HK1114878B

HK1114878B - Methods for producing polypeptides by regulating polypeptide association

Info

Publication number: HK1114878B
Application number: HK08109972.5A
Authority: HK
Inventors: 井川智之; 角田浩行
Original assignee: 中外制药株式会社
Priority date: 2005-03-31
Filing date: 2006-03-31
Publication date: 2014-09-12

Description

Method for producing polypeptides by modulating polypeptide association

Technical Field

The present invention relates to a method for producing a polypeptide by regulating intra-or intermolecular association (association), a polypeptide for regulating intra-or intermolecular association, a pharmaceutical composition containing the polypeptide as an active ingredient, and the like.

Background

Antibodies have been attracting attention as pharmaceuticals because of their high stability in blood and few side effects. Included are bispecific antibodies that recognize both antigens simultaneously. MDX-210, which has been clinically tested at present, is an IgG type bispecific antibody which retargets (retargeting) monocytes expressing Fc γ RI and the like to cancer cells expressing HER-2/neu (see non-patent document 1). Antibodies are usually prepared by genetic recombination techniques. The technology is as follows: the antibody is produced by cloning a DNA encoding an antibody protein from an antibody-producing cell such as a hybridoma or an antibody-producing sensitized lymphocyte or a phage library providing an antibody gene, inserting the DNA into an appropriate vector, and introducing the vector into a host cell. When an IgG-type bispecific antibody is produced by gene recombination, genes constituting the H chain and L chain of two target iggs, and a total of four genes, are introduced into cells, and the antibody is secreted by co-expression. In the above expression, when the constitutive genes of wild-type H chain and L chain are expressed, covalent bonding between both H chains or non-covalent bonding between H chain and L chain occurs randomly, and thus the ratio of the bispecific antibody of interest becomes very small. Specifically, only 1 of 10 antibodies was the target bispecific antibody, and the production efficiency was reduced. The production efficiency of the target antibody is lowered, and not only the purification of the target antibody is hindered, but also the unevenness such as lot-to-lot variation is increased, resulting in an increase in the production cost.

As a method for improving the production efficiency of bispecific antibodies, it has been reported that IgG having a heterogeneous combination of H chains is preferentially secreted by substituting an amino acid in the CH3 region of the H chain of IgG (see patent document 1 and non-patent documents 2 and 3). The method comprises the following specific steps: the amino acid side chains present in the CH3 region of one H-chain are substituted with larger side chains (knob), and the amino acid side chains present in the CH3 region of the other H-chain are substituted with smaller side chains (hole), whereby the knob is disposed within the void to promote the formation of a heterologous H-chain and inhibit the formation of a homologous H-chain. There are also reports of: the same "protrusions" and "voids" are used for the interface where the H chain variable region (hereinafter referred to as VH) and the L chain variable region (hereinafter referred to as VL) are associated with each other (see non-patent document 4). According to the report of Zhe et al, by substituting two (four in total in both strands) amino acids present at the interface of VH and VL, the formation of a heterologous molecule was promoted with 1.28-fold efficiency (wild type: 72%; variant: 92%). In addition, by substituting one amino acid (two for both strands), the same efficiency as the wild type is obtained. However, it cannot be said that the method of providing knob (protuberance) and hole (void) in VH and VL sufficiently promotes the formation of a heterologous molecule.

Patent document 1: international publication No. 96/27011

Non-patent document 1: segal DM et al, Current Opinion in Immunology, 1999, Vol.11, p.558-562

Non-patent document 2: ridgway JB et al, Protein Engineering, 1996, Vol.9, pp.617-621

Non-patent document 3: merchant AM et al, Nature Biotechnology, 1998, Vol.16, p.677-681

Non-patent document 4: zhe Z et al, Protein Science 1997, vol.6, p.781-788

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for regulating association of polypeptides, an associated polypeptide, and a method for producing the polypeptide. As one embodiment of the present invention, it is intended to provide a method for producing a bispecific antibody with high efficiency by regulating the association of VH and VL at the interface. Another object is to provide a process for the preparation of another conformational isomer of sc (fv)2 with high efficiency.

Means for solving the problems

The present inventors have selected VH and VL of an antibody as polypeptides subject to association modulation, and have conducted intensive studies on methods that can modulate the association of VH and VL as described above.

As a result, it was found that: by substituting an amino acid present at the interface between VH and VL with a charged amino acid, association of VH and VL can be suppressed, enabling formation of a heterologous molecule with higher efficiency than the above-described method using knob and hole.

Surprisingly, according to the method of the present invention, a heterologous molecule can be formed with high efficiency by substituting only one amino acid each present at the interface of VH and VL (two amino acids in total in VH and VL). In addition, from the viewpoint of antigenicity, it is preferable that the substitution of amino acids is small. According to one embodiment of the present invention, a heterologous molecule can be formed with high efficiency by substituting only one amino acid present at the interface between VH and VL.

That is, according to the findings of the present inventors and the like, the association of VH and VL can be modulated. The invention is useful not only for modulating the association of VH and VL, but also for modulating the association between any polypeptide.

The present inventors have also confirmed that: the bispecific antibody obtained by the association-modulating method of the present invention remains functional in nature.

As described above, the present inventors have succeeded in developing a method capable of modulating the association between arbitrary polypeptides, and have completed the present invention.

The present invention relates to a method for regulating polypeptide association and a polypeptide for regulating association, and a method for producing the polypeptide, and more specifically, the present invention provides the following [1] to [97 ]:

a method for producing a mutant polypeptide having a mutation in an amino acid residue forming an interface within the polypeptide to regulate the association of the polypeptide, the method comprising: (a) modifying nucleic acid encoding amino acid residues from the original nucleic acid to inhibit association within the polypeptide, said amino acid residues forming an interface within the polypeptide; (b) culturing a host cell to express the nucleic acid; (c) recovering the polypeptide from the host cell culture.

A method of making a heteromultimer (heteromultimer) having a mutation in an amino acid residue forming an interface between polypeptides to modulate heteromultimer association, comprising: (a) modifying nucleic acid encoding amino acid residues from the original nucleic acid to inhibit association between polypeptides, said amino acid residues forming an interface between polypeptides; (b) culturing a host cell to express the nucleic acid; (c) recovering the heteromultimer from the host cell culture.

[3] [1] A method for inhibiting the association of polypeptides forming more than one conformer by modifying a nucleic acid encoding amino acid residues forming an interface within the polypeptide from an original nucleic acid, in a polypeptide capable of forming 2 or more conformers.

[4] [2] A method of modifying a nucleic acid encoding amino acid residues forming an interface between polypeptides forming more than one multimer from an original nucleic acid to inhibit association between polypeptides forming the multimers in a heteromultimer capable of forming 2 or more multimers.

[5] The method of [1] or [2], wherein the modification of step (a) means: the original nucleic acid is modified so as to introduce mutations of amino acid residues into the interface, so that 2 or more amino acid residues forming the interface are charged with the same kind of charge.

[6] [5] the method wherein the introduced amino acid residue is glutamic acid (E).

[7] [5] the method wherein the introduced amino acid residue is aspartic acid (D).

[8] [5] the method wherein the introduced amino acid residue is lysine (K).

[9] [5] the method wherein the introduced amino acid residue is arginine (R).

[12] [11] the method according to (1), wherein the amino acid residue to be introduced is glutamic acid (E).

[13] The method of [11], wherein the introduced amino acid residue is aspartic acid (D).

[14] [11] the method wherein the introduced amino acid residue is lysine (K).

[15] [11] the method wherein the introduced amino acid residue is arginine (R).

[16] [11] the method wherein the introduced amino acid residue is histidine (H).

[17] The method of [1] or [2], wherein the interface of the polypeptide is formed by the heavy chain variable region and the light chain variable region of the antibody.

[18] The method of [1] or [2], wherein the interface of the polypeptide is formed by 2 or more variable regions.

[19] The method of [1] or [2], wherein the interface of the polypeptide is formed by a heavy chain constant region and a light chain constant region of the antibody.

[20] The method of [1] or [2], wherein the interface of the polypeptide is formed by 2 or more heavy chain constant regions.

[21] The method of [1], wherein the polypeptide is a single-chain polypeptide in which 2 or more variable regions and 2 or more variable regions of a light chain are linked by a linker.

[22] The method of [2], wherein the heteromultimer is a multispecific antibody comprising 2 or more variable regions and 2 or more variable regions of a light chain.

[23] The method of [22], wherein the heteromultimer is a bispecific antibody.

[24] A polypeptide mutant or heteromultimer produced by the method of [1] or [2 ].

[25] A mutant polypeptide comprising a modification of amino acid residues forming an interface within the original polypeptide to inhibit association within said polypeptide.

[26] A heteromultimer comprising a modification of amino acid residues forming an interface between original polypeptides to inhibit association between said polypeptides.

[27] [25] the polypeptide mutant wherein the original polypeptide is capable of forming 2 or more conformational isomers.

[28] [26] the heteromultimer, wherein the original polypeptide is capable of forming 2 or more multimers.

[29] The polypeptide mutant of [25] or the heteromultimer of [26], wherein the modification of the amino acid residues forming the polypeptide interface is: mutations of amino acid residues are introduced into the interface such that 2 or more amino acid residues forming the interface carry the same charge.

[30] [29] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is glutamic acid (E).

[31] [29] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is aspartic acid (D).

[32] [29] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is lysine (K).

[33] [29] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is arginine (R).

[34] [29] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is histidine (H).

[35] The polypeptide mutant of [25] or the heteromultimer of [26], wherein the modification of the amino acid residues forming the polypeptide interface is: mutations of amino acid residues are introduced into the interface, and the amino acid residues forming the hydrophobic core present at the interface are charged amino acid residues.

[36] [35] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is glutamic acid (E).

[37] [35] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is aspartic acid (D).

[38] [35] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is lysine (K).

[39] [35] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is arginine (R).

[40] [35] the polypeptide mutant or heteromultimer, wherein the introduced amino acid residue is histidine (H).

[41] The polypeptide mutant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by a heavy chain variable region and a light chain variable region of the antibody.

[42] The polypeptide mutant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by 2 or more variable regions.

[43] The polypeptide mutant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by a heavy chain constant region and a light chain constant region of the antibody.

[44] The polypeptide mutant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by 2 or more heavy chain constant regions.

[45] [25] the polypeptide mutant wherein the polypeptide is a single-chain polypeptide in which 2 or more variable regions and 2 or more variable regions of a light chain are linked by a linker.

[46] The heteromultimer of [26], wherein the heteromultimer is a multispecific antibody comprising 2 or more variable regions and 2 or more variable regions of a light chain.

[47] [46] the heteromultimer, wherein the heteromultimer is a bispecific antibody.

[48] A composition comprising the polypeptide mutant of [25] or the heteromultimer of [26] and a pharmaceutically acceptable carrier.

[49] A nucleic acid encoding the polypeptide mutant of [25] or the heteromultimer of [26 ].

[50] A host cell comprising the nucleic acid of [49 ].

[51] [25] the polypeptide mutant of [25] or the heteromultimer of [26], which comprises: a step of culturing the host cell of [50] and a step of recovering the polypeptide from the cell culture.

[52] A method of modulating polypeptide association, the method comprising modifying amino acid residues forming an interface within an original polypeptide to inhibit association within the polypeptide.

[53] A method of modulating heteromultimer association, the method comprising modifying amino acid residues forming an interface between the original polypeptides to inhibit association between the polypeptides.

[54] [52] A method for inhibiting the association of polypeptides forming more than one conformer by modifying amino acid residues forming an interface within the polypeptide in a polypeptide capable of forming 2 or more conformers.

[55] [53] A method for inhibiting the association between polypeptides forming more than one type of multimer by modifying the amino acid residues forming the interface between the polypeptides in a heteromultimer capable of forming 2 or more types of multimers.

[56] [52] or [53], wherein the modification of the amino acid residue forming the polypeptide interface is: mutations of amino acid residues are introduced into the interface such that 2 or more amino acid residues forming the interface carry the same charge.

[57] [56] the method according to (1), wherein the amino acid residue to be introduced is glutamic acid (E).

[58] [56] the method wherein the introduced amino acid residue is aspartic acid (D).

[59] [56] the method wherein the introduced amino acid residue is lysine (K).

[60] [56] the method wherein the introduced amino acid residue is arginine (R).

[61] [56] the method wherein the introduced amino acid residue is histidine (H).

[62] [52] or [53], wherein the modification of the amino acid residue forming the polypeptide interface is: mutations of amino acid residues are introduced into the interface, and the amino acid residues forming the hydrophobic core present at the interface are charged amino acid residues.

[63] [62] the method according to (1), wherein the amino acid residue to be introduced is glutamic acid (E).

[64] The method of [62], wherein the introduced amino acid residue is aspartic acid (D).

[65] [62] the method wherein the introduced amino acid residue is lysine (K).

[66] [62] the method wherein the introduced amino acid residue is arginine (R).

[67] [62] the method wherein the introduced amino acid residue is histidine (H).

[68] The method of [52] or [53], wherein the polypeptide interface is formed by a heavy chain variable region and a light chain variable region of the antibody.

[69] The method of [52] or [53], wherein the polypeptide interface is formed of 2 or more variable regions.

[70] The method of [52] or [53], wherein the polypeptide interface is formed by a heavy chain constant region and a light chain constant region of the antibody.

[71] The method of [52] or [53], wherein the polypeptide interface is formed by 2 or more heavy chain constant regions.

[72] The method of [52], wherein the polypeptide is a single chain polypeptide in which 2 or more variable regions and 2 or more variable regions of a light chain are linked by a linker.

[73] The method of [53], wherein the heteromultimer is a multispecific antibody comprising 2 or more variable regions and 2 or more variable regions of a light chain.

[74] The method of [73], wherein the heteromultimer is a bispecific antibody.

[75] An antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid residues of the following (1) and (2) bear the same charge:

(1) the heavy chain variable region comprises amino acid residues corresponding to SEQ ID NO: 6 (glutamic acid) at position 39 in the amino acid sequence of;

(2) the variable region of the light chain comprises amino acid residues corresponding to SEQ ID NO: 8 (glutamic acid) at position 44 in the amino acid sequence of (1).

[76] An antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid residues of the following (1) and (2) bear the same charge:

(1) the heavy chain variable region comprises amino acid residues corresponding to SEQ ID NO: 6 (leucine) at position 45 of the amino acid sequence;

(2) the variable region of the light chain comprises amino acid residues corresponding to SEQ ID NO: 8 (proline) at position 50 in the amino acid sequence of seq id No. 8.

[77] An antibody comprising a heavy chain variable region and a light chain variable region, wherein any one of the following (1) or (2) is a charged amino acid residue:

(1) the heavy chain variable region comprises amino acid residues corresponding to SEQ ID NO: 6 (leucine) at position 45 in the amino acid sequence of seq id no;

[78] [75] or [76], wherein the amino acid residue having the same charge is selected from the amino acid residues contained in any one of the following groups (a) or (b):

(a) glutamic acid (E) and aspartic acid (D);

(b) lysine (K), arginine (R) and histidine (H).

[0105] [79][77]The antibody according to (1), wherein the charged amino acid residues are: glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) or histidine (H).

[80] The antibody according to any one of [75] to [77], wherein the polypeptide is a single-chain polypeptide in which 2 or more variable regions of the multiple chain and 2 or more variable regions of the light chain are linked by a linker.

[81] The antibody according to any one of [75] to [77], wherein the polypeptide is a multispecific antibody comprising 2 or more heavy chain variable regions and 2 or more light chain variable regions.

[82] The antibody of [81], wherein the polypeptide is a bispecific antibody.

[83] A composition comprising the antibody of any one of [75] to [77] and a pharmaceutically acceptable carrier.

[84] A nucleic acid encoding the polypeptide constituting an antibody according to any one of [75] to [77 ].

[85] A host cell comprising the nucleic acid of [84 ].

[86] [75] to [77], which comprises: a step of culturing the host cell of [85] and a step of recovering the polypeptide from the cell culture.

[87] An antibody comprising 2 or more heavy chain CH3 regions, wherein amino acid residues in the 1 st heavy chain CH3 region selected from one to three groups of amino acid residues shown in the following (1) to (3) bear the same charge:

(1) amino acid residues contained in the CH3 region of the heavy chain, which are 356 to 439 according to EU numbering;

(2) amino acid residues contained in the CH3 region of the heavy chain, which are located at positions 357 and 370 according to EU numbering;

(3) heavy chain CH3 region contains amino acid residues at positions 399 and 409 according to EU numbering.

[88] [87] an antibody having one to three sets of amino acid residues in the 2 nd heavy chain CH3 region: (i) amino acid residues selected from the group consisting of amino acid residues (1) to (3) of [87 ]; (ii) the group of amino acid residues corresponding to [87] in (1) to (3); and (iii) bears an opposite charge to the corresponding amino acid residue in the heavy chain 1 CH3 region.

[89] [87] the antibody, wherein the amino acid residues having the same charge are selected from the group consisting of amino acid residues contained in any one of the following groups (a) and (b):

(a) glutamic acid (E) and aspartic acid (D);

(b) lysine (K), arginine (R) and histidine (H).

[90] [87] the antibody, wherein the above-mentioned 1 st heavy chain CH3 region and 2 nd heavy chain CH3 region are crosslinked by disulfide bond.

[91] The antibody of [87], which is an antibody having 2 or more heavy chain constant regions.

[92] The antibody of [87], which is a multispecific antibody comprising 2 or more heavy chain variable regions and 2 or more light chain variable regions.

[93] The antibody of [92], which is a bispecific antibody.

[94] A composition comprising the antibody of [87] and a pharmaceutically acceptable carrier.

[95] A nucleic acid encoding the antibody-constituting polypeptide of [87 ].

[96] A host cell comprising the nucleic acid of [95 ].

[97] [87] A method for producing an antibody, which comprises: a step of culturing the host cell of [96] and a step of recovering the polypeptide from the cell culture.

Brief Description of Drawings

FIG. 1 is a diagram modeling the Fv region of humanized SB04, with (A) representing amino acid residues H39 and L38 at the VH and VL interface and (B) representing amino acid residues H45 and L44 at the VH and VL interface.

FIG. 2 is a photograph showing the results of evaluating the association between H chain and L chain of modified antibodies to H39 and L38. The results show that: in all modified antibodies, the association ratio with the wild-type antibody was increased compared to the target antibody.

Description of electrophoretic lanes:

m: labeling with molecular weight;

1: humanized XB12H chain (Q) + humanized XB12L chain (Q);

2: humanized XB12H chain (Q) + humanized SB04L chain (Q);

3: wild type: humanized XB12H chain (Q) + humanized XB12L chain (Q) + humanized SB04L chain (Q);

4: d, modification: humanized XB12H chain (D) + humanized XB12L chain (Q) + humanized SB04L chain (D);

5: e, modification: humanized XB12H chain (E) + humanized XB12L chain (Q) + humanized SB04L chain (E);

6: r variants: humanized XB12H chain (R) + humanized XB12L chain (Q) + humanized SB04L chain (R);

7: k modification: humanized XB12H chain (K) + humanized XB12L chain (Q) + humanized SB04L chain (K).

FIG. 3 shows the results of evaluation of the clotting activity of the modified antibodies of H39 and L38. The results showed that the bispecific antibody obtained by modifying the XB12H chain (H39) and SB04L chain (L38) to Glu had a clotting activity equivalent to or higher than that of the wild type.

FIG. 4 shows the results of evaluating the factor IXa binding activity of the modified antibodies of H39 and L38. The results show that all the modified antibodies have the same binding activity as the wild type.

FIG. 5 shows the results of evaluation of factor X binding activity of modified antibodies to H39 and L38. The results show that all the modified antibodies have the same binding activity as the wild type.

FIG. 6 is a photograph showing the results of evaluating the association between the H chain and L chain of a modified antibody of L44. The results show that: in all modified antibodies, the association ratio with the wild-type antibody was increased compared to the target antibody.

Description of electrophoretic lanes:

1: wild type: humanized XB12H chain + humanized XB12L chain (P) + humanized SB04L chain (P);

2: d, modification: humanized XB12H chain + humanized XB12L chain (P) + humanized SB04L chain (D);

3: e, modification: humanized XB12H chain + humanized XB12L chain (P) + humanized SB04L chain (E);

4: r variants: humanized XB12H chain + humanized XB12L chain (P) + humanized SB04L chain (R);

5: k modification: humanized XB12H chain + humanized XB12L chain (P) + humanized SB04L chain (K).

FIG. 7 shows the results of evaluation of the clotting activity of the modified antibody of L44. The results show that all modified antibodies have higher clotting activity than the wild type.

FIG. 8 shows the results of evaluation of the factor X binding activity of the modified antibody of L44. The results show that all the modified antibodies have the same binding activity as the wild type.

FIG. 9 is a photograph showing the results of evaluating the association of H chains and L chains of modified antibodies to H39, L38, and L44. The results show that: in all modified antibodies, the association ratio with the wild-type antibody was increased compared to the target antibody.

Description of electrophoretic lanes:

1: wild type: humanized XB12H chain (H39: Q) + humanized XB12L chain (L38: Q) + humanized SB04L chain (L38: Q, L44: P);

2: e + D variants: humanized XB12H chain (H39: E) + humanized XB12L chain (L38: Q) + humanized SB04L chain (L38: E, L44: D);

3: e + E variants: humanized XB12H chain (H39: E) + humanized XB12L chain (L38: Q) + humanized SB04L chain (L38: E, L44: E);

4: e + R variants: humanized XB12H chain (H39: E) + humanized XB12L chain (L38: Q) + humanized SB04L chain (L38: E, L44: R);

5: e + K variants: humanized XB12H chain (H39: E) + humanized XB12L chain (L38: Q) + humanized SB04L chain (L38: E, L44: K); m: and (4) marking molecular weight.

FIG. 10 shows the results of evaluation of the clotting activity of the modified antibodies of H39, L38, and L44. The results showed that the bispecific antibody modified with the XB12H chain (H39) and SB04L chain (L38, L44) had a clotting activity equivalent to or higher than that of the wild type.

FIG. 11 shows the results of evaluating the factor IXa binding activity of the modified antibodies of H39, L38, and L44. The results show that all the modified antibodies have the same binding activity as the wild type.

Fig. 12 is a schematic diagram showing an example of the structure of sc (fv)2 having two types of heavy chain variable regions (VH1 and VH2) and two types of light chain variable regions (VL1 and VL 2). (a) The sc (fv)2 has a structure in which two conformational isomers represented by (b) mainly exist.

FIG. 13 shows the results of using cation exchange chromatography to separate the conformers u2-wz4, Peak 1 and Peak 2.

FIG. 14 shows peptide diagrams of Peak 1 and Peak 2 separated by cation exchange chromatography.

