Lys Gly Lys Pro Arg Lys Val Ala Leu Ile Thr Gly Ile Thr             20 25 30Gly Gln Asp Gly Ser Tyr Leu Ala Glu Phe Leu Leu Glu Lys Gly Tyr         35 40 45Glu Val His Gly Ile Val Arg Arg Ser Ser Ser Phe Asn Thr Gly Arg     50 55 60Ile Glu His Leu Tyr Lys Asn Pro Gln Ala His Ile Glu Gly Asn Met 65 70 75 80Lys Leu His Tyr Gly Asp Leu Thr Asp Ser Thr Cys Leu Val Lys Ile                 85 90 95Ile Asn Glu Val Lys Pro Thr Glu Ile Tyr Asn Leu Gly Ala Gln Ser            100 105 110His Val Lys Ile Ser Phe Asp Leu Ala Glu Tyr Thr Ala Asp Val Asp        115 120 125Gly Val Gly Thr Leu Arg Leu Leu Asp Ala Ile Lys Thr Cys Gly Leu    130 135 140Ile Asn Ser Val Lys Phe Tyr Gln Ala Ser Thr Ser Glu Leu Tyr Gly145 150 155 160Lys Val Gln Glu Ile Pro Gln Lys Glu Thr Thr Pro Phe Tyr Pro Arg                165 170 175Ser Pro Tyr Gly Ala Ala Lys Leu Tyr Ala Tyr Trp Ile Val Val Asn            180 185 190Phe Arg Glu Ala Tyr Asn Leu Phe Ala Val Asn Gly Ile Leu Phe Asn        195 200 205His Glu Ser Pro Arg Arg Gly Ala Asn Phe Val Thr Arg Lys Ile Ser    210 215 220Arg Ser Val Ala Lys Ile Tyr Leu Gly Gln Leu Glu Cys Phe Ser Leu225 230 235 240Gly Asn Leu Asp Ala Lys Arg Asp Trp Gly His Ala Lys Asp Tyr Val                245 250 255Glu Ala Met Trp Leu Met Leu Gln Asn Asp Glu Pro Glu Asp Phe Val            260 265 270Ile Ala Thr Gly Glu Val His Ser Val Arg Glu Phe Val Glu Lys Ser        275 280 285Phe Met His Ile Gly Lys Thr Ile Val Trp Glu Gly Lys Asn Glu Asn    290 295 300Glu Val Gly Arg Cys Lys Glu Thr Gly Lys Ile His Val Thr Val Asp305 310 315 320Leu Lys Tyr Tyr Arg Pro Thr Glu Val Asp Phe Leu Gln Gly Asp Cys                325 330 335Ser Lys Ala Gln Gln Lys Leu Asn Trp Lys Pro Arg Val Ala Phe Asp            340 345 350Glu Leu Val Arg Glu Met Val Gln Ala Asp Val Glu Leu Met Arg Thr        355 360 365Asn Pro Asn Ala    370<210> 62<211> 321<212> PRT<213> Cricetulus griseus<400> 62Met Gly Glu Pro Gln Gly Ser Arg Arg Ile Leu Val Thr Gly Gly Ser  1 5 10 15Gly Leu Val Gly Arg Ala Ile Gln Lys Val Val Ala Asp Gly Ala Gly             20 25 30Leu Pro Gly Glu Glu Trp Val Phe Val Ser Ser Lys Asp Ala Asp Leu         35 40 45Thr Asp Ala Ala Gln Thr Gln Ala Leu Phe Gln Lys Val Gln Pro Thr     50 55 60His Val Ile His Leu Ala Ala Met Val Gly Gly Leu Phe Arg Asn Ile 65 70 75 80Lys Tyr Asn Leu Asp Phe Trp Arg Lys Asn Val His Ile Asn Asp Asn                 85 90 95Val Leu His Ser Ala Phe Glu Val Gly Thr Arg Lys Val Val Ser Cys            100 105 110Leu Ser Thr Cys Ile Phe Pro Asp Lys Thr Thr Tyr Pro Ile Asp Glu        115 120 125Thr Met Ile His Asn Gly Pro Pro His Ser Ser Asn Phe Gly Tyr Ser    130 135 140Tyr Ala Lys Arg Met Ile Asp Val Gln Asn Arg Ala Tyr Phe Gln Gln145 150 155 160His Gly Cys Thr Phe Thr Ala Val Ile Pro Thr Asn Val Phe Gly Pro                165 170 175His Asp Asn Phe Asn Ile Glu Asp Gly His Val Leu Pro Gly Leu Ile            180 185 190His Lys Val His Leu Ala Lys Ser Asn Gly Ser Ala Leu Thr Val Trp        195 200 205Gly Thr Gly Lys Pro Arg Arg Gln Phe Ile Tyr Ser Leu Asp Leu Ala    210 215 220Arg Leu Phe Ile Trp Val Leu Arg Glu Tyr Asn Glu Val Glu Pro Ile225 230 235 240Ile Leu Ser Val Gly Glu Glu Asp Glu Val Ser Ile Lys Glu Ala Ala                245 250 255Glu Ala Val Val Glu Ala Met Asp Phe Cys Gly Glu Val Thr Phe Asp            260 265 270Ser Thr Lys Ser Asp Gly Gln Tyr