FIG. 15 is a photograph showing the results of SDS-PAGE for reducing u2-wz4 by subtilisin treatment, i.e., peak 1 and peak 2, and u2-wz4 before separation. The structure of the resulting band is shown on the right.

FIG. 16 shows the difference in degradation patterns between a bivalent scFv and a single-chain antibody by subtilisin-limited proteolysis due to the structural difference between the two. When of bivalent scFv structure, low molecular weight fragments surrounded by dotted lines are produced.

FIG. 17 shows the results of gel filtration chromatography of u2-wz4 conformers, Peak 1 and Peak 2, u2-wz4 before isolation, after limited proteolysis by subtilisin. The elution position of the low molecular weight peak is indicated by an arrow.

FIG. 18 shows the results of gel filtration chromatography of u2-wz4, variant v1 and variant v3 after purification on a column immobilized with MG10-GST fusion protein.

FIG. 19 shows the results of cation exchange chromatography for u2-wz4, variant v1, and variant v 3.

FIG. 20 is a photograph showing the results of isoelectric focusing on u2-wz4, u2-wz4 purification peak 1, u2-wz4 purification peak 2, variant v1, and variant v 3.

FIG. 21 shows the results of analysis of u2-wz4 purification peak 1, u2-wz4 purification peak 2, variant v1, and variant v3 by gel filtration chromatography after limited proteolysis.

FIG. 22 shows the results of evaluation of TPO-like agonist activity of u2-wz4 purified peak 1, u2-wz4 purified peak 2, variant v1 and variant v 3.

FIG. 23 shows the results of DSC analysis of u2-wz4 purification peak 1, u2-wz4 purification peak 2, variant v1 and variant v 3.

FIG. 24 shows the monomer residue ratios of u2-wz4 purified peak 1, u2-wz4 purified peak 2, variant v1 and variant v3 in the thermal acceleration test, which were analyzed by gel filtration chromatography.

FIG. 25 shows the conformational isomer content ratios of u2-wz4 purification peak 1, u2-wz4 purification peak 2, variant v1 and variant v3 as analyzed by cation exchange chromatography in the thermal acceleration test.

FIG. 26 shows the results of evaluation of clotting activity of a humanized bispecific antibody (humanized A69(hA 69-PFL)/humanized B26(hB 26-PF)/humanized BBA (hAL-AQ)). The results showed that the antibody had a clotting activity equivalent to or higher than that of the chimeric bispecific antibody.

Fig. 27 is a conceptual diagram of modification of the H chain constant region to increase the efficiency of formation of bispecific antibodies. The modification site numbers are given by EU numbering (Kabat EA et al, 1991.Sequences of proteins of immunologicalcalest. NIH).

Figure 28 shows a chromatogram of an IEX analysis of humanized bispecific antibody (IgG4 type) modified with CH3 interface.

FIG. 29 shows the formation ratio of A-Homo, BiAb and B-Homo by IEX analysis of a humanized bispecific antibody (IgG4 type) having a CH3 interface modified.

Figure 30 shows the monomer residual rate after 60 ℃ -1 week thermal acceleration test of BiAb purified from humanized bispecific antibody (IgG4 type) modified CH3 interface.

FIG. 31 shows the results of evaluation of the clotting activity of a humanized bispecific antibody (IgG4 type) in which the CH3 interface was modified. The results showed that the antibody had coagulation activity equivalent to that of the unmodified bispecific antibody.

FIG. 32 shows the formation ratios obtained by IEX analysis of A-Homo, BiAb, and B-Homo of a humanized bispecific antibody (IgG1 type) in which the CH3 interface has been modified.

Best Mode for Carrying Out The Invention

The present invention relates to a method of modulating the association of polypeptides or the association of heteromultimers composed of polypeptides.

In a first aspect, the present invention provides a method of modulating polypeptide association, the method comprising modifying amino acid residues forming an interface within an original polypeptide to inhibit association within the polypeptide.

The polypeptide of the present invention generally refers to peptides and proteins having a length of about 10 amino acids or more. The polypeptide is generally of biological origin, but is not particularly limited, and may be, for example, a polypeptide including an artificial sequence. May be any of natural or synthetic polypeptides, recombinant polypeptides and the like. Furthermore, fragments of the above polypeptides are also included.

In the present invention, the association of polypeptides means, in other words, the state of interaction of, for example, 2 or more polypeptide regions.

In the present invention, "adjusting the association" means adjusting to a desired association state, more specifically, means not forming an undesired association within the polypeptide.

In the present invention, the "interface" generally refers to an association plane at the time of association (interaction). The interfacial amino acid residues generally refer to one or more amino acid residues contained in the polypeptide region subject to the association, more preferably amino acid residues that are close to and participate in the interaction when associated. The interaction specifically comprises: and the formation of hydrogen bonds, electrostatic interactions, salt bridges between adjacent amino acid residues during association.

In the present invention, the "amino acid residues forming the interface" specifically means amino acid residues contained in the polypeptide region constituting the interface. Examples of polypeptide regions that constitute the interface are: antibodies, ligands, receptors, substrates, and the like, have polypeptide regions that selectively bind within or between molecules. Specifically, the antibodies include: heavy chain variable region, light chain variable region, and the like.

In the method of the present invention, "modification" of an amino acid residue specifically means: the substitution of the original amino acid residue with another amino acid residue, deletion of the original amino acid residue, addition of a new amino acid residue, and the like, preferably means the substitution of the original amino acid residue with another amino acid residue.

In the present invention, the "polypeptide" is preferably a polypeptide capable of forming 2 or more conformational isomers. The conformers are typically referred to as: proteins having the same amino acid sequence but different steric structure (three-dimensional structure). Generally, conformers often differ in at least one of chemical or physical properties.

In a preferred embodiment, the present invention relates to a method for preferentially (efficiently) obtaining a desired conformer from among 2 or more conformers that can be present. That is, as one aspect, it relates to a method for modifying amino acid residues forming an interface within a polypeptide to inhibit association between polypeptides forming more than one conformer, in a polypeptide capable of forming 2 or more conformers.

For example, first to fourth polypeptide regions are present within a polypeptide, and when any two of the above regions can associate, it is believed that the following three conformational isomers can exist predominantly: (1) the first and second polypeptide regions are associated, and the third and fourth polypeptide regions are associated; (2) the first and third polypeptide regions are associated, and the second and fourth polypeptide regions are associated; (3) the first and fourth polypeptide regions are associated, and the second and third polypeptide regions are associated.

In the above-described situation, when a polypeptide (conformational isomer) that associates as in (1) is to be preferentially obtained, for example, the amino acid residues present at the interface between the first, third, or fourth polypeptide regions may be modified so as to inhibit the association of the first polypeptide region with the third and fourth polypeptide regions.

The method of the invention also relates to a method of modulating the association of heteromultimers comprising modifying the amino acid residues forming the interface between the original polypeptides to inhibit the association between the polypeptides.

In the present invention, "heteromultimer (heteromultimer)" refers to a polymer of a protein which is composed of a plurality of polypeptides and which can associate with each other. In more detail, a "heteromultimer" has at least a first polypeptide and a second polypeptide, where the second polypeptide refers to a molecule having an amino acid sequence that differs from the first polypeptide by at least one amino acid residue. The heteromultimer is not particularly limited, but preferably has binding specificity to at least two different ligands, antigens, receptors, substrates, or the like. In addition to the "heterodimer" formed by the first and second polypeptides, other polypeptides may also be present in the heteromultimer. That is, the "heteromultimer" of the present invention is not limited to a heterodimer, but includes, for example, a heterotrimer, a heterotetramer, and the like.

The preferred embodiment of the above method is that in the heteromultimer capable of forming 2 or more multimers, the amino acid residues forming the interface between the polypeptides are modified to inhibit the association between the polypeptides forming more than one multimer.

For example, in a protein multimer composed of first to fourth polypeptides, when any two of the above polypeptides can be associated, the following multimers may be mainly present: (1) multimers of the first and second polypeptides in association and the third and fourth polypeptides in association; (2) a multimer in which the first and third polypeptides are associated and the second and fourth polypeptides are associated; or (3) a multimer in which the first and fourth polypeptides are associated and the second and third polypeptides are associated.

In the above-mentioned situation, when a multimer that associates as in (1) is to be obtained preferentially, for example, the amino acid residues contained in the first, third or fourth polypeptide may be modified so as to inhibit the association of the first polypeptide with the third and fourth polypeptides.

In a preferred embodiment of the method of the invention for modulating polypeptide association, the method comprises: for example, a method of modifying amino acid residues that form an interface of a polypeptide, the method characterized by: mutations of amino acid residues are introduced into the interface such that 2 or more amino acid residues forming the interface carry the same charge.

In the above method, it is considered that the association between the amino acid residues is suppressed by modifying 2 or more amino acid residues participating in the association at the interface to have the same charges as each other and utilizing repulsion between the charges.

Therefore, in the above method, the modified amino acid residues are preferably 2 or more amino acid residues which are close to each other when they are associated with each other in the polypeptide region forming the interface.

The amino acid residues that are close to each other in association can be identified, for example, by analyzing the steric structure of the polypeptides and examining the amino acid sequence of the polypeptide region that forms an interface when the polypeptides associate with each other. Amino acid residues that are close to each other on the interface are preferred targets for "modification" in the method of the present invention.

Among amino acids, charged amino acids are known. Generally, positively charged amino acids (positively charged amino acids) are known as follows: lysine (K), arginine (R), histidine (H). Negatively charged amino acids (negatively charged amino acids) are known: aspartic acid (D), glutamic acid (E), and the like. Therefore, in the present invention, amino acids having the same charge preferably refer to positively charged amino acids or negatively charged amino acids.

In the method of the present invention, all the amino acid residues to be mutated are preferably modified to have the same charge, but are not necessarily limited thereto. For example, when a plurality of amino acid residues are introduced by modification, a small number of uncharged amino acid residues may be included in the amino acid residues.

In the method of the present invention, the number of amino acid residues to be modified is not particularly limited, but for example, when modifying the variable region of an antibody, it is preferable that as few amino acid residues as possible be modified so as not to decrease the binding activity of the resulting antibody to an antigen and not to increase the antigenicity thereof. As described in the examples below, the methods of the invention can modulate association by modifying at least one or both of the amino acid residues that are close at the interface. The "small number" is, for example, a number of about 1 to 10, preferably about 1 to 5, more preferably about 1 to 3, and most preferably 1 or 2.

In a preferred embodiment of the present invention, the amino acid residues introduced (subjected to modification) by the modification are preferably all amino acid residues selected from the above positively charged amino acids, or all amino acid residues selected from the above negatively charged amino acids.

In the present invention, the amino acid residues to be introduced are preferably: glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), histidine (H).

Another preferred embodiment of the present invention is: when the amino acid residue (X) forming the interface in the original (unmodified) polypeptide has already been charged, the amino acid residue which is adjacent to and opposite to the amino acid residue (X) in association is modified so as to be the same amino acid residue as the amino acid residue (X) (or an amino acid residue having the same charge). In this embodiment, only one of the amino acid residues forming the interface may be modified.

In a preferred embodiment of the association modulating method of the present invention, there is included a method of modifying amino acid residues forming an interface of a polypeptide, the method being characterized in that: mutations of amino acid residues are introduced into the interface, and the amino acid residues forming the hydrophobic core present at the interface are charged amino acid residues.

Generally, a "hydrophobic core" refers to a moiety formed by aggregation of the side chains of hydrophobic amino acids inside an associated polypeptide. Hydrophobic amino acids include: alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, and the like. In addition, amino acid residues other than hydrophobic amino acids (e.g., tyrosine) may also participate in the formation of the hydrophobic core. The hydrophobic core and the hydrophilic surface together serve as a driving force for promoting the association of the water-soluble polypeptide, and the side chain of the hydrophilic amino acid is exposed to the outside on the hydrophilic surface. If two hydrophobic amino acids of different domains are present on the surface of the molecule and exposed to water molecules, the entropy increases and the free energy increases. Thus, the two domains associate with each other to reduce free energy and achieve stabilization, while the hydrophobic amino acids of the interface are buried inside the molecule, forming a hydrophobic core.

When polypeptide association occurs, hydrophobic amino acids forming a hydrophobic core are modified to charged polar amino acids, thereby suppressing formation of the hydrophobic core, and as a result, association of polypeptides is suppressed.

One skilled in the art can recognize the presence or absence of a hydrophobic core and a formation site (region), etc., by analyzing the amino acid sequence of a desired polypeptide. Namely, the present invention relates to an association regulating method characterized in that: modifying amino acid residues on the interface capable of forming a hydrophobic core to charged amino acid residues.

In the above method, the charged amino acid residues preferably include: glutamic acid (E), aspartic acid (D), lysine (K), arginine (R), histidine (H).

The association regulating method of the present invention can be used as a method for preferentially obtaining (producing) a target antibody in the production of an antibody or an antibody fragment, a polypeptide having an antibody-like activity, or the like.

In the present invention, the term "antibody" is used in its broadest sense as long as it indicates the desired biological activity, and includes: monoclonal antibodies, polyclonal antibodies, antibody mutants (chimeric antibodies, humanized antibodies, small molecule antibodies (also including antibody fragments), multispecific antibodies, etc.). In the present invention, the "antibody" may be any of a polypeptide or a heteromultimer. Preferred antibodies are: monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments. In the present invention, when the above-mentioned antibody is obtained (produced), the association regulating method of the present invention can be appropriately employed.

In the present invention, a "multispecific antibody" (in the present specification, the same as a "multispecific antibody") refers to an antibody capable of specifically binding to a plurality of different epitopes. That is, a multispecific antibody is an antibody having specificity for at least two different epitopes, and includes, in addition to antibodies that recognize different antigens, antibodies that recognize different epitopes on the same antigen. (e.g., a multispecific antibody recognizes different domains that make up a heterologous receptor when the antigen is a heterologous receptor; or multiple sites of a monomeric antigen when the antigen is a monomer). Typically, such molecules bind to two antigens (bispecific antibodies, which are meant to be the same as "dual-specific antibodies" in this specification), but may be specific for more than two (e.g., three) antigens.

In the present invention, "antibody" includes: an antibody having an amino acid sequence modified by further performing amino acid substitution, deletion, addition and/or insertion, or chimerization or humanization, etc. on the above antibody. Amino acid substitutions, deletions, additions and/or insertions, and modifications of the amino acid sequence such as humanization or chimerization, can be performed by methods known to those skilled in the art. Similarly, when the antibody of the present invention is prepared as a recombinant antibody, the amino acid sequence thereof may be modified by substituting, deleting, adding and/or inserting amino acids into the variable region and the constant region of the antibody to be used, or by chimerization or humanization.

The antibody of the present invention may be derived from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. Furthermore, for example, a modified antibody obtained by substituting an amino acid sequence, such as a chimeric antibody, particularly a humanized antibody, may be used. The antibody may be any antibody such as an antibody modification product, an antibody fragment, or a small molecule antibody to which various molecules are bound.

"chimeric antibody" refers to an antibody prepared by combining sequences derived from different animals. Examples include: an antibody comprising the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody. Methods for producing chimeric antibodies are known, and for example, a chimeric antibody can be obtained by ligating a DNA encoding a V region of an antibody and a DNA encoding a C region of a human antibody, inserting the ligated product into an expression vector, and introducing the vector into a host.

The "humanized antibody", also called a reshaped (reshaped) human antibody, is an antibody obtained by grafting Complementarity Determining Regions (CDRs) of an antibody derived from a mammal other than a human, for example, a mouse antibody, to CDRs of a human antibody. Methods for identifying CDRs are well known (Kabat et al, Sequence of Proteins of Immunological Interest (1987), national institute of Health, Bethesda, Md.; Chothia et al, Nature (1989) 342: 877). In addition, a general gene recombination method is also known (see European patent application publication Nos. EP125023 and WO 96/02576). Thus, a DNA encoding an antibody in which the CDRs of a mouse antibody and the Framework Regions (FRs) of a human antibody are ligated can be obtained by, for example, determining the CDRs of a mouse antibody by a known method, and then a humanized antibody can be produced by using a usual expression vector system. The DNA can be synthesized by PCR using a plurality of oligonucleotides as primers, and the oligonucleotides are prepared so as to have overlapping regions in the terminal regions of the CDR and the FR (see the method described in WO 98/13388). The FRs of the human antibody connected via the CDRs are selected so that the CDRs form a good antigen-binding site. Amino acids in the FR of the antibody variable region may be modified as necessary to form suitable antigen-binding sites in the CDRs of the reshaped human antibody (Sato et al, Cancer Res. (1993) 53.851-6). Among the amino acid residues in the modifiable FR are: a moiety that directly binds to an antigen by non-covalent bonds (Amit et al, Science (1986) 233: 747-53), a moiety that affects or acts on the CDR structure (Chothia et al, J.mol.biol. (1987) 196: 901-17), and a moiety involved in VH-VL interaction (EP 239400A).

In the present invention, when the antibody is a chimeric antibody or a humanized antibody, a C region derived from a human antibody is preferably used as the C region of the antibody. For example, C γ 1, C γ 2, C γ 3, C γ 4; in the L chain, C.kappa.and C.lambda.can be used. In order to improve the stability of the antibody or the production thereof, the human antibody C region may be modified as necessary. In the present invention, the chimeric antibody preferably contains a variable region derived from an antibody derived from a mammal other than human and a constant region derived from a human antibody. The humanized antibody preferably contains CDRs of an antibody derived from a mammal other than human and FRs and C regions derived from a human antibody. As for the variable regions, the descriptions are summarized in (3) -3. The constant region derived from a human antibody has an amino acid sequence unique to each of IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE equivalents. In the present invention, the constant region used for the humanized antibody may be a constant region of an antibody belonging to any isotype. The constant region of human IgG is preferably used, but not limited thereto. The FR derived from a human antibody to be used for a humanized antibody is also not particularly limited, and may be of an antibody of any isotype.

In the present invention, the variable region and the constant region of the chimeric antibody and the humanized antibody may be modified by deletion, substitution, insertion, addition or the like, as long as they show the binding specificity of the original antibody.

Chimeric antibodies and humanized antibodies obtained using human-derived sequences have been reduced in antigenicity in humans and are therefore useful when administered to humans for therapeutic purposes and the like.

Small molecule antibodies are useful as antibodies, both from the viewpoint of their in vivo kinetic properties and from the viewpoint of their ability to be produced at low cost using e.coli, plant cells, and the like.

The antibody fragment is a small molecule antibody. Small molecule antibodies also include antibodies having an antibody fragment as a portion thereof. In the present invention, the small molecule antibody is not particularly limited in its structure, production method, and the like, as long as it has an antigen binding ability. Among small-molecule antibodies, there are also antibodies with higher activity than full-length antibodies (Orita et al, Blood (2005) 105: 562-566). In the present specification, the "antibody fragment" is not particularly limited as long as it is a part of a full-length antibody (for example, full-length IgG), and preferably includes a heavy chain variable region (VH) or a light chain variable region (VL). Examples of preferred antibody fragments are: fab, F (ab ') 2, Fab', Fv, etc. The amino acid sequence of VH or VL in an antibody fragment may be modified by substitution, deletion, addition and/or insertion. Also, a part of VH and VL may be absent as long as antigen binding ability is maintained. For example, in the above antibody fragments, "Fv" is the smallest antibody fragment that includes the entire antigen recognition site and binding site. "Fv" is a dimer in which one VH and one VL are strongly bound by a non-covalent bond (VH-VL dimer). An antigen-binding site is formed on the surface of the VH-VL dimer by 3 complementary Chain Determining Regions (CDRs) of each variable region. The 6 CDRs confer an antigen binding site on the antibody. However, even a single variable region (or half of an Fv comprising only 3 antigen-specific CDRs) has the ability to recognize and bind antigen, although with a lower affinity than the full binding site. Accordingly, molecules smaller than the above-described Fv are also included in the antibody fragment of the present invention. The variable regions of antibody fragments may also be chimeric and humanized.

The small molecule antibody preferably comprises VH and VL. Examples of small molecule antibodies are: antibody fragments of Fab, Fab ', F (ab') 2 and Fv, as well as scFv (single chain Fv) made using The antibody fragments (Huston et al, Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Plickthun "The Pharmacology of Monoclonal Antibodies Pharmacology)" vol.113, Reaenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)), diabody (diabody) (Holly et al, Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP 407; WO 93/11161; Johnson et al, Methodin Enzymology (1991) 203: 88-98; Holliger et al, Protein Engineering (9: 299) 1996; Strength et al, Methodin Enzymin. 201: 26: 201, 1996) 201: 26: 32: 51: 201, Austin et al, (1996) 201: 51: 201, Austin et al, Austin Engineering et al, Protein Engineering (1996) 2000: 55: 201, Austin J.11: 14, Austin, Vol. J. 201, Shin, 2000, Three-chain antibodies (triabody) (Journal of Immunological Mtthouses (1999) 231: 177-89) and tandem diabodies (Cancer Rrsearch (2000) 60: 4336-41), and the like.

Antibody fragments can be obtained by treating an antibody with an enzyme such as a protease such as papain, pepsin, etc. (see Morimoto et al, J. biochem. Biophys. methods (1992) 24: 107-17; Brennan et al, Science (1985) 229: 81). The antibody fragment may be prepared by gene recombination based on the amino acid sequence of the antibody fragment.

Small molecule antibodies having a modified structure of an antibody fragment can be constructed using an antibody fragment obtained by enzyme treatment or gene recombination. Alternatively, genes encoding the entirety of the small molecule antibody can also be constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, e.g., Co et al, J.Immunol. (1994) 152: 2968-76; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497 515; Lamoyi, Methods Enzymol. (1986) 121: 652-63; Rousseaux et al, Methods Enzymol. (1986) 121: 663-9; Bird and Warner, Trends Biotechnol. (1991) 9: 132-7).

The "scFv" is a single-chain polypeptide in which two variable regions are linked via a linker or the like as necessary. The scFv comprises two variable regions, typically one VH and one VL, but may also comprise two VH or two VL. Typically, scFv polypeptides contain a linker between the VH and VL domains through which the paired portions of VH and VL necessary for antigen binding are formed. In general, in order to form a pair portion between VH and VL within the same molecule, the linker connecting VH and VL is generally made to be a peptide linker of 10 amino acids or more in length. However, the linker of the scFv in the present invention is not limited to the peptide linker as long as it does not inhibit the formation of scFv. For an overview of scFv, reference may be made to Pluckthun "The Pharmacology of monoclonal antibodies" Vol 113 (eds. Rosenburg and Moore, Springer Verlag, NY, p. 269-315 (1994)).

"diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Diabodies are dimers composed of two polypeptide chains, each of which is linked in the same chain by a linker as short as one cannot bind to each other, e.g., around 5 residues. The VL and VH encoded on the same polypeptide chain form a dimer because the linker between them is short and cannot form a single-chain V-region fragment (fragment), and thus the diabody has two antigen-binding sites. In this case, if a combination of VLa-VHb and VLb-VHa linked by a linker of about 5 residues is expressed simultaneously with respect to VL and VH of two different epitopes (a, b), it is secreted as a bispecific Db. In this case, the two different epitopes may be epitopes at two different sites on the same antigen, or epitopes at two sites on two different antigens, respectively.

Diabodies contain two molecules of scFv and therefore four variable regions, and as a result, diabodies have two antigen binding sites. In order to form a diabody, a linker of about 5 amino acids is generally used when the linker connecting VH and VL in each scFv molecule is a peptide linker, unlike the case of an scFv that does not form a dimer. However, the linker of the scFv that forms the diabody is not limited to the peptide linker as long as it does not interfere with the expression of the scFv and the formation of the diabody.

Preferred polypeptides or heteromultimers for use in the methods of the invention are, for example: a polypeptide or heteromultimer having an antibody heavy chain variable region and a light chain variable region. In a preferred embodiment of the present invention, more preferably the polypeptide or heteromultimer of the present invention is a method of modulating the association of 2 or more variable regions and 2 or more variable regions of a light chain. The polypeptide or heteromultimer preferably recognizes 2 or more epitopes, including, for example, multispecific antibodies.

In the present invention, a bispecific antibody, for example, can be more preferably used as the multispecific antibody.

That is, a preferred embodiment of the present invention relates to, for example, a method for modulating the association of a bispecific antibody composed of two heavy chain variable regions (a first heavy chain and a second heavy chain) and two light chain variable regions (a first light chain and a second light chain).

In further detail, in a preferred embodiment of the present invention, the "bispecific antibody" is defined such that the "first heavy chain" refers to one of the two H chains forming the antibody, and the second H chain refers to the other H chain different from the first H chain. That is, one of the two H chains may be the first H chain, and the other may be the second H chain. Similarly, a "first light chain" refers to one of the two L chains forming the bispecific antibody, and the second L chain refers to another L chain different from the first L chain, either of which may be the first L chain and the other may be the second L chain. Typically, the first L chain and the first H chain are derived from the same antibody that recognizes a rice antigen (or epitope), and the second L chain and the second H chain are also derived from the same antibody that recognizes a certain antigen (or epitope). Here, the L chain-H chain formed by the first H chain-L chain is symmetrical as a first pair, and the L chain-H chain formed by the second H chain-L chain is symmetrical as a second pair. The antigen (or epitope) used to make the antibody from the second pair is preferably different from the antigen (or epitope) used to make the antibody from the first pair. That is, the first and second pairs may recognize the same antigen, but preferably recognize different antigens (or epitopes). In this case, the H chain and the L chain of the first pair and the second pair preferably have amino acid sequences different from each other. When the first pair and the second pair recognize different epitopes, the first pair and the second pair may recognize completely different antigens or may recognize different sites (different epitopes) on the same antigen. One pair recognizes an antigen such as a protein, a peptide, a gene, or a sugar, and the other pair recognizes a cytotoxic substance such as a radioactive substance, a chemotherapeutic agent, or a cell-derived toxin. However, when preparing an antibody having a pair formed by a combination of specific H chains and L chains, the specific H chains and L chains can be arbitrarily determined as the first pair and the second pair.

It should be noted that the above-mentioned "bispecific antibody" is not necessarily limited to an antibody comprising two heavy chains and two light chains, and may be, for example, an antibody having a structure in which two heavy chain variable regions and two light chain variable regions are linked to form a single chain (e.g., sc (fv) 2).

In the method of the present invention, a gene encoding an H chain or an L chain of an antibody before mutation introduction (which may be referred to as "the antibody of the present invention" herein) may be obtained by using a known sequence or a method known to those skilled in the art. For example, the antibody can be obtained from an antibody library or a gene encoding an antibody can be cloned from a hybridoma producing a monoclonal antibody.

There are many known antibody libraries, and methods for producing antibody libraries are also known, and those skilled in the art can obtain appropriate antibody libraries. For example, with respect to phage antibody libraries, reference may be made to Clackson et al, Nature 1991, 352: 624-8; marks et al, j.mol.biol.1991, 222: 581-97; water houses et al, Nucleic Acids Res.1993, 21: 2265-6; griffiths et al, EMBO J.1994, 13: 3245-60; vaughan et al, Nature Biotechnology 1996, 14: 309-14 and JP-A No. 10-504970. Further, known methods such as a method using eukaryotic cells as a library (WO 95/15393) and a ribosome-indicating method can be used. Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, a phage capable of binding to an antigen can be selected by expressing the variable region of a human antibody as a single-chain antibody (scFv) on the surface of the phage by phage display. Analysis of the genes of the selected phage allows the determination of the DNA sequence encoding the variable region of the human antibody that binds to the antigen. Once the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector can be prepared based on the sequence to obtain a human antibody. The above-mentioned methods are well known and reference may be made to WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, WO 95/15388.

A method for obtaining a gene encoding an antibody from a hybridoma, basically, using a known technique, using a desired antigen or a cell expressing a desired antigen as a sensitizing antigen, immunizing the antigen according to a usual immunization method, fusing the obtained immunocyte with a known parent cell by a usual cell fusion method, screening a monoclonal antibody-producing cell (hybridoma) by a usual screening method, synthesizing a cDNA for an antibody variable region (V region) from the mRNA of the obtained hybridoma using a reverse transcriptase, and ligating the cDNA with a DNA encoding a desired antibody constant region (C region).

More specifically, the sensitizing antigen used for obtaining the above-mentioned antibody genes encoding H chain and L chain includes both a complete antigen having immunogenicity and an incomplete antigen having no immunogenicity, including a hapten and the like, without particularly being limited to the following examples. For example, a full-length protein or a partial peptide of the target protein, or the like can be used. It is known that a substance composed of a polysaccharide, a nucleic acid, a lipid, or the like can be used as an antigen, and the antigen of the antibody of the present invention is not particularly limited. The antigen can be prepared by a method known to those skilled in the art, for example, a method using baculovirus (for example, WO 98/46777). Hybridomas can be prepared, for example, by the method of Milstein et al (G.Kohler and C.Milstein, Methods enzymol.1981, 73: 3-46). When the immunogenicity of the antigen is low, the antigen can be immunized by combining it with an immunogenic macromolecule such as albumin. If necessary, the antigen may be bound to another molecule to become a soluble antigen. When a transmembrane molecule such as a receptor is used as an antigen, an extracellular region portion of the receptor may be used as a fragment, or a cell expressing the transmembrane molecule on the cell surface may be used as an immunogen.

The antibody-producing cell can be obtained by immunizing an animal with the above-mentioned appropriate sensitizing antigen. Alternatively, antibody-producing lymphocytes are immunized in vitro to become antibody-producing cells. Various mammals can be used as the animal to be immunized, but rodents, lagomorphs, and primates are generally used. Examples include: rodents such as mice, rats, hamsters, and the like; rabbit-shaped mesh such as rabbit; primates such as cynomolgus monkey, rhesus monkey, baboon, chimpanzee, etc. Furthermore, transgenic animals having a human antibody gene bank (reporteries) are also known, and human antibodies can be obtained by using these animals (see WO 96/34096; Mendez et al, nat. Genet.1997, 15: 146-56). Instead of using the above transgenic animal, a desired human antibody having an antigen-binding activity can also be obtained by sensitizing human lymphocytes with a desired antigen or cells expressing a desired antigen in vitro and fusing the sensitized lymphocytes with human myeloma cells such as U266 (see Japanese patent publication No. Hei 1-59878). Alternatively, a desired human antibody can be obtained by immunizing a transgenic animal having a complete human antibody gene bank with a desired antigen (see WO93/12227, WO92/03918, WO94/02602, WO96/34096, WO 96/33735).

Immunization of animals can be carried out as follows: the sensitizing antigen is diluted and suspended in a Phosphate Buffered Saline (PBS) or a physiological saline as appropriate, mixed with an adjuvant as necessary, emulsified, and then injected into the abdominal cavity or the subcutaneous space of an animal to immunize. After that, the sensitizing antigen mixed with Freund's incomplete adjuvant is preferably administered several times every 4 to 21 days. The production of antibodies can be confirmed by measuring the titer of the antibody of interest in the serum of an animal by a conventional method.

Hybridomas can be prepared as follows: antibody-producing cells obtained from animals immunized with a desired antigen (Monoclonal Antibodies): Principles and Practice, Academic Press, 1986, 59-103) or lymphocytes are fused with myeloma cells using commonly used fusion agents (e.g., polyethylene glycol). Hybridoma cells are cultured and proliferated as necessary, and the binding specificity of the antibody produced by the hybridoma is measured by a known analysis method such as immunoprecipitation, Radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Thereafter, the antibody-producing hybridomas are subcloned by a limiting dilution method or the like as necessary, and the target specificity, affinity, or activity of the antibodies are determined.

Next, the gene encoding the selected antibody can be cloned from a hybridoma or an antibody-producing cell (sensitized lymphocyte or the like) using a probe capable of specifically binding to the antibody (for example, an oligonucleotide complementary to a sequence encoding the antibody constant region or the like). Cloning from mRNA can also be performed by RT-PCR. Immunoglobulins are classified as: IgA, IgD, IgE, IgG and IgM five different classes. Moreover, these classes are divided into several subclasses (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1, IgA-2, etc.). In the present invention, the H chain and L chain used for the production of an antibody are not particularly limited, and may be derived from an antibody belonging to any of the above-mentioned classes and subclasses, and IgG is particularly preferable.

Here, the genes encoding the H chain and the L chain can also be modified by genetic engineering techniques. For example, an antibody such as a mouse antibody, a rat antibody, a rabbit antibody, a hamster antibody, a goat antibody, or a camel antibody may be appropriately modified to reduce its antigenicity against a human foreign antibody, and a genetically recombinant antibody such as a chimeric antibody or a humanized antibody may be appropriately prepared. The chimeric antibody is an antibody comprising the variable regions of the H chain and L chain of a non-human mammal such as a mouse antibody and the constant regions of the H chain and L chain of a human antibody, and can be produced by ligating a DNA encoding the variable region of a mouse antibody and a DNA encoding the constant region of a human antibody, inserting the ligated DNA into an expression vector, and introducing the resulting vector into a host. Humanized antibodies, also known as reshaped (reshaped) human antibodies, can be synthesized by PCR from a plurality of oligonucleotides that are made to have overlapping portions at the ends of DNA sequences designed to link the Complementarity Determining Regions (CDRs) of non-human mammals, such as mouse antibodies. The resulting DNA is ligated with a DNA encoding a human antibody constant region, inserted into an expression vector, and introduced into a host to produce a humanized antibody (see EP 239400; WO 96/02576). FRs of human antibodies connected via CDRs are selected from those whose complementarity determining regions form a good antigen-binding site. If necessary, amino acids in the framework region of the antibody variable region may be substituted so that the complementarity determining regions of the reshaped human antibody form an appropriate antigen-binding site (K.Sato et al, Cancer Res.1993, 53: 851-.

In addition to the above-mentioned humanization, modification may be made to improve the biological properties of an antibody, such as antigen binding properties. The modification can be carried out by site-specific mutagenesis (see, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutagenesis, cassette mutagenesis, or the like. Generally, an antibody mutant having improved biological properties has 70% or more, more preferably 80% or more, and still more preferably 90% or more (e.g., 95% or more, 97%, 98%, 99%, etc.) amino acid sequence homology and/or similarity with respect to the amino acid sequence of the variable region of the original antibody. In this specification, sequence homology and/or similarity is defined as: the proportion of amino acid residues that are identical (identical residues) or similar (classified as amino acid residues of the same group according to the nature of the amino acid side chains in general) to the original antibody residues after sequence alignment and gap introduction (gap introduction) is performed as necessary to maximize sequence homology. In general, natural amino acid residues are divided into the following groups according to the nature of their side chains: (1) hydrophobicity: alanine, isoleucine, norleucine, valine, methionine, and leucine; (2) neutral hydrophilicity: asparagine, glutamine, cysteine, threonine, and serine; (3) acidity: aspartic acid and glutamic acid; (4) alkalinity: arginine, histidine and lysine; (5) residues that influence chain orientation: glycine and proline; and (6) aromatic: tyrosine, tryptophan and phenylalanine.

In general, a total of 6 complementarity determining regions (hypervariable regions; CDRs) present in the variable regions of the H chain and the L chain interact to form an antigen-binding site of an antibody. Even one of the variable regions is known to have the ability to recognize and bind to an antigen, although it has a lower affinity than when all binding sites are included. Therefore, the antibody gene of the present invention encoding H chain and L chain may encode a fragment portion including each antigen binding site of H chain and L chain, and the polypeptide encoded by the gene may maintain the binding property to a desired antigen.

As described above, the association modulating method of the present invention, for example, can preferentially (efficiently) obtain a desired bispecific antibody. That is, the desired heteromultimeric-bispecific antibody can be efficiently formed from the monomer mixture.

Hereinafter, the case of an IgG type bispecific antibody having two heavy chain variable regions (VH1 and VH2) and two light chain variable regions (VL1 and VL2) will be described in more detail, and other heteromultimers can be similarly used in the method of the present invention.

When a bispecific antibody recognizing one of the epitopes by the first heavy chain variable region (VH1) and the first light chain variable region (VL1) and the other epitope by the second heavy chain variable region (VH2) and the second light chain variable region (VL2) is to be obtained, 10 antibody molecules can be theoretically produced if each of 4 kinds of chains is expressed in the production of the antibody.

In this case, if the adjustment is made to inhibit the association between polypeptides such as VH1 and VL2 and/or VH2 and VL1, the desired antibody molecule can be preferentially obtained.

For example, the association between the VH1 polypeptide and the VL2 polypeptide and/or the VH2 polypeptide and the VL1 polypeptide is suppressed by modifying the amino acid residues forming the interface as described above.

In addition, with the association regulating method of the present invention, it is also possible to regulate the association between heavy chains (VH1 and VH2) or between light chains (VL1 and VL 2).

As mentioned above, the heavy chain variable region is generally composed of 3 CDR regions and FR regions. In a preferred embodiment of the present invention, the amino acid residue to be subjected to "modification" may be appropriately selected from, for example, amino acid residues located in a CDR region or an FR region. In general, amino acid residues in CDR regions are modified, sometimes to reduce binding energy to antigen. Therefore, the amino acid residues to be subjected to "modification" in the present invention are not particularly limited, and are preferably appropriately selected from the amino acid residues located in the FR region.

With respect to the desired polypeptide to be associated by the method of the present invention, the skilled person can appropriately find the kind of amino acid residues which are close on the FR interface when associated.

The sequences of FRs useful as antibody variable regions in organisms such as humans or mice can be obtained appropriately by those skilled in the art using public databases and the like. More specifically, the amino acid sequence information of the FR region can be obtained by the method described in the examples below.

For example, specific examples of amino acid residues that are close to the FR interface during association in bispecific antibodies shown in the following examples are: glutamine (Q) at position 39 (FR2 region) (e.g., position 39 of the amino acid sequence shown in SEQ ID NO: 6) in the heavy chain variable region and glutamine (Q) at position 38 (FR2 region) (e.g., position 44 of the amino acid sequence shown in SEQ ID NO: 8) in the opposite (contacting) light chain variable region. Mention may also be made, where appropriate, of: leucine (L) at position 45 (FR2) on the heavy chain variable region (e.g., position 45 of the amino acid sequence set forth in SEQ ID NO: 6) and proline (P) at position 44 (FR2) on the opposite light chain variable region (e.g., position 50 of the amino acid sequence set forth in SEQ ID NO: 8). Reference is made to Kabat et al (Kabat EA et al, 1991.Sequence of Proteins of immunologica interest. NIH) for the numbering of the above-mentioned sites.

As shown in the following examples, by modifying these amino acid residues and carrying out the method of the present invention, a desired antibody can be preferentially obtained.

It is known that these amino acid residues are highly conserved in human and mouse (J.mol.Recognit.2003; 16: 113-120), and therefore, for the association of VH and VL of antibodies other than those shown in the examples, the association of the variable regions of the antibodies can also be regulated by modifying the amino acid residues corresponding to the above amino acid residues.

That is, in a preferred embodiment, the present invention provides an antibody (polypeptide (e.g., sc (fv)2), heteromultimer (e.g., IgG-type antibody), etc.) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid residues of the following (1) and (2), or (3) and (4) have the same charge.

(1) The heavy chain variable region comprises amino acid residues corresponding to SEQ ID NO: 6 at position 39 in the amino acid sequence described in (6);

(2) the variable region of the light chain comprises amino acid residues corresponding to SEQ ID NO: 8 at amino acid residue position 44 in the amino acid sequence according to [8 ];

(3) the heavy chain variable region comprises amino acid residues corresponding to SEQ ID NO: 6 at position 45 in the amino acid sequence described in (6);

(4) the variable region of the light chain comprises amino acid residues corresponding to SEQ ID NO: 8 in the amino acid sequence of SEQ ID NO. 50.

It is to be noted that, the above-mentioned SEQ ID NO: the amino acid sequence shown in 6 or 8 is used to more specifically exemplify the position of the amino acid residue to be modified in the present invention, and the present invention is not limited to the case where the heavy chain variable region or the light chain variable region is the above amino acid sequence.

As shown in the following examples and FIG. 1, the amino acid residues described in the above (1) and (2), (3) and (4) are close to each other in association. The desired heavy chain variable region or light chain variable region can be appropriately modified by finding the position corresponding to the amino acid residues described in (1) to (4) above by performing homology modeling using commercially available software, etc.

In the above antibody, the "charged amino acid residue" is preferably selected from amino acid residues contained in any one of the following groups (a) and (b), for example.

(a) Glutamic acid (E), aspartic acid (D);

(b) lysine (K), arginine (R), histidine (H).

The present invention also provides an antibody (polypeptide, heteromultimer, etc.) comprising a heavy chain variable region and a light chain variable region, wherein the antibody has a charged amino acid residue as an amino acid residue in any one of the following (3) and (4). The side chains of the amino acid residues shown in the following (3) and (4) can approach each other to form a hydrophobic core.

In the above antibody, the "charged amino acid residue" is preferably, for example: glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) or histidine (H).

In general, the amino acid residues described in the above (1) to (4) are, in humans and mice: (1) glutamine (Q), (2) glutamine (Q), (3) leucine (L), and (4) proline (P). Thus, in a preferred embodiment of the invention, these amino acid residues are subjected to modification (e.g. substitution to charged amino acids). The types of the amino acid residues (1) to (4) are not necessarily limited to the amino acid residues described above, and may be other amino acids corresponding to the amino acids. For example, when human, the light chain variable region is a region corresponding to SEQ ID NO: the amino acid at position 44 in the amino acid sequence of [8] may be, for example, histidine (H). Those skilled in the art can find a nucleotide sequence corresponding to SEQ ID NO: 8, the amino acid residue may be appropriately modified (for example, substituted with a charged amino acid).

The preferred scheme of the invention is as follows: the method for producing the antibody and the method for regulating association of the present invention are characterized in that: modifying the amino acid residues of the above (1) to (4).

Another embodiment of the present invention is a method for modulating the association between heavy chains or between heavy and light chains by introducing electrostatic repulsion at the interface of the constant regions of the heavy or light chains. The amino acid residues that contact each other at the interface of the heavy chain constant region are, for example: regions of 377 bits (356 bits) and 470 bits (439 bits), 378 bits (357 bits) and 393 bits (370 bits), 427 bits (399 bits) and 440 bits (409 bits) corresponding to the CH3 regions. The amino acid residues that contact each other at the interface between the heavy chain constant region and the light chain constant region are, for example: a region of 221 bits (213 bits) corresponding to the CH1 region and 123 bits of the CL region. For the numbering of the constant regions of antibodies, reference is made to the Kabat et al reference (Kabat EA et al, 1991.Sequence of Proteins of Immunological interest. NIH) and the EU numbering of the constant regions of the heavy chain is shown in parentheses.

As shown in the examples below, by modifying the amino acid residues described above and performing the methods of the invention, the association of the heavy chains of an antibody can be modulated to preferentially obtain the desired antibody.

That is, in a preferred embodiment, the present invention provides an antibody which is an antibody comprising 2 or more heavy chain CH3 regions wherein 1 to 3 groups of amino acid residues in the 1 st heavy chain CH3 region have the same charge and are selected from the group of amino acid residues shown in the following (1) to (3), and an Fc region-binding protein (e.g., IgG type antibody, small molecule antibody (AltM et al, FEBS Letters 1999; 454: 90-94), immunoadhesin (non-patent document 2), etc.).

(3) amino acid residues contained in heavy chain CH3 region, located at positions 399 and 409 according to EU numbering;

in a more preferred embodiment, the invention provides an antibody in which one to three sets of amino acid residues in the 2 nd heavy chain CH3 region are: (i) amino acid residues selected from the group consisting of the amino acid residues represented by the above (1) to (3); (ii) the amino acid residues corresponding to the amino acid residue groups represented by (1) to (3) above; and (iii) bears an opposite charge to the corresponding amino acid residue in the heavy chain 1 CH3 region.

As shown in the following examples and FIG. 27, the amino acid residues in the above-mentioned (1) to (3) are close to each other in association. The desired heavy chain CH3 region or heavy chain constant region can be appropriately modified by finding a site corresponding to the amino acid residues described in (1) to (3) above by homology modeling using commercially available software, or the like, by a person skilled in the art.

(a) Glutamic acid (E), aspartic acid (D);

(b) lysine (K), arginine (R), histidine (H).

In the above antibody, "having the same charge" means that, for example, 2 or more amino acid residues each have an amino acid residue contained in any one of the above groups (a) or (b). "oppositely charged" means, for example, that when at least one of 2 or more amino acid residues has an amino acid residue contained in any one of the above-mentioned groups (a) or (b), the remaining amino acid residues have amino acid residues contained in different groups.

In a preferred embodiment, the 1 st heavy chain CH3 region and the 2 nd heavy chain CH3 region of the above antibody may be cross-linked by a disulfide bond.

In the present invention, the amino acid residue subjected to "modification" is not limited to the amino acid residues of the above-mentioned antibody variable region or antibody constant region. With respect to polypeptide mutants or heteromultimers, those skilled in the art can find the amino acid residues forming the interface by performing homologous modeling or the like using commercially available software, and the amino acid residues at that site can be subjected to modification so as to adjust the association.