Lys Lys Thr Ala Ser Asn Gly Lys        275 280 285Leu Arg Ala Tyr Leu Pro Asp Phe Arg Phe Thr Pro Phe Lys Gln Ala    290 295 300Val Lys Glu Thr Cys Ala Trp Phe Thr Asp Asn Tyr Glu Gln Ala Arg305 310 315 320Lys   <210> 63<211> 590<212> PRT<213> Cricetulus griseus<400> 63Met Ala Ser Leu Arg Glu Ala Ser Leu Arg Lys Leu Arg Arg Phe Ser  1 5 10 15Glu Met Arg Gly Lys Pro Val Ala Thr Gly Lys Phe Trp Asp Val Val             20 25 30Val Ile Thr Ala Ala Asp Glu Lys Gln Glu Leu Ala Tyr Lys Gln Gln         35 40 45Leu Ser Glu Lys Leu Lys Arg Lys Glu Leu Pro Leu Gly Val Asn Tyr     50 55 60His Val Phe Thr Asp Pro Pro Gly Thr Lys Ile Gly Asn Gly Gly Ser 65 70 75 80Thr Leu Cys Ser Leu Gln Cys Leu Glu Ser Leu Tyr Gly Asp Lys Trp                 85 90 95Asn Ser Phe Thr Val Leu Leu Ile His Ser Gly Gly Tyr Ser Gln Arg            100 105 110Leu Pro Asn Ala Ser Ala Leu Gly Lys Ile Phe Thr Ala Leu Pro Leu        115 120 125Gly Glu Pro Ile Tyr Gln Met Leu Asp Leu Lys Leu Ala Met Tyr Met    130 135 140Asp Phe Pro Ser Arg Met Lys Pro Gly Val Leu Val Thr Cys Ala Asp145 150 155 160Asp Ile Glu Leu Tyr Ser Ile Gly Asp Ser Glu Ser Ile Ala Phe Glu                165 170 175Gln Pro Gly Phe Thr Ala Leu Ala His Pro Ser Ser Leu Ala Val Gly            180 185 190Thr Thr His Gly Val Phe Val Leu Asp Ser Ala Gly Ser Leu Gln His        195 200 205Gly Asp Leu Glu Tyr Arg Gln Cys His Arg Phe Leu His Lys Pro Ser    210 215 220Ile Glu Asn Met His His Phe Asn Ala Val His Arg Leu Gly Ser Phe225 230 235 240Gly Gln Gln Asp Leu Ser Gly Gly Asp Thr Thr Cys His Pro Leu His                245 250 255Ser Glu Tyr Val Tyr Thr Asp Ser Leu Phe Tyr Met Asp His Lys Ser            260 265 270Ala Lys Lys Leu Leu Asp Phe Tyr Glu Ser Val Gly Pro Leu Asn Cys        275 280 285Glu Ile Asp Ala Tyr Gly Asp Phe Leu Gln Ala Leu Gly Pro Gly Ala    290 295 300Thr Ala Glu Tyr Thr Lys Asn Thr Ser His Val Thr Lys Glu Glu Ser305 310 315 320His Leu Leu Asp Met Arg Gln Lys Ile Phe His Leu Leu Lys Gly Thr                325 330 335Pro Leu Asn Val Val Val Leu Asn Asn Ser Arg Phe Tyr His Ile Gly            340 345 350Thr Thr Glu Glu Tyr Leu Leu His Phe Thr Ser Asn Gly Ser Leu Gln        355 360 365Ala Glu Leu Gly Leu Gln Ser Ile Ala Phe Ser Val Phe Pro Asn Val    370 375 380Pro Glu Asp Ser His Glu Lys Pro Cys Val Ile His Ser Ile Leu Asn385 390 395 400Ser Gly Cys Cys Val Ala Pro Gly Ser Val Val Glu Tyr Ser Arg Leu                405 410 415Gly Pro Glu Val Ser Ile Ser Glu Asn Cys Ile Ile Ser Gly Ser Val            420 425 430Ile Glu Lys Ala Val Leu Pro Pro Cys Ser Phe Val Cys Ser Leu Ser        435 440 445Val Glu Ile Asn Gly His Leu Glu Tyr Ser Thr Met Val Phe Gly Met    450 455 460Glu Asp Asn Leu Lys Asn Ser Val Lys Thr Ile Ser Asp Ile Lys Met465 470 475 480Leu Gln Phe Phe Gly Val Cys Phe Leu Thr Cys Leu Asp Ile Trp Asn                485 490 495Leu Lys Ala Met Glu Glu Leu Phe Ser Gly Ser Lys Thr Gln Leu Ser            500 505 510Leu Trp Thr Ala Arg Ile Phe Pro Val Cys Ser Ser Leu Ser Glu Ser        515 520 525Val Ala Ala Ser Leu Gly Met Leu Asn Ala Ile Arg Asn His Ser Pro    530 535 540Phe Ser Leu Ser Asn Phe Lys Leu Leu Ser Ile Gln Glu Met Leu Leu545 550 555 560Cys Lys Asp Val Gly Asp Met Leu Ala Tyr Arg Glu Gln Leu Phe Leu                565 570 575Glu Ile Ser Ser Lys Arg Lys Gln Ser Asp Ser Glu Lys Ser            580 585 590