The process of the present invention may be carried out in combination with known techniques, although this is not essential. For example, in addition to promoting the association of VH1 and VL1 and/or VH2 and VL2 by "modification" of the present invention, the association of VH1 and VL1 and/or VH2 and VL2 may be promoted by substituting amino acid side chains present in the variable region of one H chain into larger side chains (knob, protuberance), substituting amino acid side chains present in the opposite variable region of the other H chain into smaller side chains (hole, void), and disposing the protuberance in the gap, and as a result, the association between polypeptides of VH1 and VL2 and/or VH2 and VL1 may be further inhibited.

The association modulation method of the present invention may suitably be carried out while preferentially (efficiently) obtaining the desired sc (fv) 2. Hereinafter, sc (fv)2 having two types of heavy chain variable regions (H1 and H2) and two types of light chain variable regions (L1 and L2) will be described in more detail as an example.

Generally, sc (fv)2 is a single chain polypeptide in which two heavy chain variable regions (VH1 and VH2) and two light chain variable regions (VL1 and VL2) are linked by a linker. That is, sc (fv)2 is a single-chain small-molecule antibody in which four antibody variable regions are connected by a linker or the like. Generally, sc (fv)2 is a single-chain antibody in which four variable regions, two light chain variable regions and two heavy chain variable regions, are linked by a linker or the like (Hudson et al, J Immunol. Methods 1999; 231: 177-189).

sc (fv)2 can be prepared by a method known to those skilled in the art, for example, by linking an scFv to a linker. The scFv includes VH and VL of the antibody, and these regions are present in one polypeptide chain (for a review of scFv see Pluckthun "the Pharmacology of Monoclonal Antibodies" Vol.113 (edited by Rosenburg and Moore (Springer Verlag, New York) p.269-315, 1994)).

Preferably, the antibody is characterized in that: two VH and two VL are arranged in the order of VH, VL, VH ([ VH ] linker [ VL ] linker [ VH ] linker [ VL ]), with the N-terminal side of the single-chain polypeptide as a base point.

The order of the two VH and the two VL is not particularly limited to the above arrangement, and may be arranged in any order. The following arrangements may also be enumerated:

[ VL ] linker [ VH ] linker [ VL ]

[ VH ] linker [ VL ] linker [ VH ]

[ VH ] linker [ VL ]

[ VL ] linker [ VH ]

[ VL ] linker [ VH ] linker [ VL ] linker [ VH ]

sc (fv)2 may contain amino acid sequences other than the antibody variable region and the linker.

The variable region of the antibody may be the entire length of the variable region, but may be a partial sequence of the variable region as long as the antigen-binding activity is maintained. The amino acid sequence in the variable region may be substituted, deleted, added, inserted, or the like. For example, it may be chimeric or humanized in order to reduce antigenicity.

The linker linking the antibody variable regions may be: any peptide linker that can be introduced by genetic Engineering or chemically synthesized linker (for example, see the linkers disclosed in Protein Engineering, 9(3), 299-305, 1996), etc., and a peptide linker is preferable in the present invention. The length of the peptide linker is not particularly limited, and may be appropriately selected by those skilled in the art according to the purpose, but the preferred length is 12 amino acids or more (the upper limit is not particularly limited, and is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. When three peptide linkers are included in sc (fv)2, all of the peptide linkers may have the same length, or peptide linkers having different lengths may be used.

Examples of peptide linkers are:

Ser

Gly·Ser

Gly·Gly·Ser

Ser·Gly·Gly

Gly·Gly·Gly·Ser

Ser·Gly·Gly·Gly

Gly·Gly·Gly·Gly·Ser

Ser·Gly·Gly·Gly·Gly

Gly·Gly·Gly·Gly·Gly·Ser

Ser·Gly·Gly·Gly·Gly·Gly

Gly·Gly·Gly·Gly·Gly·Gly·Ser

Ser·Gly·Gly·Gly·Gly·Gly·Gly

(Gly·Gly·Gly·Gly·Ser)n

(Ser·Gly·Gly·Gly·Gly)n

examples thereof include [ n is an integer of 1 or more ]. However, the length or sequence of the peptide linker may be appropriately selected by those skilled in the art according to the purpose.

Preferred examples of sc (fv)2 include the following sc (fv) 2:

[ VH ] peptide linker (15 amino acids) [ VL ] peptide linker (15 amino acids) [ VH ] peptide linker (15 amino acids) [ VL ].

Chemically synthesized linkers (chemical cross-linkers) are cross-linkers commonly used for peptide cross-linking, such as: n-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl suberate) (BS)³) Dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethyleneglycol bis (succinimidyl succinate) (EGS), ethyleneglycol bis (sulfosuccinimidyl succinate) (Sulfo-EGS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (Sulfo-DST), bis [2- (succinimidocarbonyloxy) ethyl ] bis]Sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl]Sulfone (Sulfo-BSOCOES), and the like, and the above-mentioned crosslinking agents are commercially available.

In linking four antibody variable regions, three linkers are typically required, and these linkers may be the same or different.

Examples of conformational isomers in sc (fv)2 include: single chain diabody (single chain diabody) and bivalent scFv types.

When sc (fv)2 is arranged in the order of [ variable region 1] (linker 1) [ variable region 2] (linker 2) [ variable region 3] (linker 3) [ variable region 4], the bivalent scFv type in the present invention refers to sc (fv)2 having the following structure: variable region 1 and variable region 2 are associated and variable region 3 and variable region 4 are associated. In the present invention, the single-chain diabody type refers to sc (fv)2 having the following structure: variable region 1 and variable region 4 are associated and variable region 2 and variable region 3 are associated.

Examples of single chain diabody types are: sc (fv)2 having the structure shown on the right side of FIG. 12 (b); bivalent scFv types are for example: sc (fv)2 having the structure shown on the left side of FIG. 12 (b).

For example, sc (fv)2 can be analyzed by limited proteolysis for a single-chain diabody-type or bivalent scFv-type structure. The analysis can be performed, for example, by the following method.

The test sc (fv)2 was subjected to limited degradation using subtilisin A, a protease that partially limited degrades the linker moiety of sc (fv) 2.

When sc (fv)2 is of the single-chain diabody type, the apparent molecular weight does not change due to the interaction between VH and VL regardless of which of the three linkers of sc (fv)2 is cleaved.

When sc (fv)2 is a bivalent scFv type, a half molecular weight of a molecular species is generated when the central linker is cleaved.

Therefore, by analyzing the reaction product, the bivalent scFv type and the single-chain diabody type can be discriminated.

For example, the reaction product can be analyzed by gel filtration chromatography. The ratio of the structure of the divalent sc (fv)2 and the structure of the single-chain diabody contained in sc (fv)2 can also be quantitatively evaluated by chromatography on the basis of the peak area.

When the sc (fv)2, i.e., the single-chain diabody type or the bivalent scFv type, which is a desired type is to be preferentially obtained, the association regulating method of the present invention can be appropriately used.

More specifically, when sc (fv)2 has a structure of VH1- (linker) -VL1- (linker) -VH2- (linker) -VL2 type, if a bivalent scFv type sc (fv)2 is to be preferentially obtained by the association-modulating method of the present invention, for example, it is sufficient to suppress the association between VH1 and VL2 and/or VH2 and VL1 (for example, mutation is introduced so that the amino acid residues forming the interface between VH1 and VL2 have the same charge).

In addition, if it is intended to preferentially obtain the single-chain diabody type sc (fv)2, for example, it is sufficient to suppress the association between VH1 and VL1 and/or VH2 and VL2 (for example, mutation is introduced so that the amino acid residues forming the interface between VH1 and VL1 have the same charge).

The present invention can be similarly carried out when sc (fv)2 is a monospecific antibody (monospecific antibody).

In addition to the above techniques, the respective domains of VH and VL may be cross-linked by disulfide bonds (Clin Cancer Res.1996 Fed: 2 (2): 245-52).

By using the association-modulating method of the present invention, for example, an antibody or polypeptide having activity can be efficiently produced. Examples of the above activities include: binding activity, neutralizing activity, cytotoxic activity, agonist activity, antagonist activity, enzymatic activity, and the like. Agonist activity refers to an activity of inducing a change in a certain physiological activity by binding an antibody to an antigen such as a receptor, transmitting a signal into a cell, and the like. Examples of the physiological activity include: proliferation activity, survival activity, differentiation activity, transcription activity, membrane transport activity, binding activity, proteolysis activity, phosphorylation/dephosphorylation activity, redox activity, transfer activity, nucleolysis activity, dehydration activity, cell death-inducing activity, apoptosis-inducing activity, and the like, but is not limited thereto.

According to the method of the present invention, an antibody or polypeptide recognizing a desired antigen or binding to a desired receptor can be efficiently produced.

The antigen is not particularly limited, and may be any antigen. Examples of antigens are: a receptor or a fragment thereof, a cancer antigen, an MHC antigen, a differentiation antigen, and the like, but is not particularly limited thereto.

Examples of such receptors are: receptors belonging to receptor families such as hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine/threonine kinase type receptor family, TNF receptor family, G-protein coupled type receptor family, GPI-anchored type receptor family, tyrosine phosphatase type receptor family, adhesion factor family, and hormone receptor family. Many reports have been made on receptors belonging to the above receptor family and their characteristics, for example: cooke BA., King RJB., van der Molen HJ. eds, New comprehensive biochemistry 18B volume "hormons and the Actions Part II (Hormones and their Actions Part II)" pages 1-46 (1988) Elsevier Science Publishers BV., New York, USA; pattern l. (1990) Cell, 61: 13-14.; ullrich A. et al (1990) Cell, 61: 203-; massagul j. (1992) Cell, 69: 1067, 1070; miyajima a. et al, (1992) annu. rev. immunol., 10: 295 and 331; taga T, and Kish imoto T, (1992) FASEB j, 7: 3387-; fantl Wl. et al, (1993) annu, rev, biochem, 62: 453 ion 481; smith CA. et al, (1994) Cell, 76: 959. supplement 962; flower DR (1999) biochim. biophysis. acta, 1422: 207- > 234; the modification of Osaka, the pamphlet of cell engineering, series "adhesion factor booklet" (1994) (Xiu Ruo, Tokyo, Japan), and the like. Specific receptors belonging to the above receptor family include, for example: human or mouse Erythropoietin (EPO) receptor, human or mouse granulocyte colony-stimulating factor (G-CSF) receptor, human or mouse Thrombopoietin (TPO) receptor, human or mouse insulin receptor, human or mouse Flt-3 ligand receptor, human or mouse platelet-derived growth factor (PDGF) receptor, human or mouse Interferon (IFN) - α, β receptor, human or mouse leptin receptor, human or mouse Growth Hormone (GH) receptor, human or mouse Interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse Leukemia Inhibitory Factor (LIF) receptor, human or mouse ciliary neurotrophic factor (CNTF) receptor, etc. (hEmon: Simon, S. et al (1990) Blood 76, 31-35.; mEPOR' Andrea, AD. et al (1989) 57, Cell 277; hG-285: Fukuga; CSuna R285 FR, r, et al (1990) proc.natl.acad.sci.usa.87, 8702-; mG-CSFR: fukunaga, R. et al (1990) Cell 61, 341-350.; hTPOR: vigon, I. et al (1992)89, 5640-; mTPOR: Skoda.RC. et al (1993)12, 2645. supplement 2653; hInsR: ullrich, A. et al (1985) Nature 313, 756-761.; hFlt-3: small, D.et al (1994) Proc.Natl.Acad.Sci.USA.91, 459-463: hPDGFR: gronwald, RGK, et al (1988) Proc.Natl.Acad.Sci.USA.85, 3435-; hIFN α/β R: uze, G.et al (1990) Cell 60, 225-234 and Novick, D.et al (1994) Cell 77, 391-400).

Cancer antigens are antigens that are expressed as cells deteriorate, and are also called tumor-specific antigens. In addition, abnormal sugar chains appearing on the cell surface or protein molecules at the time of canceration of cells also serve as cancer antigens, and are particularly called cancer sugar chain antigens. Examples of cancer antigens include: CA19-9, CA15-3, sialic acid SSEA-1(SLX), etc.

MHC antigens are broadly classified into: MHC class I antigens and MHC class II antigens, MHC class I antigens including: HLA-A, -B, -C, -E, -F, -G, -H; MHC class II antigens include: HLA-DR, -DQ, -DP.

Differentiation antigens include: CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD 3649 a, CD a.

The invention also provides mutants or heteromultimers of the associated polypeptides modulated by the methods of the invention. That is, the present invention relates to a polypeptide or heteromultimer obtained by the association regulating method of the present invention.

A preferred embodiment of the present invention provides a polypeptide mutant having modifications of amino acid residues forming an interface within the original polypeptide to inhibit association within the above polypeptide.

Another aspect of the present invention provides a heteromultimer having modifications of amino acid residues forming an interface between original polypeptides to inhibit association between the polypeptides.

In the present invention, the "original polypeptide" refers to a polypeptide before being modified by the method of the present invention to adjust the association.

An example of a mutant of the above-described polypeptide of the invention is, for example, a mutant in which the original polypeptide is capable of forming two conformational isomers. An example of such a heteromultimer is a multimer in which the original polypeptide is capable of forming 2 or more multimers.

The invention also includes: polypeptide mutants or heteromultimers whose association is modulated by the above-described association modulation method of the present invention. That is, in the above-described preferred embodiment of the association-regulating method, a polypeptide or heteromultimer whose association is regulated is also one of the preferred embodiments of the present invention.

The invention also provides a method for producing a polypeptide or multisource multimer in which the association of the polypeptide or multisource multimer is modulated.

A preferred embodiment of the production method of the present invention provides a method for producing a polypeptide mutant in which a mutation is present in an amino acid residue forming an internal interface of a polypeptide to regulate association of the polypeptide, the method comprising: (a) modifying nucleic acid encoding amino acid residues from the original nucleic acid to inhibit association within the polypeptide, said amino acid residues being amino acid residues that form an interface within the polypeptide; (b) culturing the host cell to express the nucleic acid; (c) recovering the polypeptide from the host cell culture.

Another embodiment of the production method of the present invention provides a method for producing a heteromultimer in which a mutation is present in an amino acid residue forming an interface between polypeptides to regulate the association of the heteromultimer, the method comprising: (a) modifying nucleic acid encoding amino acid residues from original nucleic acid to inhibit association between polypeptides, said amino acid residues being amino acid residues forming an interface between polypeptides; (b) culturing the host cell to express the nucleic acid; (c) recovering the heteromultimer from the host cell culture.

The following method is also one of the preferable embodiments of the above preparation method of the present invention, and the method comprises the steps of: the association-regulating method of the present invention is used to regulate the association of polypeptides by modifying nucleic acids encoding amino acid residues, which are amino acid residues forming an interface within (between) the polypeptides, from the original nucleic acids.

In the above-mentioned method of the present invention, the term "modified nucleic acid" means that the nucleic acid is modified so as to correspond to the amino acid residue introduced by the "modification" of the present invention. More specifically, it refers to a nucleic acid in which a nucleic acid encoding an original (before modification) amino acid residue is modified to encode an amino acid residue introduced by the modification. Generally refers to a genetic manipulation or mutation process in which at least one nucleotide is inserted, deleted or substituted into the original nucleic acid to form a codon encoding an amino acid residue of interest. That is, the codon encoding the original amino acid residue is replaced with a codon encoding an amino acid residue introduced by modification. The modification of the nucleic acid can be appropriately performed by a person skilled in the art using a known technique, for example, site-specific mutagenesis, PCR mutagenesis, or the like.

In addition, the nucleic acid of the present invention is usually carried (inserted) on an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as the inserted nucleic acid is stably retained, and for example, when Escherichia coli is used as the host, pBluescript vector (Stratagene) is preferable as the cloning vector, but various commercially available vectors can be used. Expression vectors are particularly useful when vectors are used for the production of the polypeptides of the invention. The expression vector is not particularly limited as long as it is a vector for expressing a polypeptide in vitro, in Escherichia coli, in cultured cells, or in an individual organism, and for example, when a polypeptide is expressed in vitro, a pBEST vector (manufactured by Promega corporation) is preferable; when the polypeptide is expressed in E.coli, pET vector (manufactured by Invitrogen) is preferable; when the polypeptide is expressed in cells, pME18S-FL3 vector (GenBank accession No. AB009864) is preferred; when the polypeptide is expressed in an individual organism, the pME18S vector (Mol Cell biol.8: 466-472(1988)) and the like are preferred. The insertion of the DNA of the present invention into a vector can be carried out by a conventional method such as ligase reaction using restriction enzyme sites (published by Current protocols in Molecular Biology edge. Ausubel et al (1987). John Wiley & sons. section 11.4-11.11).

The host cell is not particularly limited, and various host cells can be used according to the purpose. Examples of cells for expressing a polypeptide include: bacterial cells (e.g., streptococci (Streptococcus), staphylococci (Staphylococcus), escherichia coli (e.coli), Streptomyces (Streptomyces), Bacillus subtilis (Bacillus subtilis)); fungal cells (e.g.yeast (Yeast), Aspergillus (Aspergillus)); insect cells (e.g., Drosophila S2(Drosophila S2), Spodoptera SF9(Spodoptera SF 9)); animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. The vector can be introduced into the host cell by a known method such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology apparatus. Ausubel. et al (1987), John Wiley & sons. section 9.1-9.9), lipofectamine (Lipofectamine) method (GIBCO-BRL), microinjection, or the like.

Appropriate secretion signals can be inserted into the polypeptide of interest to cause secretion of the polypeptide expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. The signal may be endogenous or heterologous with respect to the polypeptide of interest.

Recovery of the polypeptide in the above production method, if the polypeptide of the present invention is secreted into the medium, the medium is recovered. If the polypeptide of the invention is produced intracellularly, the cells are first lysed and the polypeptide is then recovered.

When the polypeptide of the present invention is recovered and purified from a recombinant cell culture, the following known methods can be used: ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and clusterin chromatography.

The present invention also relates to a composition (medicament) comprising: a polypeptide mutant of the invention or a heteromultimer of the invention and a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutical composition generally refers to a drug used for the treatment or prevention of a disease or for examination or diagnosis.

The pharmaceutical composition of the present invention can be formulated according to methods known to those skilled in the art. For example, parenteral administration may be carried out in the form of injections, which may be prepared as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle (vehicle), preservative, binder, and the like may be suitably combined to be mixed in a unit dosage form satisfying the requirements for pharmaceutical practice to prepare a preparation. The amount of the active ingredient in the above preparation is set to an appropriate volume to obtain a predetermined range.

The sterile composition for injection can be prepared according to a usual preparation procedure using a vehicle such as distilled water for injection.

Examples of the aqueous solution for injection include: physiological saline and isotonic solutions including glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable cosolvents such as alcohols (ethanol, etc.), polyols (propylene glycol, polyethylene glycol, etc.), nonionic surfactants (Tween 80(TM), HCO-50, etc.) may be used in combination.

The oily liquid comprises: sesame oil and soybean oil, and benzyl benzoate and/or benzyl alcohol may be used together as cosolvent. It can also be mixed with buffers (e.g., phosphate buffer and sodium acetate buffer), demulcents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection is usually filled into an appropriate ampoule.

The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, the composition can be prepared into an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, systemic or local administration can be carried out by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

The method of administration may be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody can be set, for example, as follows: each time, the weight of the medicine is 0.0001 mg-1000 mg. Alternatively, the amount to be administered may be, for example, 0.001 to 100000mg per patient, but the present invention is not necessarily limited to the above values. The dose and the administration method vary depending on the body weight, age, symptoms, and the like of the patient, and one skilled in the art can set an appropriate dose and administration method in consideration of the above conditions.

The polypeptide or heteromultimer of the present invention can be combined with other pharmaceutical ingredients to prepare a preparation as required.

The invention also provides a nucleic acid encoding a mutant polypeptide of the invention or a heteromultimer of the invention. Moreover, a vector carrying the nucleic acid is also included in the present invention.

The present invention also provides a host cell having the above nucleic acid. The host cell is not particularly limited, and examples thereof include Escherichia coli and various animal cells. The host cell may be used, for example, as a production system for producing and expressing the antibody or polypeptide of the present invention. A production system for producing a polypeptide comprising: in vitro (in vitro) and in vivo (in vivo) production systems. Examples of in vitro production systems are: production systems using eukaryotic cells and production systems using prokaryotic cells.

Examples of eukaryotic cells that can be used as host cells are: animal cells, plant cells, fungal cells. The animal cells include: mammalian cells such as CHO (J.Exp.Med. (1995) 108: 945), COS, 3T3, myeloma cells, BHK (baby hamster kidney), HeLa, Vero, etc.; amphibian cells such as Xenopus laevis oocytes (Valle et al, Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21, Tn 5. CHO-DG44, CHO-DX11B, COS7 cells, and BHK cells are suitably used for the expression of the antibody of the present invention. Among animal cells, CHO cells are particularly preferable for the purpose of large-scale expression. The introduction of the vector into the host cell can be carried out, for example, by the following method: the calcium phosphate method, the DEAE-dextran method, the method using cationic liposome DOTAP (manufactured by Boehringer Mannheim), the electroporation method, the lipofection method, and the like.

Plant cells include, for example, tobacco-derived cells, which are known as protein production systems, and the antibody of the present invention can be produced by a method of culturing the cells using callus. Protein expression systems using fungal cells are well known and can be used as hosts for producing the antibodies of the invention, such as: yeasts such as cells of the genus Saccharomyces (Saccharomyces) (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Saccharomyces pombe), and the like); and filamentous fungi such as cells of Aspergillus (Aspergillus) (Aspergillus niger) and the like).

Prokaryotic cells for use in production systems may employ bacterial cells. Bacterial cells other than the above-mentioned Escherichia coli (E.coli) are known, and Bacillus subtilis is known, and these bacterial cells can be used for producing the antibody of the present invention.

When the host cell of the present invention is used to produce an antibody, the polynucleotide can be expressed by culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody of the present invention. The culture can be carried out according to a known method. For example, when animal cells are used as hosts, DMEM, MEM, RPMI1640, or IMDM can be used as a culture medium, for example. In this case, a serum supplement such as FBS or Fetal Calf Serum (FCS) may be used in combination, or the cells may be cultured in serum-free culture. The pH during the culture is preferably about 6 to 8. Usually, the culture is carried out at about 30 to 40 ℃ for about 15 to 200 hours, and the medium may be exchanged or aerated or stirred as necessary.

On the other hand, examples of systems for producing a polypeptide in vivo include: production systems using animals or production systems using plants. The target polynucleotide is introduced into these animals or plants, and the polypeptide is produced in the animals or plants and recovered. The "host" of the present invention includes such animals and plants as mentioned above.

Animals used for the production system include mammals and insects. The mammal may use: goat, pig, sheep, mouse, cow, etc. (Vicki Glaser, SPECTRUM Biotechnology applications (1993)). In addition, the mammal may be a transgenic animal.

For example, a polynucleotide encoding an antibody of the present invention is prepared as a fusion gene with a gene encoding a polypeptide inherently produced in milk such as goat β -casein. Then, a polynucleotide fragment comprising the fusion gene is injected into a goat embryo, and the embryo is transferred into a female goat. The goat which received the embryo gives rise to a transgenic goat from which the antibody of interest can be obtained or from the milk produced in its offspring. In order to increase the amount of antibody-containing milk produced by the transgenic goat, the above transgenic goat may be administered with an appropriate hormone (Ebert et al, Bio/Technology (1994) 12: 699-).

As insects for producing the antibody of the present invention, silkworms can be used, for example. When silkworms are used, the target antibody can be obtained from the body fluid of the silkworms by infecting them with baculovirus into which a polynucleotide encoding the target antibody has been inserted (Susumu et al, Nature (1985) 315: 592-4).

Tobacco (tabaco) can be used as a plant for producing the antibody of the present invention. When tobacco is used, a polynucleotide encoding an antibody of interest is inserted into a plant expression vector such as pMON530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). Infection of tobacco such as tobacco (Nicotiana tabacum) with this bacterium can produce the desired antibody from the leaves of this tobacco (Ma et al, Eur.J. Immunol. (1994) 24: 131-8).

The antibody thus obtained can be isolated from the inside or outside of the host cell (medium, milk, etc.) and purified to be a substantially pure and homogeneous antibody. The antibody can be isolated and purified by a method of isolation and purification generally used for purification of a polypeptide, but is not limited thereto. For example, the antibody can be separated and purified by appropriately combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, and the like.

Examples of chromatography include: affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and characterization guide: A Laboratory Corsemoual. Ed Daniel R. Marshark et al (1996) Cold Spring Harbor Laboratory Press). The above chromatography can be performed using liquid chromatography such as HPLC, FPLC, and the like. The affinity chromatography column has: protein A column and protein G column. Examples of protein a columns are: hyper D, POROS, Sepharose F.F (Pharmacia), and the like.

If necessary, an appropriate protein-modifying enzyme may be allowed to act on the antibody to arbitrarily modify or partially remove the peptide before or after purifying the antibody. Protein-modifying enzymes used are, for example: trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glycosidase, and the like.

A further preferred embodiment of the present invention is a method for the preparation of a mutant polypeptide or heteromultimer of the present invention, which comprises the steps of: the host cell of the invention is cultured in the manner described above and the polypeptide is recovered from the cell culture.

All prior art documents cited in the present specification are incorporated herein by reference.

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

EXAMPLE 1 preparation of non-neutralizing anti-factor IXa (F.IXa)

1-1 immunization and preparation of hybridomas

8 BALB/c mice (male, 6 weeks old at the start of immunization, Charles River Japan) and 5 MRL/lpr mice (male, 6 weeks old at the start of immunization, Charles River Japan) were immunized with factor IXa β (Enzyme Research Laboratories, Inc.) in the following manner. Factor IXa β emulsified with FCA (freund's complete adjuvant H37Ra (Difco laboratories)) was administered subcutaneously at 40 μ g/mouse as a primary immunization. After two weeks, factor IXa β emulsified with FIA (freund incomplete adjuvants) was administered subcutaneously at 40 μ g/mouse. Thereafter, the booster immunization is performed 3 to 7 times every other week. After confirming the increase in serum antibody titer against factor IXa beta by ELISA (enzyme-linked immunosorbent assay) as shown in 1-2, factor IXa beta diluted with PBS (-) (phosphate-buffered saline solution containing no calcium ion or magnesium ion) was administered at a dose of 40. mu.g/vein as a final immunization. 3 days after the final immunization, mouse spleen cells and mouse myeloma cells P3X63Ag8U.1 (designated P3U1, ATCC CRL-1597) were fused according to a conventional method using PEG1500(Roche Diagnostics). Fused cells suspended in RPMI1640 medium (Invitrogen) containing 10% FBS (Invitrogen) (hereinafter referred to as 10% FBS/RPMI1640) were seeded in a 96-well culture plate, and after 1, 2, 3, and 5 days of fusion, the medium was replaced with HAT selection medium (10% FBS/RPMI 1640/2% HAT50x concentrate (Dainippon pharmaceutical)/5% BM-conditioned H1(Roche diagnostics)), and selection culture of hybridomas was performed. The binding activity to factor IXa was measured by ELISA as shown in 1-2 using culture supernatants collected on day 8 or day 9 after the fusion, thereby selecting hybridomas having factor IXa binding activity. Next, the neutralizing activity of the enzymatic activity of factor IXa was measured by the method shown in 5-3, and a hybridoma having no neutralizing activity against factor IXa was selected. This limiting dilution was performed twice by seeding one cell per well in a 96-well culture plate to clone hybridomas, and hybridoma XB12 producing an anti-factor IXa antibody was established.

1-2 factor IXa ELISA

Factor IXa beta diluted to 1. mu.g/mL with coating buffer (100mM sodium bicarbonate, pH9.6, 0.02% sodium azide) was dispensed at 100. mu.L/well into Nunc-Immuno plates (Nunc-Immuno)^TM 96MicroWell^TM plates MaxiSorp^TM(Nalge Nunc International)), followed by incubation at 4 ℃ overnight. With compositions containing Tween^(R)After washing 3 times with 20 PBS (-), the plates were washed with dilution buffer (50mM Tris-HCl, pH8.1, 1% bovine serum albumin, 1mM MgCl) at room temperature₂，0.15M NaCl，0.05％Tween^(R)20, 0.02% sodium azide) for 2 hours. After removing the buffer, 100. mu.L/well of mouse antiserum or hybridoma culture supernatant diluted with the dilution buffer was added to the plate, and the plate was incubated at room temperature for 1 hour. After washing the plate 3 times, 100. mu.L/well of alkaline phosphatase-labeled goat anti-mouse IgG (H + L) (Zymed Laboratories) diluted with dilution buffer 1/2000 was added and incubated at room temperature for 1 hour. After washing the plate 6 times, the chromogenic substrate Blue-Phos was added at 100. mu.L/well^TMPhosphate substrate (Kirkegaard)&Perry Laboratories), incubation at room temperature for 20 minutes. Blue-Phos was added at 100. mu.L/well^TMStopping liquid (Kirkegaard)&Perry Laboratories), after which the absorbance at 595nm is determined with a Microplate Reader Model 3550 (Bio-Rad Laboratories) Model 3550.

1-3 determination of neutralizing Activity of factor IXa

Phospholipid (Sigma-Aldrich) was dissolved in distilled water for injection and sonicated to prepare a 400. mu.g/mL solution of phospholipid. In a 96-well plate, 40. mu.L of a Tris-buffered saline solution (hereinafter referred to as TBSB) containing 0.1% bovine serum albumin, 10. mu.L (30ng/mL) of factor IXa. beta. (Enzyme Research Laboratories), 5. mu.L (400. mu.g/mL) of a phospholipid solution, and 5. mu.L of a solution containing 100mM CaCl₂、20mM MgCl₂The resulting mixture was mixed with 10. mu.L of hybridoma culture supernatant and incubated at room temperature for 1 hour. To the mixed solution were added 20. mu.L (50. mu.g/mL) of factor X (enzyme Research laboratories) and 10. mu.L (3U/mL) of factor VIIIa (Amrican diagnostics)tica) at room temperature for 30 minutes. To this, 10. mu.L (0.5M) of EDTA was added to stop the reaction. To the reaction solution, 50. mu.L of S-2222 solution (Chromogenix) was added, incubated at room temperature for 30 minutes, and then the absorbance at a detection wavelength of 405nm and a control wavelength of 655nm was measured using a 3550 type microplate reader (Bio-Rad Laboratories, Inc.).

EXAMPLE 2 preparation of non-neutralizing anti-factor X (F.X) antibody

2-1 immunization and hybridoma preparation

8 BALB/c mice (male, 6 weeks old at the start of immunization, Charles River Japan) and 5 MRL/lpr mice (male, 6 weeks old at the start of immunization, Charles River Japan) were immunized using factor X (enzyme Research laboratories) in the following manner. Factor X emulsified with FCA was administered subcutaneously at 40. mu.g/mouse as a primary immunization. After two weeks, factor X emulsified with FIA was administered subcutaneously at 20 or 40 μ g/mouse. The booster immunization is performed 3 to 6 times every other week. After confirming the increase in anti-factor X serum antibody titer by ELISA shown in 2-2, factor X diluted with PBS (-) was administered intravenously at 20 or 40. mu.g/mouse as a final immunization. 3 days after the final immunization, mouse splenocytes and mouse myeloma cells P3U1 were fused according to the conventional method using PEG 1500. The fused cells suspended in 10% FBS/RPMI1640 medium were seeded in a 96-well culture plate, and after 1, 2, 3, and 5 days of fusion, the culture medium was replaced with HAT selection medium, and selection culture of hybridomas was performed. The binding activity to factor X was determined by ELISA as shown in 2-2 using culture supernatants collected on day 8 after fusion. Hybridomas having factor X-binding activity were selected, and the neutralizing activity of the enzyme activity of factor Xa was measured by the method shown in FIGS. 2 to 3. Hybridoma SB04 producing an anti-factor X antibody was established by cloning hybridomas that did not have neutralizing activity against factor Xa by performing 2 limiting dilutions.

2-2 factor X ELISA

Factor X diluted to 1. mu.g/mL with coating buffer was dispensed at 100. mu.l/well into Nunc-Immuno plates, followed by incubation at 4 ℃ overnight. Applying the plate with Tween^(R)After washing 3 times with 20 PBS (-), the cells were blocked with dilution buffer at room temperature for 2 hours. After removing the buffer, the plate was added with mouse antiserum or hybridoma culture supernatant diluted with a dilution buffer and incubated at room temperature for 1 hour. After washing the plate 3 times, alkaline phosphatase-labeled goat anti-mouse IgG (H + L) diluted with dilution buffer 1/2000 was added and incubated at room temperature for 1 hour. After washing the plate 6 times, the chromogenic substrate Blue-Phos was added at 100. mu.L/well^TMPhosphate substrate (Kirkegaard)&Perry Laboratories), incubation at room temperature for 20 minutes. Blue-Phos was added at 100. mu.L/well^TMStopping liquid (Kirkegaard)&Perry Laboratories), the absorbance at 595nm is measured with a model 3550 microplate reader (Bio-Rad Laboratories).

2-3. determination of factor Xa neutralizing Activity

mu.L hybridoma culture supernatant diluted with TBSB1/5 and 40. mu.L TBCP (containing 2.78mM CaCl)₂22.2 μ M of TBSB phospholipid (phosphatidyl choline: phosphatidyl serine 75: 25, Sigma-Aldrich) containing 250pg/mL factor Xa (enzyme Research laboratories) was mixed and incubated at room temperature for 1 hour. To the mixed solution, 50. mu.L of TBCP containing 20. mu.g/mL of prothrombin (Enzyme Research Laboratories) and 100ng/mL of activated blood coagulation factor V (factor Va (Haematologic technologies)) was added and reacted at room temperature for 10 minutes. The reaction was stopped by adding 10. mu.L (0.5M) of EDTA. To the reaction solution, 50. mu.L (1mM) of S-2238 solution (Chromogenix) was added, incubated at room temperature for 30 minutes, and then absorbance at 405nm was measured using a 3550 microplate reader (Bio-Rad laboratories).

EXAMPLE 3 construction of chimeric bispecific antibody expression vector

3-1. preparation of DNA fragment encoding antibody variable region derived from hybridoma

Using QIAGEN^(R)RNeasy^(R)Total RNA was extracted from hybridoma XB12 producing anti-f.ixa antibody or hybridoma SB04 producing anti-F.X antibody using the method described in the specification with a mini kit (QIAGEN). Total RNA was dissolved in 40. mu.L of sterile water. In 1 &Mu.g of the purified RNA was used as a template, and single-stranded cDNA was synthesized by RT-PCR using SuperScript cDNA Synthesis System (Invitrogen) according to the method described in the specification.

3-2 amplification of antibody H chain variable region by PCR and sequence analysis

An HB primer mixture and an HF primer mixture described in a report of Krebber et al (J.Immunol. methods 1997; 201: 35-55) were prepared as primers for amplification of mouse antibody H chain variable region (VH) cDNA. Using 0.5. mu.L (100. mu.M) of the HB primer mixture and 0.5. mu.L (100. mu.M) of the HF primer mixture, 25. mu.L of a reaction solution (2.5. mu.L of the cDNA solution prepared in 3-1, KOD plus buffer (Toyo Boseki Co., Ltd.), 0.2mM dNTPs, 1.5mM MgCl₂0.75units DNA polymerase KOD plus (Toyo Boseki)). PCR was performed as follows: the amplification of cDNA fragments was carried out under either conditions of A (after heating at 98 ℃ for 3 minutes; 1 cycle for reactions at 98 ℃ for 20 seconds, 58 ℃ for 20 seconds, and 72 ℃ for 30 seconds; 32 cycles) or B (after heating at 94 ℃ for 3 minutes; 1 cycle for reactions at 94 ℃ for 20 seconds, 46 ℃ for 20 seconds, and 68 ℃ for 30 seconds; 5 cycles for reactions at 94 ℃ for 20 seconds, 58 ℃ for 20 seconds, and 72 ℃ for 30 seconds; 1 cycle for 30 cycles) using a thermal cycler GeneAmp PCR system 9700(Perkin Elmer) according to the amplification efficiency of cDNA fragments. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the target size (about 400bp) was purified using QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. The nucleotide sequence of each DNA fragment was determined by the method described in the appendix, using BigDye Terminator cycle sequencing Kit (Applied Biosystems) using a DNA sequencer ABI PRISM3100 genetic analyzer (Applied Biosystems). The set of sequences determined by the method was analyzed by comparison using the analysis software GENETYX-SV/RC version 6.1 (Genetyx) to select DNA fragments having different sequences.

3-3 preparation of antibody variable region DNA fragment for cloning

In order to add restriction enzyme Sfi I cleavage sites for cloning to both ends of the antibody variable region amplified fragment, the following operation was performed.

To amplify the VH segment with the added Sfi I cleavage site (Sfi I-VH), a primer (primer VH-5' end) was prepared which was a change of the (Gly4Ser) 2-linker sequence of primer HB to a sequence with the Sfi I cleavage site. Using 0.5. mu.L (10. mu.M) of the 5' end of the sequence-specific primer VH and 0.5. mu.L (10. mu.M) of the primer scfor (J.Immunol.Mthods 1997; 201: 35-55), 20. mu.L of the reaction solution (1. mu.L of the purified VH cDNA amplified fragment solution prepared in 3-2, KOD plus buffer (Toyo Boseki), 0.2mM dNTPs, 1.5mM MgCl, 0.5 mM dNTPs, etc.) was prepared₂0.5 units of DNA polymerase KOD plus (Toyo Boseki)). PCR was performed as follows: the amplification was carried out under either conditions of A (after heating at 98 ℃ for 3 minutes; 1 cycle for reactions at 98 ℃ for 20 seconds, 58 ℃ for 20 seconds, and 72 ℃ for 30 seconds; 32 cycles) or B (after heating at 94 ℃ for 3 minutes; 1 cycle for reactions at 94 ℃ for 20 seconds, 46 ℃ for 20 seconds, and 68 ℃ for 30 seconds; 5 cycles for reactions at 94 ℃ for 20 seconds, 58 ℃ for 20 seconds, and 72 ℃ for 30 seconds; 1 cycle for reactions at 94 ℃ for 30 seconds; 30 cycles) using a thermal cycler GeneAmp PCR system 9700(Perkin Elmer) according to the amplification efficiency of the fragment. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the desired size (about 400bp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water.

For amplification of mouse antibody L chain variable region (VL) cDNA fragment, first, 25. mu.L of reaction solution (2.5. mu.L of 3-1 prepared cDNA solution, KOD plus buffer solution (Toyo Boseki), 0.2mM dNTPs, 1.5mM MgCl) was prepared using 0.5. mu.L (100. mu.M) of LB primer mixture and 0.5. mu.L (100. mu.M) of LF primer mixture as described in Krebber et al report (J.Immunol. Mthods 1997; 201: 35-55)₂0.75units of DNA polymerase KOD plus (Toyo Boseki)). PCR was performed as follows: after heating at 94 ℃ for 3 minutes using a thermal cycler GeneAmp PCR system 9700(Perkinelmer) according to the amplification efficiency of the fragments; the reaction at 94 ℃ for 20 seconds, at 46 ℃ for 20 seconds and at 68 ℃ for 30 seconds is circulated for 1 time and 5 times; then the reaction at 94 ℃ for 20 seconds, at 58 ℃ for 20 seconds and at 72 ℃ for 30 seconds is 1 cycle, and 30 cycles are carried out. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the target size (about 400bp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. The state of the fragment is: to the C-terminal thereof was added a (Gly4Ser) 3-linker sequence derived from primer LF. To add an Sfi I cleavage site to the C-terminus of this fragment, a primer (primer VL-3' terminus) was prepared in which the (Gly4Ser) 3-linker sequence of primer LF was changed to a sequence having an Sfi I cleavage site. To amplify VL fragment (Sfi I-VL) added with Sfi I cleavage site, 20. mu.L of reaction solution (1. mu.L of purified VL cDNA amplified fragment solution, KOD plus buffer (Toyo Boseki), 0.2mM dNTPs, 1.5mM MgCl. sub.L) was prepared using 0.5. mu.L (10. mu.M) of VL-3' end primer mixture and 0.5. mu.L (10. mu.M) of scback primer₂0.5 units of DNA polymerase KOD plus (Toyo Boseki)). PCR was performed as follows: after heating at 94 ℃ for 3 minutes using a thermal cycler GeneAmp PCR System 9700(Perkin Elmer); the reaction at 94 ℃ for 20 seconds, at 46 ℃ for 20 seconds and at 68 ℃ for 30 seconds is circulated for 1 time and 5 times; the reaction was repeated 1 cycle at 94 ℃ for 20 seconds, 58 ℃ for 20 seconds and 72 ℃ for 30 seconds, and 30 cycles were repeated. After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the target size (about 400bp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water.

Reaction solutions in which purified Sfi I-VH and Sfi I-VH fragments were digested with Sfi I (Takara Bio) overnight at50 ℃ were prepared according to the method described in the appendix. Thereafter, the reaction solution was purified by the method described in the appendix, using a QIAquick PCR purification kit (QIAGEN), and eluted with 30. mu.L of EB buffer solution contained in the kit. 3-4 plasmid for expressing human IgG 4-mouse chimeric bispecific IgG antibody

In order to form a heterologous molecule in each H chain when a bispecific IgG antibody of interest is produced, an amino acid substitution in which the CH3 portion of IgG4 is substituted is prepared with reference to the nanobs-into-holes technique of IgG1 (non-patent document 3). Type a (IgG4 γ a) is the Y349C, T366W substituent, and type b (IgG4 γ b) is the E356C, T366S, L368A, Y407V substituent. Furthermore, a substitution (-ppcpSCp- - > -ppcpPcp) was introduced into the hinge region of both substituents. Although almost all of the techniques can form a heterozygote, the L chain is not necessarily so, and the subsequent activity measurement may be affected by the unnecessary production of antibody molecules. Therefore, in this protocol, in order to express antibody molecules with various specificities in one arm (called HL molecules) separately and efficiently produce bispecific IgG antibodies of the target type in cells, vectors induced with different drugs are used as expression vectors for the respective HL molecules.

Each H chain or L chain region (pcDNA4-g4H or pcDNA4-g4L) was made as an expression vector for a single arm (one arm) of an antibody molecule (conventionally referred to as a right arm HL molecule), and this region was inserted into a tetracycline-inducible vector pcDNA4(Invitrogen), i.e., an appropriate mouse antibody variable region (VH or VL) and a human IgG4 γ a constant region (SEQ ID NO: 9) or a κ constant region (SEQ ID NO: 10) were inserted downstream of a signal sequence (IL3ss) for animal cells (Proc. Natl. Acad. Sci. USA.1984; 81: 1075). First, pcDNA4 was digested with restriction enzyme cleavage sites Eco RV and Not I (Takara Bio) present in its multiple cloning site. The right-arm H chain or L chain expression unit (about 1.6kb or about 1.0kb, respectively) of the chimeric bispecific antibody having an appropriate antibody variable region was digested with Xho I (Takara Bio), purified by the method described in the attached manual using QIAquick PCR purification kit (QIAGEN), and reacted at 72 ℃ for 10 minutes using DNA polymerase KOD (eastern ocean textile) according to the reaction composition described in the attached manual to smooth the ends. The blunt-ended fragment was purified using QIAquick PCR purification kit (QIAGEN) according to the method described in the appendix and digested with Not I (Takara Bio). Using Ligation High (Toyo Boseki), pcDNA4 was ligated and pcDNA4 was digested with the Not I-blunt fragment (about 1.6kb or 1.0kb, respectively) and the Eco RV-Not I, according to the method described in the attached manual. Coli DH5 α strain (complent high DH5 α (eastern ocean textiles)) was transformed with the reaction solution. From the obtained ampicillin resistant clones, each plasmid DNA was isolated using QIAprep SpinMiniprep kit (QIAGEN).

For the other single-arm (conventionally called left-arm HL molecule), each H chain or L chain region (pIND-g4H or pIND-g4L) was made according to the above method, inserted into an ecdysone-analogue inducible vector pIND (Invitrogen), i.e., the appropriate mouse antibody variable region (VH or VL) and human IgG 4. gamma.b constant region (SEQ ID NO: 11) or kappa constant region were inserted downstream of the signal sequence for animal cells (IL3ss) (EMBO. J.1987; 6: 2939), and each plasmid DNA was isolated.

3-5. construction of bispecific antibody expression vector

The tetracycline-inducible expression plasmid (pcDNA4-g4H or pcDNA4-g4L) prepared in 3-4 was digested with Sfi I, and the reaction solution was subjected to 1% agarose gel electrophoresis. The fragment (about 5kb) from which the variable region portion (VH or VL) of the original antibody was removed was purified by QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. The above fragments and the corresponding 3-3 prepared Sfi I-VH or Sfi I-VL fragments derived from Sfi I digested anti-F.IXa antibody XB12 were subjected to Ligation reaction using a Rapid Ligation Kit (Quick Ligation Kit) (New Egland Biolabs) according to the method described in the appendix. Coli strain DH5 alpha (Competent high DH5 alpha (Toyo Boseki)) was transformed with the reaction mixture. The antibody variable region portion (VH or VL) was removed from the 3-4 prepared Sfi I-digested ecdysone analogue-inducible expression plasmid (pIND-g4H or pIND-g4L) according to the same method as described above, and the resulting fragments were combined with the corresponding 3-3 prepared Sfi I-VH or Sfi I-VL fragment, respectively, derived from the Sfi I-digested anti-F.X antibody SB04, according to the same method.