权利要求:
Claims (48)
[1" claim-type="Currently amended] A composition consisting of an antibody molecule that specifically binds to CD20 and has an N-glycosidic conjugated sugar chain in an Fc region, wherein the composition comprises a total of N-glycosidic conjugated sugar chains that bind to an Fc region included in the composition. And a cell producing an antibody composition in which the proportion of sugar chains in which fucose is not bonded to N-acetylglucosamine at the sugar chain reducing end is 20% or more.
[2" claim-type="Currently amended] The cell according to claim 1, wherein the sugar chain to which the fucose is not bound is a sugar chain that is not α-bonded on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end of the fucose. .
[3" claim-type="Currently amended] The method according to claim 1 or 2, wherein the activity of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose or 1 of the fucose on 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminus. Cells in which the activity of enzymes involved in sugar chain modification to which the stomach binds is reduced or deleted.
[4" claim-type="Currently amended] The cell according to claim 3, wherein the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose is an enzyme selected from the group consisting of the following (a), (b) and (c).
(a) GMD (GDP-mannose 4,6-dehydratase);
(b) Fx (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase);
(c) GFPP (GDP-beta-L-fucose pyrophosphorylase).
[5" claim-type="Currently amended] The cell of Claim 4 whose GMD is a protein which the DNA of following (a) or (b) codes.
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41;
(b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41 under stringent conditions and which encodes a protein having GMD activity.
[6" claim-type="Currently amended] The cell according to claim 4, wherein the GMD is a protein selected from the group consisting of the following (a), (b) and (c).
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 61;
(b) the amino acid sequence represented by SEQ ID NO: 61, wherein the at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has GMD activity;
(c) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 61 and having GMD activity.
[7" claim-type="Currently amended] The cell according to claim 4, wherein Fx is a protein encoded by DNA having the following (a) or (b).
(a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48;
(b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48 under stringent conditions and which encodes a protein having Fx activity.
[8" claim-type="Currently amended] The cell according to claim 4, wherein Fx is a protein selected from the group consisting of the following (a), (b) and (c).
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 62;
(b) the amino acid sequence represented by SEQ ID NO: 62, wherein the protein comprises one or more amino acid sequences deleted, substituted, inserted and / or added, and further has Fx activity;
(c) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 62 and having Fx activity.
[9" claim-type="Currently amended] The cell according to claim 4, wherein the GFPP is a protein encoded by the DNA (a) or (b) below.
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51;
(b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51 under stringent conditions and which encodes a protein having GFPP activity.
[10" claim-type="Currently amended] The cell according to claim 4, wherein GFPP is a protein selected from the group consisting of the following (a), (b) and (c).
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 63;
(b) the amino acid sequence represented by SEQ ID NO: 63, wherein the protein comprises one or more amino acid sequences deleted, substituted, inserted and / or added, and also has GFPP activity;
(c) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 63 and having GFPP activity.
[11" claim-type="Currently amended] 4. The enzyme according to claim 3, wherein the enzyme involved in the sugar chain modification in which the first position of the fucose binds to 6 of the N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is α-1,6-fucosyl. Cells that are transferases.
[12" claim-type="Currently amended] The cell according to claim 11, wherein the α-1,6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a), (b), (c) and (d).
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1;
(b) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2;
(c) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity;