The nucleotide sequence of each DNA fragment was determined by the method described in the appendix, using BigDye Terminator cycle sequencing kit (Applied Biosystems) and using an ABI PRISM3100 genetic Analyzer (Applied Biosystems) using a DNA sequencer. The set of sequences determined by the method was analyzed using the analysis software GENETYX-SV/RC version 6.1 (Genetyx).

From the objective clone, each plasmid DNA was isolated using QIAprep Spin Miniprep kit (QIAGEN), and dissolved in 100. mu.L of sterilized water. The anti-f.ixa antibody chimeric H chain expression vector, the anti-f.ixa antibody chimeric L chain expression vector, the anti-F.X antibody chimeric H chain expression vector, and the anti-F.X antibody chimeric L chain expression vector were designated as: pcDNA4-g4XB12H, pcDNA4-g4XB12L, pIND-g4SB04H and pIND-g4SB 04L.

EXAMPLE 4 preparation of chimeric bispecific antibody

Preparation of DNA solution

The expression vector (pcDNA4-g4XB12H and pcDNA4-g4XB12L) of the HL molecule on the right arm of the antibody is subjected to expression induction by using tetracycline. In the absence of tetracycline, plasmid pcDNA6/TR (Invitrogen) encoding the tetracycline repressor (Tet-reducer) is required for complete inhibition of expression. Expression vectors for the antibody left-arm HL molecule (pIND-g4SB04H and pIND-g4SB04L) were induced by expression of insect hormone-ecdysone analog (ponasterone A). At this time, plasmid pvgrxr (invitrogen) encoding ecdysone receptor and retinoid X receptor, both of which were induced by the reaction with ponasterone a, was required. Therefore, in order to transfect animal cells, a total of 6 plasmid DNA mixtures were prepared. 10mL of cell culture medium was prepared using 3. mu.g each of pcDNA4-g4XB12H, pcDNA4-g4XB12L, pIND-g4SB04H and pIND-g4SB04L, and 18. mu.g each of pcDNA6/TR and pVgRXR.

4-2 transfection of animal cells

The HEK293H strain (Invitrogen) derived from human fetal kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% FCS (MOLEATE) and cultured at 5X 10⁵Cell density per mL 10mL of each cell was seeded in a cell-adhesive dish (10 cm diameter, CORNING)₂Incubator (37 ℃, 5% CO)₂) Culturing for a day and night. The plasmid DNA mixture prepared in 4-1 was added to a mixture containing: transfection reagent, 75.8. mu.L Lipofectamine 2000(Invitrogen) and 2708. mu.L Opti-MEM I medium (Invitrogen), before room temperatureStanding for 20 min, adding the mixture to the cells in each well, and adding the mixture to the cells in CO₂Incubator (37 ℃, 5% CO)₂) Internal culture is carried out for 4-5 hours.

4-3 expression induction of bispecific IgG antibodies

The medium was aspirated from the culture of cells transfected in the previous manner and 10mL of CHO-S-SFM-II (Invitrogen) medium containing 1. mu.g/mL tetracycline (Wako pure chemical industries, Ltd.) was added in CO₂Incubator (37 ℃, 5% CO)₂) The antibody was cultured for 1 day for the first induction of expression of HL molecules in the right arm. Thereafter, the medium was aspirated, and once washed with 10mL of CHO-S-SFM-II medium, 10mL of CHO-S-SFM-II medium containing 5. mu.M pinosterone A (Invitrogen) was added thereto under CO₂Incubator (37 ℃, 5% CO)₂) The cells were cultured for 3 days, and the second expression induction of HL molecules in the left arm of the antibody was performed to secrete the bispecific IgG antibody into the medium. After recovery of the culture supernatant, the cells were removed by centrifugation (about 2000g, 5 min, room temperature) and then passed through a 0.22 μm filter MILLEX^(R)GV (Millipore) for sterilization. The sample was stored at 4 ℃ until use.

4-4. antibody purification

To 10mL of the culture supernatant obtained in example 4-3, 100. mu.L of rProtein A Sepharose was added^TMFast Flow (Amersham Biosciences) was mixed at 4 ℃ for 4 hours or more by tumbling. The solution was transferred to a 0.22 μm filter cup Ultrafree^(R)in-MC (Millipore), 500. mu.L of Tween 0.01% was used^(R)20 TBS 3 times after washing, rProtein A Sepharose^TMThe resin was suspended in 100. mu.L of a suspension containing 0.01% Tween^(R)20 in 10mM HCl (pH2.0), and left to stand for 2 minutes, the antibody was dissolved. 5. mu.L of 1M Tris-HCl (pH8.0) was immediately added for neutralization.

4-5 quantification of human IgG concentration

Goat anti-human IgG (Biosource International) was adjusted to 1. mu.g/mL with coating buffer and immobilized on Nunc-Immuno plates (Nunc). After blocking treatment with dilution buffer (D.B.),culture supernatant samples diluted appropriately with d.b. were added. In addition, human IgG4 (humanized anti-TF antibody, see WO99/51743) was added in the same manner, and as a standard for calculating the antibody concentration, human IgG4 was diluted 11 stages by 3-fold stepwise dilution from 2000ng/mL using D.B. as a standard. After 3 washes, goat anti-human IgG was reacted with alkaline phosphatase (biosource international). After 5 washes, Sigma 104^(R)Phosphatase substrate (Sigma-Aldrich) was used as a substrate for color development, and absorbance at 405nm was measured using an absorbance reader 3550 (Bio-Rad laboratories) at a reference wavelength of 655 nm. The concentration of human IgG in the culture supernatant was calculated from the standard curve using the Microplate Manger III (Bio-Rad Laboratories) software.

EXAMPLE 5 plasma coagulation analysis

To determine whether bispecific antibodies are able to correct the clotting ability of hemophilia a blood, the effect of the bispecific antibodies on the thromboplastin time of the activated moiety (APTT) was studied using factor VIII deficient plasma. A mixture of 50. mu.L of antibody solutions at various concentrations, 50. mu.L of factor VIII deficient plasma (Biomerieux) and 50. mu.L of APTT reagent (Dade Behring) was heated at 37 ℃ for 3 minutes. To the mixture was added 50. mu.L of 20mM CaCl₂(DadeBehring) to initiate the coagulation reaction. The time required for coagulation was determined using KC10A (Amelung) with CR-A (Amelung) attached.

A calibration curve was prepared with the coagulation time of factor VIII-deficient plasma as 0% and the coagulation time of normal plasma as 100%, and the factor VIII-like activity (%) of the bispecific antibody was calculated from the coagulation time when the bispecific antibody was added.

[ example 6] humanization of bispecific antibody

The anti-factor IXa antibody XB12 and anti-factor X antibody SB04, which had the strongest effect of shortening the clotting time, were humanized as described below.

6-1. homology search of human antibodies

Human antibody amino acid sequence data were obtained from the generally disclosed Kabat database (ftp:// ftp. ebi. ac. uk/pub/databases/Kabat /) and IMGT database (http:// IMGT. cines. fr /), and using the constructed databases, they were divided into a mouse XB12-H variable region, a mouse XB-12L variable region, a mouse SB04-H variable region, and a mouse SB04-L variable region, and homology searches were performed. As a result, it was confirmed that the sequences have high homology with the human antibody sequences shown below, and thus the framework regions (hereinafter referred to as FR) for the humanized antibody were determined.

(1) XB12-H variable region: KABATID-020619(Kabat database)

(Mariette et al, Arthritis Rheum. 1993; 36: 1315-

(2) XB12-L variable region: EMBL Access No. X61642(IMGT database)

(Mark et al, J Mol biol. 1991; 222: 581-597.)

(3) SB04-H chain variable region: KABATID-025255(Kabat database)

(Demaison et al, Immunogetetics 1995; 42: 342-)

(4) SB04-L chain variable region: EMBL Access No. AB064111(IMGT database)

(unpublished data)

A humanized antibody was prepared by grafting complementary epitope regions (hereinafter referred to as CDRs) of each mouse antibody to the human antibody FRs of (1) to (4).

Secretion signal sequences of human antibodies having high homology to the human antibodies of (1) to (4) were searched using the homology search website published by NCBI (http:// www.ncbi.nlm.nih.gov/BLAST /). The following secretion signal sequence obtained by the search was used.

(1) XB12-H variable region: GenBank Accession No. AF062120

(2) XB12-L variable region: GenBank Accession No. M74019

(3) SB04-H chain variable region: GenBank Accession No. BC019337

(4) SB04-L chain variable region: GenBank Accession No. AY204756

6-2 construction of humanized antibody Gene expression vector

12 synthetic oligo DNAs of about 50 bases are alternately prepared in the nucleotide sequence encoding the amino acid sequence from the secretion signal sequence to the antibody variable region, and about 20 bases are annealed to the 3' -end. Then, a primer having an XhoI cleavage sequence that anneals to the 5 '-end of the antibody variable region gene and a primer having an SfiI cleavage sequence that anneals to the 3' -end of the antibody variable region gene were prepared.

mu.L of each synthetic oligo DNA adjusted to 2.5. mu.M was mixed, and 1 XTaKaRa ExTaq buffer, 0.4mM dNTPs, and 0.5 unit TaKaRa Ex Taq (all Takara Shuzo Co., Ltd.) were added to prepare 48. mu.L of a reaction solution. After 5 minutes of incubation at 94 ℃, the reaction was cycled 2 times at 94 ℃ for 2 minutes, 55 ℃ for 2 minutes, and 72 ℃ for 2 minutes for assembly and extension reactions of each synthetic oligo DNA. Then, 1. mu.L of each 10. mu.M primer annealing to the 5 '-end and 3' -end of the antibody gene was added, and the reaction was cycled 35 times at 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute, and then reacted at 72 ℃ for 5 minutes to amplify the antibody variable region gene. After PCR, the entire reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the desired size (about 400bp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. The fragment was cloned using pGEM-T Easy vector system (Promega) according to the method described in the attached manual. The nucleotide sequence of each DNA fragment was determined by using BigDye Terminator cycle sequencing kit (Applied Biosystems) by using ABI PRISM 3700DNA Seguencer (Applied Biosystems) according to the method described in the appendix.

The plasmid confirmed to have the correct humanized antibody variable region gene sequence was digested with EcoRI and SfiI, and the reaction mixture was subjected to 1% agarose gel electrophoresis. DNA fragments of the desired size (about 400bp) were purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. The tetracycline-inducible expression plasmids (pcDNA4-g4H, pcDNA4-g4L) and ecdysone analog-inducible expression plasmids (pIND-g4H, pIND-g4L) prepared in example 3-3 were digested with EcoRI and SfiI, and the fragment containing the antibody constant region (about 5kp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. A gene fragment (H chain variable region or L chain variable region) of the humanized XB12 digested with EcoRI and SfiI and a tetracycline-inducible expression plasmid (pcDNA4-g4H, pcDNA4-g4L) digested with EcoRI and SfiI were ligated using a Rapid DNA Ligation Kit (Rapid DNA Ligation Kit) (Roche Diagnostics) according to the method described in the appendix. In addition, the EcoRI and SfiI digested humanized SB04 antibody gene fragment (H chain variable region or L chain variable region) and EcoRI and SfiI digested ecdysone analogue inducible expression plasmids (pIND-g4H, pIND-g4L) were ligated using a Rapid DNA ligation kit (Roche Diagnostics) according to the method described in the appendix. Coli DH5 a strain (eastern ocean textiles) was transformed using a part of each reaction solution.

In addition, for expression as a general humanized antibody other than bispecific antibody, an expression vector was prepared as follows. Plasmids (pCAG-g4H, pCAG-g κ) having a chicken β -actin promoter, in which a wild-type antibody constant region was inserted into pCAGGS (Niwa et al, 1991Gene, 108: 193-199.) having a chicken β -actin promoter, and a humanized XB12 antibody Gene fragment (H chain variable region or L chain variable region) or a humanized SB04 antibody Gene fragment (H chain variable region or L chain variable region) which was recovered by XhoI and SfiI digestion, were prepared. The DNA ligation reaction was performed using a rapid DNA ligation kit (Roche Diagnostics) and E.coli strain DH5 alpha (Toyo Boseki) was transformed.

6-3 preparation of humanized bispecific antibody

Genes were introduced into HEK293H using 4 humanized bispecific antibody expression vectors, pcDNA6/TR and pVgRXR, and expression was induced by the methods described in examples 4-2 and 4-3. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

6-4 preparation of humanized antibody

In order to express a general humanized antibody other than bispecific antibody, genes were introduced into HEK293H by the method described in example 4-2 using the humanized H chain antibody expression vector and the humanized L chain antibody expression vector prepared in example 6-3. After the gene was introduced, 10mL of CHO-S-SFM-II medium (Invitrogen) was added thereto, the medium was removed again to wash the cells, 10mL of CHO-S-SFM-II was added again, and the cells were washed with CO₂Incubator (37 ℃, 5% CO)₂) Cultured for 3 days to secrete humanized antibody.

6-5 evaluation of Activity of humanized bispecific antibody and modification of antibody sequence

To evaluate the plasma coagulation energy of the humanized bispecific antibody and the chimeric bispecific antibody (XB12/SB04) prepared, the effect of the antibody on APTT was investigated using f.viii deficient plasma according to the method of example 5. For a humanized bispecific antibody that reduces blood coagulation ability, the amino acids of the human antibody FR are modified with the aim of increasing the activity. In addition, cysteine residues of CDR3 of the XB12 antibody VH, which may reduce thermostability and the like, are also modified to alanine residues. Specifically, a mutation was introduced into the variable region of the humanized antibody by the method described in the attached manual using a QuikChange site-Directed Mutagenesis Kit (Stratagene). By repeating amino acid modification of the FR sequence and evaluation of blood coagulation ability, a humanized bispecific antibody (humanized XB12 antibody (VH: hXB12F-A, VL: hXBVL)/humanized SB04 antibody (VH: hSB04e, VL: hSBVL-F3F)) having the same activity as XB12/SB04 was obtained. The variable region sequence of each antibody is shown below as SEQ ID NO.

(1) Humanized XB12 antibody VH (hXB12f-a) SEQ ID NO: 1 (nucleotide sequence), SEQ ID NO: 2 (amino acid sequence)

(2) Humanized XB12 antibody vl (hxbvl) SEQ ID NO: 3 (nucleotide sequence), seq id NO: 4 (amino acid sequence)

(3) Humanized SB04 antibody VH (hSB04e) SEQ ID NO: 5 (nucleotide sequence), SEQ ID NO: 6 (amino acid sequence)

(4) Humanized SB04 antibody VL (hSBVL-F3F) SEQ ID NO: 7 (nucleotide sequence), SEQ ID NO: 8 (amino acid sequence)

EXAMPLE 7 modeling of humanized antibody

To confirm the amino acid residues at the VH and VL interfaces of the humanized SB04 antibody, an antibody Fv region model was created by homology modeling using MOE software (Chemical Computing Group Inc.). It was confirmed that at the interface between VH and VL, the amino acids H39 and L38 were both glutamine (Gln), and hydrogen bonds were formed by the side chains of both residues (FIG. 1 (A)). In addition, it was confirmed that the amino acids H45 and L44 were leucine (Leu) and proline (Pro), respectively, and that the side chains of both residues were very close and formed a hydrophobic core (fig. 1 (B)). Amino acid residues at these two positions have been reported to be highly conserved in human antibodies (Vargas-Madrazo E et al, J.mol.Recognit.2003, 16: 113-120). For the numbering of the antibodies H39, L38, H45, L44, etc., reference is made to the Kabat et al literature (Kabat EA et al, 1991.Sequences of Proteins of immunological Interest NIH).

EXAMPLE 8 preparation and evaluation of humanized antibody modified with H39 and L38 amino acids

8-1 construction of H39 and L38 modified antibody expression vectors

Based on the findings of example 7, glutamine of H39 of the humanized XB12H chain and glutamine of L38 of the humanized SB04L chain were substituted to suppress association of the H chain of the humanized XB12 and the L chain of the humanized SB 04. Specifically, two amino acids (H39, L38) are substituted with lysine (Lys) or arginine (Arg) whose side chains are positively charged, or glutamic acid (Glu) or aspartic acid (Asp) whose side chains are negatively charged, to suppress hydrogen bonding of glutamine side chains, thereby causing electrostatic repulsion. The humanized antibody gene was substituted by introducing a mutation using QuikChange site-directed mutagenesis kit (Stratagene) according to the method described in the attached manual. Each humanized antibody gene fragment whose amino acid had been substituted was inserted into the bispecific antibody expression vector used in example 6-2 or a usual antibody expression vector.

8-2 preparation of antibody for evaluation of Association Regulation and evaluation of Association Regulation

To evaluate the regulation of the association between the H chain and the L chain, three antibody expression vectors, i.e., the humanized XB12H chain (modified H39), the humanized SB04L chain (modified L38), and the wild-type humanized XB12L chain, were prepared, and genes were introduced into HEK293H by the method described in example 4-2, and the antibody was secreted in the culture supernatant. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

200ng of purified antibody was subjected to reduction treatment in sample buffer (TEFCO), and injected into a 14% SDS-PAGE minigel (TEFCO) for electrophoresis. After electrophoresis, the gel was immersed in a 7% acetic acid solution containing 10% methanol for 30 minutes for fixation, and then subjected to SYPRO^(R)Staining was carried out by immersing the Ruby protein gel stain (BIO-RAD) in the stain for one day and night. Subsequently, the resultant was immersed in a 7% acetic acid solution containing 10% methanol for 1 hour for decolorization treatment, and image analysis was performed using a fluorescence detection apparatus FluorImagerSI (Amersham biosciences) to obtain a developed image. Using the obtained images, the band fluorescence intensities of the H chain and the L chain were calculated by Image Quant ver4.2(Amersham biosciences).

The results are shown in FIG. 2. Using the calculated fluorescence intensity values, the ratio (%) of the target XB12-L chain was calculated by "XB 12-L chain/total amount of L chain (XB12-L chain + SB04-L chain) × 100". When the amino acids of the humanized XB12H chain (H39) and the humanized SB04L chain (L38) were wild-type glutamine (Gln), the ratio was 50%; in contrast, when H39 and L38 were substituted, the ratio of the humanized XB12L chain increased; when glutamic acid (Glu) was substituted, the ratio was found to increase 1.6-fold and reach 82%.

8-3 preparation of bispecific antibody for evaluation of clotting Activity and evaluation of clotting Activity

To evaluate the clotting activity, genes were introduced into HEK293H and the expression was induced by using the prepared bispecific antibody expression vector composed of the humanized XB12H chain (modified H39) and the humanized SB04L chain (modified L38), the wild-type bispecific antibody expression vector composed of the humanized XB12L chain and the humanized SB04H chain, pcDNA6/TR and pVgRXR, in accordance with the methods described in examples 4-2 and 4-3. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

The clotting activity was evaluated in the same manner as in example 5. The results are shown in FIG. 3. In the association regulation evaluation, it was confirmed that: the glutamic acid (Glu: E) modified antibody whose ratio was increased to 82% showed the same or higher clotting activity as compared with the wild type.

8-4 preparation of antibody for evaluation of binding Activity

To evaluate the binding activity of factor IXa and factor X, an antibody was secreted in the culture supernatant by introducing a gene into HEK293H according to the method shown in example 4-2 using an antibody expression vector of a humanized XB12H chain (H39 modification) and a wild-type humanized XB12L chain or an antibody expression vector of a wild-type humanized SB04H chain and a humanized SB04L (L38 modification). Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

The binding activity of factor IXa and factor X was evaluated according to the methods described in examples 1-2 and 2-2. The results are shown in FIGS. 4 and 5. Confirming that: the amino acids substituted for H39 and L38 did not alter the binding activity.

The above results show that: by modifying H39 of the XB12H chain and L38 of the SB04L chain, the ratio of the bispecific antibody of interest can be increased without reducing biological activities such as binding activity to an antigen and coagulation activity in place of factor VIII. To date, including the methods using knob and hole, there has been no report on an example in which association can be regulated without reducing the function by introducing a mutation into only one site amino acid in a polypeptide. Therefore, the present invention can be said to be unprecedented.

EXAMPLE 9 preparation and evaluation of humanized antibody modified with L44 amino acid

9-1 construction of L44-modified antibody expression vector

Based on the findings of example 7, proline of L44 of the humanized SB04L chain was substituted with an amino acid having a charged side chain to suppress association of the H chain of the humanized XB12 and the L chain of the humanized SB 04. Specifically, proline present in the hydrophobic core at the interface between VH and VL is substituted with lysine (Lys) or arginine (Arg) whose side chain is positively charged, or glutamic acid (Glu) or aspartic acid (Asp) whose side chain is negatively charged. The humanized antibody gene was substituted by introducing a mutation using QuikChange site-directed mutagenesis kit (Stratagene) according to the method described in the attached manual. Each humanized antibody gene fragment whose amino acid had been substituted was inserted into the bispecific antibody expression vector used in example 6-2 or a usual antibody expression vector.

9-2 preparation of antibody for evaluation of Association Regulation and evaluation of Association Regulation

To evaluate the regulation of the association between the H chain and the L chain, three antibody expression vectors, i.e., the humanized SB04L chain (modified with L44), the wild-type humanized XB12H chain and the wild-type humanized XB12L chain, were prepared, and genes were introduced into HEK293H by the method described in example 4-2, and the antibodies were secreted in the culture supernatant. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

200ng of the purified antibody was subjected to reduction treatment in a sample buffer (TEFCO), and the resulting solution was injected into a 14% SDS-PAGE minigel (TEFCO) and subjected to electrophoresis. After electrophoresis, the gel was immersed in a 7% acetic acid solution containing 10% methanolFixing for 30 min, and performing SYPRO^(R)Staining was carried out by immersing the Ruby protein gel stain (BIO-RAD) in the stain for one day and night. Subsequently, the resultant was immersed in a 7% acetic acid solution containing 10% methanol for 1 hour for decolorization treatment, and image analysis was performed using a fluorescence detection apparatus FluorImagerSI (Amersham biosciences) to obtain a developed image. Using the obtained images, the band fluorescence intensities of the H chain and the L chain were calculated by Image Quant ver4.2(Amersham biosciences).

The results are shown in FIG. 6. Using the calculated fluorescence intensity values, the ratio (%) of the target XB12-L chain was calculated by "total XB12-L chain/L chain (XB12-L chain + SB04-L chain) × 100". When the amino acid of the humanized SB04L chain (L44) was wild-type proline (Pro), the ratio of the XB12-L chain was 47%; on the other hand, when L44 was substituted, the ratio of the humanized XB12L chain was found to increase by 1.8 to 1.9 times and to be 86 to 90%.