(d) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[13" claim-type="Currently amended] 12. The α-1,6-fucosyltransferase is selected from the group consisting of the following (a), (b), (c), (d), (e) and (f). Cells that are proteins.
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 23;
(b) a protein consisting of the amino acid sequence represented by SEQ ID NO: 24;
(c) the amino acid sequence represented by SEQ ID NO: 23, wherein at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has α-1,6-fucosyltransferase activity ;
(d) the amino acid sequence represented by SEQ ID NO: 24, wherein at least one amino acid consists of an amino acid sequence deleted, substituted, inserted and / or added, and also has α-1,6-fucosyltransferase activity ;
(e) a protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 23, and having α-1,6-fucosyltransferase activity;
(f) A protein consisting of an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 24 and having α-1,6-fucosyltransferase activity.
[14" claim-type="Currently amended] The activity according to any one of claims 3 to 13, wherein the activity of the enzyme is lowered by a method selected from the group consisting of the following (a), (b), (c), (d) and (e): Deleted cells.
(a) a method of gene disruption targeting a gene of an enzyme;
(b) a method of introducing a dominant negative of the gene of the enzyme;
(c) a method of introducing a mutation for an enzyme;
(d) techniques for inhibiting the transcription or translation of an enzyme's genes;
(e) A method of selecting a strain that is resistant to a lectin that recognizes a sugar chain structure in which the 6th position of N-acetylglucosamine and the 1st position of fucose at the N-glycoside-linked sugar chain reduction terminal are recognized.
[15" claim-type="Currently amended] The lectin according to any one of claims 1 to 14, wherein at least the 6th position of N-acetylglucosamine at the N-glycosidic linkage sugar-reducing end and the 1st position of fucose are resistant to lectin that recognizes an α-linked sugar chain structure. cell.
[16" claim-type="Currently amended] The cell according to any one of claims 1 to 15, wherein the cell is a cell selected from the group consisting of the following (a) to (j).
(a) CHO cells derived from Chinese hamster ovary tissue;
(b) rat myelomer cell line YB2 / 3HL. P2. G11. 16Ag. 20 cells;
(c) mouse myelomer cell line NSO cells;
(d) mouse myelomer cell line SP2 / O-Ag 14 cells;
(e) Syrian hamster kidney tissue derived BHK cells;
(f) monkey COS cells;
(g) hybridoma cells producing antibodies;
(h) human leukemia cell line Namalba cells;
(i) embryonic stem cells;
(j) fertilized egg cells.
[17" claim-type="Currently amended] A composition consisting of an antibody molecule that specifically binds to CD20 and has an N-glycosidic conjugated sugar chain in an Fc region, wherein the composition comprises a total of N-glycosidic conjugated sugar chains that bind to an Fc region included in the composition. A transgenic non-human animal or plant, into which a gene encoding the antibody molecule is introduced, which produces an antibody composition in which the proportion of sugar chains in which fucose is not bonded to N-acetylglucosamine at the sugar chain reducing end is 20% or more; descendants.
[18" claim-type="Currently amended] The sugar chain according to claim 17, wherein the sugar chain to which the fucose is not bonded is a sugar chain which is not α-bonded on 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end of the fucose. Genic non-human animals or plants, or descendants thereof.
[19" claim-type="Currently amended] 19. The position of the fucose according to claim 17 or 18, wherein the activity of an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the 6th position of the fucose is above 6 of N-acetylglucosamine at the N-glycosidic linking sugar chain reducing end. A transgenic non-human animal or plant, or a progeny thereof, whose genome has been modified so that the activity of enzymes involved in α-binding sugar chain modification is reduced.
[20" claim-type="Currently amended] 19. The fucose 1st position according to claim 17 or 18, wherein the gene of the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose or the 6th position of the fucose is on the 6th position of N-acetylglucosamine at the N-glycoside-linked sugar chain reducing end. A transgenic non-human animal or plant, or a progeny thereof, in which a gene of an enzyme involved in α-binding sugar chain modification is knocked out.
[21" claim-type="Currently amended] The transgenic non-human human according to claim 19 or 20, wherein the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose is an enzyme selected from the group consisting of the following (a), (b) and (c). Animals or plants, or their descendants.
(a) GMD (GDP-mannose 4,6-dehydratase);
(b) Fx (GDP-keto-6-deoxymannose, 3,5-epimerase, 4-reductase);
(c) GFPP (GDP-beta-L-fucose pyrophosphorylase).
[22" claim-type="Currently amended] The transgenic non-human animal or plant, or the progeny thereof according to claim 21, wherein GMD is a protein encoded by DNA having the following (a) or (b).
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41;
(b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 41 under stringent conditions and which encodes a protein having GMD activity.
[23" claim-type="Currently amended] The transgenic non-human animal or plant, or its progeny according to claim 21, wherein Fx is a protein encoded by DNA which is (a) or (b) below.
(a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48;
(b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48 under stringent conditions and which encodes a protein having Fx activity.
[24" claim-type="Currently amended] The transgenic non-human animal or plant, or the progeny thereof according to claim 21, wherein GFPP is a protein encoded by DNA which is (a) or (b) below.
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51;
(b) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 51 under stringent conditions and which encodes a protein having GFPP activity.
[25" claim-type="Currently amended] The enzyme according to claim 19 or 20, wherein the enzyme involved in sugar chain modification wherein the first position of the fucose is on α- 6 of N-acetylglucosamine at the N-glycoside-linked sugar chain-reducing terminal is α-1,6- Fucosyltransferase, a transgenic non-human animal or plant, or progeny thereof.
[26" claim-type="Currently amended] The trans | transformer of Claim 25 whose alpha-1, 6- fucosyl transferase is a protein which the DNA chosen from the group which consists of the following (a), (b), (c), and (d) codes for. Genic non-human animals or plants, or descendants thereof.
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1;
(b) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2;
(c) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
(d) DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and which encodes a protein having α-1,6-fucosyltransferase activity.
[27" claim-type="Currently amended] 27. The transgenic non-human animal of any one of claims 17-26, wherein the transgenic non-human animal is an animal selected from the group consisting of cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys and rabbits. Or plants, or their descendants.
[28" claim-type="Currently amended] The cell according to any one of claims 1 to 16, wherein the antibody molecule is a molecule selected from the group consisting of the following (a), (b), (c) and (d).
(a) human antibodies;
(b) humanized antibodies;
(c) an antibody fragment comprising the Fc region of (a) or (b);
(d) a fusion protein having the Fc region of (a) or (b).
[29" claim-type="Currently amended] The cell of claim 1, wherein the class of antibody molecules is IgG.
[30" claim-type="Currently amended] The method according to any one of claims 1 to 16, 28 or 29, wherein the complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 of the light chain variable region of the antibody molecule are SEQ ID NOs: 5, 6, 7, and / or a cell comprising the amino acid sequence of complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 of heavy chain variable region represented by SEQ ID NOs: 8, 9, 10.
[31" claim-type="Currently amended] The amino acid according to any one of claims 1 to 16, 28, 29, or 30, wherein the light chain variable region of the antibody molecule is SEQ ID NO: 12, and / or the heavy chain variable region is the amino acid of SEQ ID NO: 14. A cell comprising a sequence.
[32" claim-type="Currently amended] The transgenic non-human animal or plant according to any one of claims 17 to 27, wherein the antibody molecule is a molecule selected from the group consisting of the following (a), (b), (c) and (d). , Or descendants thereof.
(a) human antibodies;
(b) humanized antibodies;
(c) an antibody fragment comprising the Fc region of (a) or (b);
(d) a fusion protein having the Fc region of (a) or (b).
[33" claim-type="Currently amended] 33. The transgenic non-human animal or plant, or progeny thereof, according to any one of claims 17 to 27 or 32, wherein the class of the antibody molecule is IgG.