9-3 preparation of bispecific antibody for evaluation of clotting Activity and evaluation of clotting Activity

To evaluate the clotting activity, genes were introduced into HEK293H and expression induction was performed by the method shown in examples 4-2 and 4-3 using the prepared humanized SB04L chain (L44 modified) bispecific antibody expression vector and wild-type humanized XB12H chain, humanized XB12L chain and humanized SB04H chain bispecific antibody expression vector, pcDNA6/TR and pVgRXR. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

The coagulation activity was evaluated in accordance with the method described in example 5. The results are shown in FIG. 7. In the evaluation of association control, it was confirmed that: all antibodies with increasing ratios showed clotting activities that exceeded that of the wild type.

9-4 preparation of antibody for evaluation of binding Activity

To evaluate the binding activity of factor X, genes were introduced into HEK293H using antibody expression vectors of a wild-type humanized SB04H chain and a humanized SB04L chain (L44 modified) according to the method shown in example 4-2, and the antibody was secreted in the culture supernatant. The antibody concentration in the culture supernatant was then quantified in accordance with the method described in examples 4 to 5.

The binding activity of factor X was evaluated by the method described in example 2-2 using the culture supernatant. The results are shown in FIG. 8. Confirming that: the amino acid substitution of L44 did not alter the binding activity.

The above results show that: by modifying the amino acid at the L44 site of SB04L chain, the ratio of the target bispecific antibody can be increased without reducing biological activities such as binding activity to an antigen and coagulation activity instead of factor VIII. To date, no example has been reported including the method using knob and hole, in which association can be regulated without reducing the function by introducing a mutation into only one site amino acid in the polypeptide. Therefore, the present invention can be said to be unprecedented.

EXAMPLE 10 preparation and evaluation of humanized antibody modified with H39, L38 amino acid and L44 amino acid

10-1 construction of antibody expression vectors modified with H39, L38 and L44

Based on the findings of examples 8 and 9, H39 of the humanized XB12H chain and L38 and L44 of the humanized SB04L chain were substituted with amino acids having charged side chains to suppress association of the H chain of the humanized XB12 and the L chain of the humanized SB 04. Specifically, two amino acids, H39 of the humanized XB12H chain and L38 of the humanized SB04L chain, were substituted with glutamic acid (Glu) having the most significant effect in example 8, and proline present in L44 of the humanized SB04L chain was substituted with lysine (Lys) or arginine (Arg) having a positively charged side chain, or glutamic acid (Glu) or aspartic acid (Asp) having a negatively charged side chain. The humanized antibody gene was substituted by introducing a mutation using QuikChange site-directed mutagenesis kit (Stratagene) according to the method described in the attached manual. Each humanized antibody gene fragment whose amino acid had been substituted was inserted into the bispecific antibody expression vector used in example 6-2 or a usual antibody expression vector.

10-2 preparation of antibody for evaluation of Association Regulation and evaluation of Association Regulation

To evaluate the regulation of the association between the H chain and the L chain, three antibody expression vectors, i.e., a variant humanized SB04L chain, a variant humanized XB12H chain and a wild-type humanized XB12L chain, were used to introduce genes into HEK293H by the method described in example 4-2, and the antibody was secreted in the culture supernatant. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

The results are shown in FIG. 9. Using the calculated fluorescence intensity values, the ratio (%) of the target XB12-L chain was calculated by "XB 12-L chain/total amount of L chain (XB12-L chain + SB04-L chain) × 100". The ratio was 82% when two amino acids of the humanized XB12H chain (H39) and the humanized SB04L chain (L38) were modified to glutamic acid (Glu), and the humanized SB04L chain (L44) was wild-type proline (Pro); on the other hand, when two amino acids, i.e., the humanized XB12H chain (H39) and the humanized SB04L chain (L38), were modified to glutamic acid (Glu), and L44 was substituted in addition, the ratio of the humanized XB12L chain increased to 94 to 96%. The ratio was increased to 86 to 90% as compared with the case of example 9 in which L44 was independently substituted.

10-3 preparation of bispecific antibody for evaluation of clotting Activity and evaluation of clotting Activity

In order to evaluate the clotting activity, genes were introduced into HEK293H and expression induction was performed by using the prepared bispecific antibody expression vector of the variant humanized XB12H chain, the humanized XB12L chain and the humanized SB04H chain, the bispecific antibody expression vector of the wild-type humanized XB12H chain, the humanized XB12L chain and the humanized SB04H chain, pcDNA6/TR and pVgRXR, in accordance with the methods described in examples 4-2 and 4-3. Antibody purification and antibody concentration quantification were performed according to the methods described in examples 4-4 and 4-5.

The coagulation activity was evaluated in accordance with the method described in example 5. The results are shown in FIG. 10. In the evaluation of association control, it was confirmed that: all of the modified antibodies with increased ratios showed clotting activities equivalent to those of the wild type.

10-4 preparation of antibody for evaluation of binding Activity

To evaluate the binding activity of factor X, genes were introduced into HEK293H using antibody expression vectors of wild-type humanized SB04H chain and variant humanized SB04L chain according to the method shown in example 4-2, and the antibody was secreted in the culture supernatant. The antibody concentration in the culture supernatant was then quantified in accordance with the method described in examples 4 to 5.

The binding activity of factor X was evaluated by the method described in example 2-2 using the culture supernatant. The results are shown in FIG. 11. Confirming that: the two amino acids substituted for L38 and L44 did not alter the binding activity.

The above results show that: by modifying the amino acids of H39 of the XB12H chain and L38, L44 of the SB04L chain, the ratio of the bispecific antibody of interest can be increased without reducing biological activities such as binding activity to an antigen and coagulation activity of the substitute factor VIII. Confirming that: the ratio of bispecific antibodies increased with increasing number of amino acid modifications at the interface.

EXAMPLE 11 separation and structural characterization of conformational isomers of hVB22B u2-wz4sc (fv)2

11-1 preparation of humanized anti-human Mpl antibody hVB22B u2-wz4sc (Fv)2

A method for producing a humanized anti-Mpl antibody hVB22B u2-wz4sc (fv)2 (hereinafter referred to as u2-wz4) is shown in WO 2005/56604. The gene is made as follows: the PCR method was performed using a nucleotide sequence encoding a linker sequence (GlyGlyGlyGlySer) x3, and the PCR method was performed so as to have a nucleotide sequence consisting of VH-linker sequence-VL-linker sequence-VH-linker sequence-VL (SEQ ID NO: 12; see SEQ ID NO: 286 of WO 2005/56604). After confirming the nucleotide sequence of the gene, the DNA fragment was cloned into an expression vector pCXND3 to construct an expression vector, and the gene was introduced into CHO-DG44 cells to prepare a cell line with stable expression. Specifically, 20. mu.g of the expression vector and 0.75mL of CHO-DG44 cells (1X 10) suspended in PBS were added⁷cells/mL), the resulting mixture was cooled on ice for 10 minutes and transferred to a tube, after which it was pulsed at 1.5kV, 25 μ FD using Gene Pulser Xcell (BioRad). After 10 minutes of recovery at room temperature, the electroporated cells were added to CHO-S-SFMII medium (Invitrogen) containing 500. mu.g/mL Genetin (Invitrogen) and selected, establishing a CHO cell line producing u2-wz 4.

Humanized antibody hVB22B u2-wz4sc (fv)2, which was not tagged with FLAG tag (FLAG tag), was purified from the culture supernatant using a fusion protein recognizing epitope MG10 (Gln 213 to Ala231 in the amino acid sequence of human MpI) and GST. For purification of the MG10 and GST fusion protein, Glutathione-Sepharose 4B (Amersham biosciences) was used, and purification was performed according to the manufacturer's protocol. The purified MG10 and GST fusion protein were immobilized on HiTrap NHS-activated HP (Amersham biosciences) according to the manufacturer's protocol to prepare an affinity column. Culture supernatant of CHO cells expressing humanized antibody hVB22B u2-wz4sc (Fv)2 was applied to a column on which MG10-GST fusion protein was immobilized, so that humanized antibody hVB22B u2-wz4sc (Fv)2 was adsorbed on the column, followed by elution with 100mM glycine-HCl (pH3.5), 0.01% Tween 80. The eluted fractions were neutralized with 1M Tris-HCl (pH7.4) and subjected to gel filtration chromatography using HiLoad 16/60Superdex200pg (Amersham biosciences) to purify the monomer. The buffer for gel filtration chromatography was 20mM citric acid buffer (pH7.5) containing 300mM NaCl and 0.01% Tween 80.

Separation and purification of conformational isomers of 11-2, hVB22B u2-wz4sc (Fv)2

hVB22B u2-wz4sc (Fv)2 has the sequence VH₁-linker-VL₂-linker-VH₃-linker-VL₄Sc (Fv)2 of (a), it is therefore considered that, like VB22B sc (Fv)2, the following two conformational isomers exist in the structure according to the combination of Fv (molecules that are not covalently bound between VH and VL): bivalent scFv type, wherein VH₁And VL₂、VH₃And VL₄Fv are formed respectively; and the single-chain diabody type, in which VH₁And VL₄、VH₂And VL₃Fv were formed separately (FIG. 12).

The separation of the conformational isomers of hVB22B u2-wz4sc (Fv)2 was studied and shown to be: using cation exchange chromatography BioAssist S (TOSOH), the various components of hVB22B u2-wz4sc (Fv)2 were separated under the following elution conditions.

Mobile phase A: 20mM sodium phosphate, pH7.5

Mobile phase B: 20mM sodium phosphate, 500mM NaCl, pH7.5

Flow rate: 0.8 ml/min

Gradient: b0% → B35% (30 minutes)

Under the above conditions, hVB22B u2-wz4sc (fv)2 separated into two peaks. The chromatogram shown in FIG. 13 was obtained, starting from the peak with a short retention time and being designated as peak 1 and peak 2, respectively.

The molecular weights of peak 1 and peak 2 were determined using a Q-TOF Mass spectrometer (Q T of Ultima, Micro Mass). The sample solution was injected into Q-TOF, and the resulting multivalent ion spectrum (+) was deconvoluted with the attached software (MassLynx), resulting in: peak 1 has a molecular weight of 53768Da, and peak 2 has a molecular weight of 53769 Da. From this, it was found that peak 1 and peak 2 have the same molecular weight.

Peptide maps were made for peak 1 and peak 2. After reductive denaturation, carboxymethylation, it was broken down into peptide fragments with trypsin, and a peptide map was obtained by reverse phase chromatography (YMC-Pack-ODS). When the peptide patterns of peak 1 and peak 2 are compared, as shown in FIG. 14, the peptide patterns of peak 1 and peak 2 are identical, and it is found that the primary amino acid structures are identical.

Since hVB22B u2-wz4sc (Fv)2 has no sugar chain, the molecular weights of peak 1 and peak 2 measured by TOF-MASS are the same, and the peptide patterns of peak 1 and peak 2 are the same, as can be seen from the above: peaks 1 and 2 are conformational isomers that differ from each other in steric structure.

hVB22B u2-wz4sc (Fv)2 has the sequence VH₁-linker-VL₂-linker-VH₃-linker-VL₄According to the combination of Fv (molecules that are not covalently bound between VH and VL), two conformational isomers exist in the structure, as shown in fig. 12: bivalent scFv type, wherein VH₁And VL₂、VH₃And VL₄Fv are formed respectively; and the single-chain diabody type, in which VH₁And VL₄、VH₂And VL₃Fv were formed separately. Peak 1 and peak 2 are considered to be a certain structure of the bivalent scFv type and the single chain diabody type, respectively.

Analytical methods for identifying two conformers have developed limited proteolytic methods. The linker moiety of sc (Fv)2 has a relatively free structure and low resistance to proteases, and therefore, subtilisin A, which is one of proteases, was reacted with Peak 1, Peak 2, and hVB22B u2-wz4sc (Fv)2 (Peak 1: Peak 2. about.1: 4) under the following conditions.

20mM sodium citrate, 150mM NaCl, pH7.5

hVB22B u2-wz4sc (Fv)2 peak 1 or peak 2: 0.15mg/mL

Subtilisin A: 10 μ g/mL

30 minutes at 37 DEG C

After the reaction, reduced SDS-PAGE was performed using 12.5% Phastgel Homogeneous. As a result, as shown in fig. 15, hVB22B u2-wz4sc (Fv)2 showed the same band pattern for all of (bulk), peak 1, and peak 2. Since the three linker moieties of hVB22B u2-wz4sc (Fv)2 are cleaved, the resulting fragments have specific bands, and thus the linker moiety of hVB22B u2-wz4sc (Fv)2 can be partially and limitedly cleaved by using the above reaction conditions.

In the bivalent scFv-type and single-chain diabody-type structures, when 1 of 3 linkers is cleaved, as shown in fig. 16, in the undenatured state, neither of the 3 linkers is cleaved, and the apparent molecular weight is changed in the single-chain diabody-type structure, due to the non-covalent bond between VH and VL; in the bivalent scFv type, when the central linker is cleaved, a molecular species having a half molecular weight is generated. Therefore, the linker was partially cleaved under the above reaction conditions, and the obtained hVB22B u2-wz4sc (Fv)2 as a whole, peak 1 and peak 2 were subjected to gel filtration chromatography using TSK SuperSW2000 (TOSOHO). Gel filtration chromatography was performed under the following conditions.

Mobile phase: DPBS (-), pH7.4

Flow rate: 0.2 ml/min

As a result, as shown in fig. 17, no low molecular weight peak was observed at all in peak 2; in contrast, a low molecular weight (about half molecular weight) peak was observed in peak 1. A low molecular weight peak was observed in the whole of the mixture of peak 1 and peak 2-hVB 22B u2-wz4sc (Fv)2, the amount of the peak corresponding to the existence ratio of peak 1. Thus, it was determined by this result that: peak 1 is a bivalent scFv type, and peak 2 is a single-chain diabody type.

EXAMPLE 12 preparation of VH/VL interface-modified sc (fv)2, analysis and characterization of conformational isomer

Preparation of VH/VL interface-modified sc (fv)2

The VH/VL interface-modified sc (fv)2 was prepared in the following manner to confirm whether the small molecule antibody sc (fv)2 can form a conformational isomer of sc (fv)2 when the association is regulated by modifying the VH/VL interface.

Gln at position 39 of the amino acid-VH (position 39 in the amino acid sequence shown in SEQ ID NO: 13; see SEQ ID NO: 289 of WO 2005/56604) and Gln at position 38 of VL (position 43 in the amino acid sequence shown in SEQ ID NO: 14; see SEQ ID NO: 289 of WO 2005/56604) which form the VH/VL interface of u2-wz4 were modified as follows. First, gene hVB22 (designated as v1, the nucleotide sequence is shown as SEQ ID NO: 15; and the amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO: 16) 8922 22B u2-wz4(v1) sc (fv)2 (hereinafter, referred to as v1) was prepared, in which Gln at position 39 of VH1 (genetic codon CAG) was changed to Glu (genetic codon GAG), Gln at position 38 of VL2 (genetic codon CAG) was changed to Glu (genetic codon GAG), Gln at position 39 of VH3 (genetic codon CAG) was changed to Lys (genetic codon AAG), and Gln at position 38 of VL4 (genetic codon CAG) was changed to Lys (genetic codon AAG). Further, gene hVB22 (designated as v3 hereinafter, the nucleotide sequence is shown as SEQ ID NO: 17; and the amino acid sequence encoded by this nucleotide sequence is shown as SEQ ID NO: 18) 8922 22B u2-wz4(v3) sc (fv)2 (designated as v3 hereinafter) was prepared, in which Gln at position 39 (genetic codon CAG) of VH1 was changed to Glu (genetic codon GAG), Gln at position 38 (genetic codon CAG) of VL2 was changed to Lys (genetic codon AAG), Gln at position 39 (genetic codon CAG) of VH3 was changed to Lys (genetic codon AAG), and Gln at position 38 (genetic codon CAG) of VL4 was changed to Glu (genetic codon AAG). Gene modification was carried out by introducing a point mutation using a QuikChange site-directed mutagenesis kit (manufactured by STRATAGENE) according to the manufacturer's protocol. After confirming the nucleotide sequence of each gene, the DNA fragment was cloned into an expression vector pCXND3 to construct an expression vector, and the gene was introduced into CHO-DG44 cells to prepare a cell line stably expressing the gene. The V1-producing CHO cell line and the V3-producing CHO cell line were established according to the method described in example 11.

For variants v1, v3, the monomer molecules were purified using columns immobilized with MG10-GST fusion protein, according to the method described in example 11. From the results of gel filtration chromatography shown in FIG. 18, it can be seen that: for variants v1, v3, there was a reduction in aggregates above the dimer in the culture supernatant; the monomer ratio was higher than that of u2-wz4 before modification (59%), the monomer ratio of v1 was higher than that of 89%, and the monomer ratio of v3 was higher than that of 77%. It is assumed that in variants v1 and v3, the amino acid at the VH/VL interface is modified to suppress undesired association by electrostatic repulsion and promote desired association. From the above, it was found that the monomer molecule was successfully and efficiently expressed by the association regulation.

Analysis and identification of conformational isomers of VH/VL interface-modified sc (fv)2

The conformational isomer existence ratio of the obtained VH/VL interfacial variants v1, v3 and the unmodified u2-wz4 was analyzed by cation exchange chromatography and isoelectric point electrophoresis. In addition, structural identification was performed by limited proteolysis.

Cation exchange chromatography was performed as follows.

Column: TSK-gel Bioassist S, 4.6 mm. phi. times.50 mm (manufactured by TOSOH Co., Ltd.)

Flow rate: 0.8 mL/min

Detection wavelength: 220nm

Elution conditions:

eluent A: 20mmol/L phosphate buffer (pH7.0)

Eluent B: 20mmol/L phosphate buffer/500 mmol/L NaCl (pH7.0)

Gradient:

time (min) B%

0 0

5 0

25 30

25.1 100

35 100

35.1 0

Isoelectric point electrophoresis was performed as follows. PhastGel Dry IEF gel (Amersham biosciences) was swollen with the following gel swelling solution for 30 minutes or more. The sample was added to the gel that had been swollen previously, and electrophoresis was performed using the PhastSystem under the following electrophoresis conditions. After electrophoresis, the sample was immersed in a 20% TCA solution for 30 minutes, washed with Milli-Q water for 5 minutes X3 times or more, and then subjected to Coomassie staining or silver staining depending on the protein concentration of the sample. In Coomassie staining, a staining solution containing 0.1% CuSO was used₄(w/v) 0.02% CBB was used as a staining solution for staining, and then destaining was performed with 30% methanol containing 10% acetic acid. For silver staining, silver staining was performed using a silver staining kit and Protein (Amersham Biosciences), according to the standard protocol attached to the kit.

< gel swelling solution >

Pharmalyte 8.5-10 80μL

Biolyte 7-9 10μL

Biolyte 3-9 10μL

20% Glycerol 2.0mL

< electrophoresis procedure >

SAMPLE APPLICATION DOWN AT step2 0Vh

SAMPLE APPLICATION UP AT step2 0Vh

Step 12000V 2.5mA 3.5W 15 ℃ 75Vh

Step 2200V 2.5mA 3.5W 15 ℃ 15Vh

Step 32000V 2.5mA 3.5W 15 ℃ 410Vh

The structure was identified by the limited protease method under the following conditions. Subtilisin A was reacted with u2-wz4 purification peak 1, u2-wz4 purification peak 2, variant v1 and variant v3 under the following conditions.

20mM sodium citrate, 150mM NaCl, pH7.5

hVB22B u2-wz4sc (Fv)2 peak 1 or peak 2: 0.15mg/mL

Subtilisin A: 10 μ g/mL

30 minutes at 37 DEG C

The obtained reaction solution was analyzed by gel filtration chromatography under the following conditions.

Column: TSKgel Super2000sw (TOSOH)

Eluent: 50mM sodium phosphate, 300mM KCl, pH7.0

Flow rate: 0.2 mL/min

And (3) detection: 220nm

Conformational isomers were analyzed by cation exchange chromatography and isoelectric point electrophoresis, and from the analysis results shown in fig. 19 and 20: u2-wz4 was expressed as a mixture of two conformers, 24% of which were of the bivalent scFv type and 76% of which were of the single chain diabody type; in contrast, 100% of variant v1 was expressed as single chain diabody-type conformers; 100% of the variant v3 was expressed as bivalent scFv-type conformers. In addition, as shown in fig. 21, from the results of limited proteolysis: the variant v3 shows a low molecular peak as with the u2-wz4 purification peak 1; as with u2-wz4 purified peak 2, no low molecular peak was observed in variant v1, indicating that: variant v1 is expressed as a single chain diabody-type conformer; variant v3 is expressed as a bivalent scFv-type conformer.

Example 13 evaluation of Activity and evaluation of stability of VH/VL interface-modified sc (fv)2

Evaluation of biological Activity of VH/VL interface-modified sc (fv)2

It has been reported in the literature (Blood 2005; 105: 562-. Thus, isolated conformers were evaluated for TPO-like agonist activity using BaF 3-human Mpl or BaF 3-monkey Mpl that showed TPO-dependent proliferation.

Each cell was washed 2 times with RPMI1640 containing 1% fetal bovine serum (Invitrogen), and then suspended in RPMI1640 containing 10% fetal bovine serum to a concentration of 4X 10⁵cells/mL, 60. mu.L/well into 96-well plates. To each well was added 40. mu.L of rhTPO (R) at various concentrations&D) Or a sample of conformer at 37 ℃ with 5% CO₂Incubated under conditions for 24 hours. WST-8 Reagent (Cell Count Reagent SF, NakalaiTesque) was added at 10. mu.L/well, absorbance at 450nm was measured immediately with Benchmark Plus (control 655nm), and after 2 hours of incubation, absorbance at 450nm was measured again (control 655 nm). The WST-8 reagent exhibits a color reaction at 450nm depending on the number of living cells, and therefore, TPO-like agonist activity was evaluated using a change in absorbance for 2 hours as an index.

The purified VB22B sc (fv)2 conformer was used to evaluate the TPO-like agonist activity of BaF 3-human and BaF 3-monkey mpls, respectively, as shown in FIG. 22. Comparing the agonist activity of the conformers of peak 1 and peak 2 found: peak 2 showed significantly higher activity. The above shows that: in order to exert TPO-like agonist activity, the anti-Mpl antibody sc (fv)2 must form a single-chain diabody-type structure.

Agonist activity of VH/VL interface variants v1 and v3 was assessed as described in example 1. Agonist activity was very different between conformers, as shown in figure 12, peak 2 of the single chain diabody structure showed very high agonist activity; whereas peak 1 of the bivalent scFv structure had very low activity. As shown in FIG. 22, variant v1 showed the same activity as peak 2, and variant v3 showed the same activity as peak 1. Thus, biological activity also indicates: variant v1 forms a single chain diabody structure, and variant v3 forms a bivalent scFv structure.