[34" claim-type="Currently amended] 34. The composition of any one of claims 17 to 27, 32, or 33, wherein the complementarity determining region 1, complementarity determining region 2, and complementarity determining region 3 of the light chain variable region of the antibody molecule are SEQ ID NOs: 5, 6, respectively. A transgenic non-human animal or plant, wherein the complementarity determining region 1, the complementarity determining region 2, the complementarity determining region 3 of the 7, and / or heavy chain variable regions each comprise an amino acid sequence represented by SEQ ID NOs: 8, 9, and 10, or descendants.
[35" claim-type="Currently amended] 35. The amino acid according to any one of claims 17 to 27, 32, 33 or 34, wherein the light chain variable region of the antibody molecule is SEQ ID NO: 12, and / or the heavy chain variable region is amino acid set forth in SEQ ID NO: 14. Transgenic non-human animal or plant, or progeny thereof, comprising a sequence.
[36" claim-type="Currently amended] An antibody composition produced by the cell according to any one of claims 1 to 16 or 28 to 31.
[37" claim-type="Currently amended] The antibody composition produced by the animal or plant which raised and bred the transgenic non-human animal or plant of any one of Claims 17-27, or 32-35, or its offspring.
[38" claim-type="Currently amended] A composition consisting of an antibody molecule that specifically binds to CD20 and has an N-glycoside-binding complex sugar chain in an Fc region, and among all N-glycoside complex sugar chains that bind to an Fc region included in the composition, The antibody composition which has the ratio of the sugar chain in which the fucose is not bonded to N-acetylglucosamine of the sugar chain reduction terminal is 20% or more.
[39" claim-type="Currently amended] The antibody according to claim 38, wherein the sugar chain to which the fucose is not bonded is a sugar chain that is not α-bonded onto 6 of N-acetylglucosamine at the N-glycosidic bond complex sugar chain reducing end of the fucose. Composition.
[40" claim-type="Currently amended] The antibody composition according to claim 38, wherein the antibody molecule is a molecule selected from the group consisting of the following (a), (b), (c) and (d).
(a) human antibodies;
(b) humanized antibodies;
(c) an antibody fragment comprising the Fc region of (a) or (b);
(d) a fusion protein having the Fc region of (a) or (b).
[41" claim-type="Currently amended] 41. The antibody composition of any one of claims 38-40, wherein the class of antibody molecules is IgG.
[42" claim-type="Currently amended] 42. The composition of any one of claims 38-41, wherein the complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 of the light chain variable region of the antibody molecule are SEQ ID NOs: 5, 6, 7, and / or heavy chain variable, respectively. Complementarity determining region 1, complementarity determining region 2, complementarity determining region 3 of the region comprises an amino acid sequence represented by SEQ ID NO: 8, 9, 10, respectively.
[43" claim-type="Currently amended] 43. The antibody composition of any one of claims 38-42, wherein the light chain variable region of the antibody molecule comprises SEQ ID NO: 12 and / or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14.
[44" claim-type="Currently amended] A cell according to any one of claims 1 to 16 or 28 to 31 is cultured in a medium, and the antibody composition according to any one of claims 36 or 38 to 43 in a culture. A method of producing the antibody composition, which comprises the step of producing and accumulating lysine and extracting the antibody composition from the culture.
[45" claim-type="Currently amended] A tissue obtained by breeding a transgenic non-human animal or plant according to any one of claims 17 to 27 or 32 to 35, or a progeny thereof, and acquiring tissue or body fluid from the animal or plant. Or the method of manufacturing this antibody composition containing the process of extracting the antibody composition as described in any one of Claim 36 or 38-43 from a body fluid.
[46" claim-type="Currently amended] A pharmaceutical comprising the antibody composition according to any one of claims 36 to 43 as an active ingredient.
[47" claim-type="Currently amended] A therapeutic drug for a CD20-related disease, comprising as an active ingredient the antibody composition according to any one of claims 36 to 43.
[48" claim-type="Currently amended] 48. The therapeutic agent according to claim 47, wherein the CD20 related disease is cancer or an immune disease.

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同族专利:

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公开号 | 公开日

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EP1469065A1|2004-10-20|

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US20040093621A1|2004-05-13|

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EP1469065A4|2006-05-10|

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WO2003055993A1|2003-07-10|

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AU2002360029A1|2003-07-15|

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JPWO2003055993A1|2005-05-12|

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CN1617922A|2005-05-18|

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CA2471647A1|2003-07-10|

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CN100471948C|2009-03-25|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-12-25|Priority to JP2001392753

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2001-12-25|Priority to JPJP-P-2001-00392753

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2002-04-09|Priority to JP2002106948

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2002-04-09|Priority to JPJP-P-2002-00106948

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2002-11-01|Priority to JPJP-P-2002-00319975

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2002-11-01|Priority to JP2002319975

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2002-12-25|Application filed by 교와 핫꼬 고교 가부시끼가이샤

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2002-12-25|Priority to PCT/JP2002/013534

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2004-08-11|Publication of KR20040071254A

优先权:

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申请号 | 申请日 | 专利标题

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JP2001392753|2001-12-25|

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JPJP-P-2001-00392753|2001-12-25|

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JP2002106948|2002-04-09|

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JPJP-P-2002-00106948|2002-04-09|

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JP2002319975|2002-11-01|

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JPJP-P-2002-00319975|2002-11-01|

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PCT/JP2002/013534|WO2003055993A1|2001-12-25|2002-12-25|Composition of antibody specifically binding to cd20|