Evaluation of stability of VH/VL interface-modified sc (fv)2

The stability of u2-wz4 purification peak 1, u2-wz4 purification peak 2, and variant v1 and variant v3 was evaluated by differential scanning calorimetry by measuring the denaturation middle temperature (Tm value) under the following conditions.

DSC: N-DSCII (manufactured by Applied Thermodynamics Co., Ltd.)

Solution conditions: 20mM sodium citrate, 300mM NaCl, pH7.0

Protein concentration: 0.1mg/mL

Scanning speed: 1 deg.C/min

The results of each DSC measurement are shown in fig. 23. Therefore, the following steps are carried out: the Tm values of u2-wz4 purified peak 2 and variant v1 were approximately equal to those of the unmodified form, and the stability was also the same. u2-wz4 purified peak 1 had slightly lower stability than variant v3 for variant v 3. In adjusting the interface according to the method using the knob-into-hole technique, for example, it has been reported (Acta Pharmacol sin.200526 (6): 649-58): in the hetero-association of the CH3 domains of IgG, the unmodified CH3 domain had a Tm of 80.4 ℃ and the modified CH3 domain had a Tm of 69.4 ℃, with significantly lower Tm and reduced stability. In contrast, in the present invention, it was confirmed that: the association can be adjusted without decreasing stability.

Next, a thermal acceleration test was carried out under the following conditions to evaluate the stability of u2-wz4 purification peak 1, u2-wz4 purification peak 2, and VH/VL interface modifier-variants v1 and v 3.

< thermal acceleration Condition >

Solution conditions: 20mM sodium citrate, pH6.0

Protein concentration: 0.25mg/mL

And (3) acceleration conditions: 40-6 days, 12 days

The heat accelerated sample was analyzed by gel filtration chromatography and cation exchange chromatography under the following conditions.

As shown in fig. 24, from the analysis result of gel filtration chromatography: the monomer residue ratios of u2-wz4 purified peak 2 and variant v1 were approximately the same, and the association stability was approximately the same. Further, the monomer residue ratios of u2-wz4 purified peak 1 and variant v3 were also approximately the same, and the association stabilities were approximately the same for both conformers.

As shown in fig. 25, from the analysis result of the cation exchange chromatography: the purification peak 1 of the unmodified body was isomerized into the peak 2 by isomerization reaction, and the purification peak 2 of the unmodified body was isomerized into the peak 1 by isomerization reaction; in contrast, VH/VL interface variants v1 and v3 did not isomerize even after thermal acceleration. Thus, it can be seen that: by modifying the VH/VL interface, only one of the two conformers can be expressed in 100% state; in addition, each conformational isomer obtained does not undergo isomerization reaction and can be stored stably.

In this example, by using VH/VL interfacial modifications appropriate for v1 and v3, it was found that: only one of the two conformers may be expressed in a state in which 100% is present. As a method for regulating the VH/VL interface of a single-chain antibody to obtain a target structure, a method of regulating the structure of a bispecific diabody using the knob-to-inteo-hole technique is known (Protein Sci.1997 Apr; 6 (4): 781-8, remodelling domain interfaces to exchange heterologous heterodimerization. (the reconstructed domain interface promotes heterodimer formation), Zhu Z, Presta LG, Zapatata G, Carter P.). It was reported that in this method a total of 4 amino acids were modified in each VH/VL interface, thereby increasing the rate of formation of the desired heterodimeric structure from 72% to 92%. In contrast, the present invention successfully obtains a target structure at a rate of 100% without decreasing thermal stability and stability of conformers by modifying amino acids at 4 positions.

EXAMPLE 14 humanization of bispecific antibody with hybrid L chain

A bispecific antibody (Japanese patent application No. 2005-112514) comprising: the combination of anti-factor IXa antibody A69-VH, anti-factor X antibody B26-VH, and hybrid L chain (BBA) was most effective in shortening clotting time.

14-1. homology search of human antibodies

Human antibody amino acid sequence data were obtained from the generally disclosed Kabat database (ftp:// ftp. ebi. ac. uk/pub/databases/Kabat /) and IMGT database (http:// IMGT. cines. fr), and homology searches were performed using the constructed databases, divided into a mouse A69-H variable region (amino acid sequence: SEQ ID NO: 57), a mouse B26-H variable region (amino acid sequence: SEQ ID NO: 58), and a mouse BBA-L variable region (amino acid sequence: SEQ ID NO: 59). As a result, it was confirmed that: since they have high homology with the human antibody sequences shown below, the framework regions (hereinafter referred to as FR) used as a humanized antibody are determined.

(1) A69-H variable region: KABATID-000064(Kabat database)

(Kipps et al, J Clin invest.1991; 87: 2087-

(2) B26-H chain variable region: EMBL Access No. AB063872(IMGT database)

(unpublished data)

(3) BBA-L variable region: KABATID-024300(Kabat database)

(Welschof et al, JImmunol method.1995; 179: 203-

A humanized antibody was prepared by grafting complementary epitope regions (hereinafter referred to as CDRs) of each mouse antibody to the human antibody FRs of (1) to (3).

The human antibody having high homology to the human antibodies of (1) to (3) was searched for a secretion signal sequence using a homology search website (http:// www.ncbi.nlm.nih.gov/BLAST /) published by NCBI. The following secretion signal sequence obtained by the search was used.

(1) A69-H variable region: GenBank Accession No. AF062257

(2) B26-H chain variable region: GenBank Accession No. AAC18248

(3) BBA-L variable region: GenBank Accession No. AAA59100

14-2 construction of humanized antibody Gene expression vector

12 synthetic oligo DNAs of about 50 bases are prepared alternately in the nucleotide sequence encoding the amino acid sequence from the secretion signal sequence to the antibody variable region, and about 20 bases are annealed to the 3' -end side. Further, two kinds of primers were prepared, one of which was annealed to the 5 ' -end of the antibody variable region gene and had an XhoI cleavage sequence, and the other of which was annealed to the 3 ' -end of the antibody variable region gene and had an SfiI cleavage sequence, and also encoded the 5 ' -end sequence of the intron sequence.

Mu.l each of the synthetic oligo DNAs adjusted to 2.5. mu.M was mixed, and 1 XTaKaRa ExTaq buffer, 0.4mM dNTPs, and 0.5 unit TaKaRa Ex Taq (all Takara Shuzo Co., Ltd.) were added to prepare 48. mu.L of a reaction solution. After the reaction solution was incubated at 94 ℃ for 5 minutes, the reaction was cycled 2 times at 94 ℃ for 2 minutes, 55 ℃ for 2 minutes, and 72 ℃ for 2 minutes to carry out the assembly and extension reactions of each synthetic oligo DNA. Subsequently, 1. mu.L of primers annealing to the 5 '-end and 3' -end sides of the antibody gene, each at 10. mu.M, were added, and the reaction was cycled 35 times at 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 minute, and further at 72 ℃ for 5 minutes to amplify the antibody variable region gene. After PCR, the entire reaction solution was subjected to 1% agarose gel electrophoresis. The amplified fragment of the target size (about 400bp) was purified by the method described in the appendix, using QIAquick gel recovery kit (QIAGEN), and eluted with 30. mu.L of sterile water. The above fragment was cloned by the method described in the appendix, using pGEM-T Easy Vector Systems (Promega). The nucleotide sequence of each DNA fragment was determined by using BigDye Terminator cycle sequencing kit (Applied Biosystems) by using ABI PRISM 3730xL DNA Seguencer (Applied Biosystems) according to the method described in the appendix.

The plasmid having the H chain variable region fragment inserted therein was digested with XhoI and SfiI, and the plasmid having the L chain variable region fragment inserted therein was digested with EcoRI, and it was confirmed that all of the plasmids had the correct humanized antibody variable region gene sequence. Thereafter, the reaction solution was subjected to 1% agarose gel electrophoresis. The DNA fragment of the desired size (about 400bp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. Then, an expression vector for animal cells was prepared as follows. For preferential expression of IgG4 in which the H chain is heterogeneously combined, IgG4 in which the CH3 moiety is substituted with an amino acid is used with reference to the Nanbs-into-holes technique for IgG1 (non-patent document 3). Substituted amino acids (-ppcpsscp- → -ppcpPcp-) are further introduced into the hinge to promote the formation of dimers of the H chain. An expression vector was obtained by inserting a constant region Gene substituted with Y349C or T366W into pCAGGS (Niwa et al, 1991Gene, 108: 193-199.) having a chicken β -actin promoter, and a humanized A69H chain expression vector was prepared by inserting a humanized A69H chain variable region antibody Gene fragment into the obtained expression vector. Furthermore, an expression vector was obtained by inserting a constant region gene substituted with E356C, T366S, L368A, and Y407V into pCAGGS, and a humanized B26H variable region antibody gene fragment was inserted into the obtained expression vector to prepare a humanized B26H chain expression vector. A plasmid (pCAG-g. kappa.DNA) obtained by inserting a wild-type antibody L chain constant region into pCAGGS was digested with EcoRI to prepare an expression vector into which a humanized BBA L chain variable region antibody gene fragment was inserted. Coli strain DH5 a (eastern textiles) was transformed using a rapid DNA ligation kit (Roche Diagnostics) for ligation.

14-3 preparation of humanized bispecific antibody

The humanized bispecific antibody was expressed using the method described in example 4-2 or the following method. HEK293H strain (Invitrogen) derived from human fetal kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% FCS (Invitrogen) at 5-6 × 10⁵Cell density per mL, 10mL of each cell-adhering dish (10 cm diameter, CORNING) was seeded in CO₂Incubator (37 ℃, 5% CO)₂) After internal culture for one day and night, the medium was aspirated, and 6.9mL of CHO-S-SFM-II (Invitrogen) was added) And (4) a culture medium. A total of 13.8. mu.g of plasmid DNA mixture prepared in 14-2, 20.7. mu.L of 1. mu.g/mL polyethyleneimine (Polysciences Inc.) and 690. mu.L of CHO-S-SFMII medium were mixed, left to stand at room temperature for 10 minutes, and the resulting mixture was added to the cells of each dish in CO₂Incubator (37 ℃, 5% CO)₂) Internal culture is carried out for 4-5 hours. Thereafter, 6.9mL of CHO-S-SFM-II medium was added in CO₂Culturing in an incubator for 3 days. The culture supernatant was recovered, followed by centrifugation (about 2000g, 5 min, room temperature) to remove cells, and then by passing through a 0.22 μm filter, MILLEX^(R)-GV (Millipore) sterilization. The sample was stored at 4 ℃ until use.

Next, antibody purification was performed according to the method shown in example 4-4, and the antibody concentration was quantified according to the method shown in example 4-5 or the following method. Protein A was immobilized on Sensor Chip CM5(BIACORE) using BIAcore3000 (BIACORE). Specifically, a protein A solution diluted to 50. mu.g/mL with 10mM sodium acetate aqueous solution (pH4.0, BIACORE) was reacted with the activated sensor chip at a flow rate of 5. mu.L/min for 30 minutes according to the manufacturer's protocol. Then, a sealing operation was performed to produce a sensor chip on which protein a was immobilized. Using the sensor chip, the concentrations of the culture supernatant and the purified product were measured using BIAcore Q. HBS-EP Buffer (BIACORE) was used in the immobilization and concentration determination of the sensor chip. As a standard for concentration measurement, human IgG4 (humanized anti-TF antibody, see WO99/51743) was used, and the human IgG4 was diluted 6 stages in HBS-EP buffer in 2-fold stepwise dilution from 2000 ng/mL.

14-4 evaluation of Activity of humanized bispecific antibody and modification of antibody sequence

To evaluate the plasma clotting energies of the humanized bispecific antibody and the chimeric bispecific antibody prepared (A69/B26/BBA), the effect of the antibodies on APTT was investigated using F.VIII deficient plasma according to the method of example 5. For a humanized bispecific antibody that reduces blood coagulation ability, the amino acids of the human antibody FR are modified with the aim of increasing the activity. In addition, upon expression secretion, the following 3 antibodies were expressed: humanized a 69/humanized BBA antibody, humanized B26/humanized BBA antibody, humanized a 69/humanized B26/humanized BBA bispecific antibody. These 3 antibodies were isolated and the bispecific antibody alone was purified by modifying the amino acids such that the isoelectric point of the humanized a69H chain variable region was decreased and the isoelectric point of the humanized B26H chain variable region was increased. Specifically, mutations were introduced into the variable regions of the humanized antibody by the method described in the attached manual using QuikChange site-directed mutagenesis kit (Stratagene). The plasmid having the H chain variable region fragment inserted therein was digested with XhoI and SfiI, and the plasmid having the L chain variable region fragment inserted therein was digested with EcoRI, and it was confirmed that all of the plasmids had the target humanized antibody variable region gene sequence. Thereafter, the reaction solution was subjected to 1% agarose gel electrophoresis. The DNA fragment of the desired size (about 400bp) was purified using QIAquick gel recovery kit (QIAGEN) according to the method described in the appendix, and eluted with 30. mu.L of sterile water. Thereafter, an expression vector for animal cells was prepared in the same manner as in example 14-2. Humanized bispecific antibodies were prepared according to the method shown in example 14-3, and the blood coagulation activity was evaluated according to the method shown in example 5.

Humanized bispecific antibody (humanized A69(hA 69-PFL)/humanized B26(hB 26-PF)/humanized BBA (hAL-AQ)) having the same activity as the chimeric bispecific antibody (A69/B26/BBA) was obtained by repeating amino acid modification of FR sequence and evaluation of blood clotting ability (FIG. 26). The variable region sequence of each antibody is shown below as SEQ ID NO.

(1) Humanized a69 antibody VH (hA69-PFL) SEQ ID NO: 19 (nucleotide sequence), SEQ ID NO: 20 (amino acid sequence)

(2) Humanized B26 antibody VH (hB26-PF) SEQ ID NO: 21 (nucleotide sequence), seq id NO: 22 (amino acid sequence)

(3) Humanized BBA antibody VL (hAL-AQ) SEQ ID NO: 23 (nucleotide sequence), seq id NO: 24 (amino acid sequence)

Example 15 selection of amino acid modification sites of constant region to increase the efficiency of formation of bispecific antibody

The amino acids present at the constant region CH3 interface were modified to study the enhancement of heterodimer-bispecific antibody formation efficiency by electrostatic repulsion. First, starting from the crystal structure of the CH3 region (Protein Data bank, PDB code 1OQX), amino acid pairs that form electrostatic interactions when forming CH3 homodimers were sought. As a result, it was found that: in the interface where CH3 homodimers were formed, three pairs of amino acids (numbers in EU numbering order (Kabat EA et al, 1991.Sequences of Proteins of immunologica interest. nih)) at positions 356 and 439, 357 and 370, 399 and 409 of the H chain were positively and negatively charged, respectively, and electrostatic interactions occurred, and thus they were selected as sites for modification. The charges of the positively and negatively charged amino acid pairs are switched, and heterodimer formation is believed to be promoted by this approach. The principle of this adjustment is shown in fig. 27. Modification by introducing a disulfide bond into the CH3 interface was also attempted. The modified amino acid positions are summarized in table 1.

EXAMPLE 16 modification of amino acids at the constant region CH3 interface of humanized bispecific antibody

To modify the amino acids at the interface of the H chain constant region CH3 selected in example 15, the following procedure was performed. Amplifying each H chain constant region by PCR using H chain constant region genes of human IgG1 and human IgG4 as templates, using a5 'end primer and another primer, the 5' end primer being designed such that a nucleotide sequence encoding two amino acids (Ala-Ser) on the N-terminal side of the H chain constant region becomes a NheI recognition sequence (GCTAGC); the other primer was designed to anneal at the 3' end and has a NotI recognition site. Thereafter, pBCH (including IgG1 constant region gene) and pBCH4 (including IgG4 constant region gene) were prepared, which were ligated to a vector obtained by digesting pBluescriptKS + vector (Toyobo) with Nhel and NotI (both Takara Shuzo). PCR was performed using two primers, one of which was complementary to the 5' terminal nucleotide sequence of the H chain variable region of humanized a69 antibody and humanized B26 antibody and had Kozak sequence (CCACC) and EcoRI recognition sequences; the other primer is located on the 3' terminal nucleotide sequence having the NheI recognition sequence. The resulting PCR product was inserted into pBCH or pBCH4 digested with EcoRI and NheI (both Takara Shuzo), and the variable region and the constant region were ligated. Next, a mutation was introduced into the H chain constant region by the method described in the appendix, using the QuikChange site-directed mutagenesis kit (Stratagene), to modify the amino acids present at the CH3 interface of the H chain constant region. The plasmid into which the H chain gene fragment was inserted was digested with EcoRI and NotI (both prepared from Takara Shuzo) and confirmed to have the target H chain constant region gene sequence. Thereafter, the reaction solution was subjected to 1% agarose gel electrophoresis. The H chain gene fragment of the desired size (about 1400bp) was purified by the method described in the appendix, using QIAquick gel recovery kit (QIAGEN), and eluted with 30. mu.L of sterile water. Then, this fragment was inserted into EcoRI and NotI digested pCAGGS to prepare an expression plasmid. Humanized bispecific antibody preparation according to the embodiment of 14-3 shows the method. The modified amino acid positions are summarized in table 1. The number of modification sites in Table 1 is ordered by EU numbering (Kabat EA et al, 1991.Sequences of Proteins of immunological interest. NIH)). The letter before the modification site number is a letter mark of the amino acid before modification, and the letter after the modification site number is a letter mark of the amino acid after modification.

[ Table 1]

In the above table, KiH represents the use of non patent literature 3 described in the Knobs-into-holes technology obtained variants.

EXAMPLE 17 evaluation of Forming efficiency and stability of bispecific antibody (IgG4 type) having CH3 interface modified

The wild type IgG4, KiH, s1, s2, s3, w1, w2, w3, s1C, s2C, s3C, w3C, and w3C2 were analyzed by cation exchange chromatography (IEX) to evaluate the efficiency of formation of bispecific antibody (hereinafter referred to as BiAb). The peak area ratios of the homodimer a-Homo of the humanized a69 antibody, the heterodimer BiAb of the humanized a69 antibody and the humanized B26 antibody, and the homodimer B-Homo of the humanized B26 antibody were calculated under the following conditions for cation exchange chromatography analysis.

Column: ProPac WCX-10, 4X 250mm, (Dionex)

Mobile phase: a: 10mmol/L NaH₂PO₄/Na₂HPO₄，pH6.25

B：10mmol/L NaH₂PO₄/Na₂HPO₄，500mmol/L NaCl，pH6.25

Flow rate: 1.0 mL/min

Gradient: 10% B (5 minutes) → (40 minutes) → 60% B → (5 minutes) → 100% B (5 minutes)

And (3) detection: 220nm

When wild-type, KiH, s2, s3, s1C, s2C, s3C, w3C, w3C2 were analyzed by IEX as described above, the BiAb was purified by collecting the BiAb peak components. The BiAb fraction was concentrated with AmiconUltra, MWCO 10000(Millipore), dialyzed overnight against 20mM sodium acetate, 150mM NaCl (pH6.0) with cooling, and then recovered. BiAb concentration was adjusted to 0.1mg/mL, and the resulting mixture was dispensed into vials as initial samples and 60 to 1 week samples, 2 vials were prepared for each sample, and stability tests were conducted for 60 to 1 week. The residue of the monomer peak (monomer peak area of the sample at 60 ℃ to 1 week/monomer peak area of the initial sample. times.100) was calculated by analysis using gel filtration chromatography (SEC). The gel filtration chromatography analysis conditions were as follows.

Column: super3000(TOSOH)

Mobile phase: 50mM sodium phosphate, 300mM KCl, pH7.0

Flow rate: 0.2 mL/min

And (3) detection: 220nm

IEX chromatography of wild type IgG4, s1, s2, s3, w1 is shown in figure 28; the formation ratios of A-Homo, BiAb and B-Homo in wild type, KiH, s1, s2, s3, w1, w2, w3, s1C, s2C, s3C, w3C and w3C2 are shown in FIG. 29. The residual monomer ratio after 60 ℃ to 1 week is shown in FIG. 30.

As shown in fig. 28 and 29, the target BiAb formation efficiency of the CH3 interface modified form found in this example was significantly improved as compared with the wild type. Since CH3 is located in the constant region, when a natural amino acid is modified, it is desirable that the number of modification sites is small from the aspect of antigenicity. In KiH, 4 sites in both H chains are modified in order to introduce knob and hole; in addition, in order to introduce disulfide bonds, 2 sites were modified, so that 6 sites were modified in total. Therefore, as shown in fig. 29, the BiAb formation efficiency is high. However, as is clear from the stability test results shown in fig. 30: although a disulfide bond was introduced, thermostability was significantly reduced compared to the wild type. In order to develop an antibody into a medical drug, a stable preparation is required, and therefore, high thermal stability is desired.

On the other hand, the CH3 interface modified forms found in this example successfully improved the target BiAb formation efficiency significantly as compared with the wild type. Among the above variants, for example, s2, s3, w1, w2, w3, and s1C, the high BiAb formation efficiency of 90% or more was obtained by modification of less than KiH (6-site modification) at 2 or 4 sites in total, and the antigenic risk was considered to be smaller. In addition, from the stability test results shown in fig. 30, it is clear that: among mutants, for example, s2, s3, w3, w3C, and w3C2 have a high BiAb formation efficiency of 90% or more and have a high thermal stability (a high monomer residue rate) as compared with KiH; s3, s2C, s3C, w3C and w3C2 have higher thermal stability than wild type, and are useful for developing stable pharmaceutical preparations.

In this example, it is found that: the efficiency of formation of the desired BiAb can be greatly improved by modifying the H chain at positions 356, 357, 370, 399, 409 and 439 of the CH3 interface to introduce molecular repulsion due to charges. It was also found that: by introducing the above modifications alone or in combination and introducing a disulfide bond, the efficiency of BiAb formation can be greatly improved, with fewer modifications than in KiH; the efficiency of BiAb formation can be greatly improved while maintaining stability higher than KiH, even higher than the thermal stability of wild type.

EXAMPLE 18 evaluation of coagulation Activity of bispecific antibody having CH3 interface modified

The clotting activity was evaluated by the method shown in example 5 using the IgG 4-type bispecific antibody (s1, s2, s3, w1, w2, and w3) modified at the CH3 interface purified in example 16. As shown in FIG. 31, even if the amino acid at the CH3 interface of the constant region was modified, the clotting activity did not change, indicating that the amino acid modification at the CH3 interface did not affect the structure of the variable region involved in the reaction with the antigen.

EXAMPLE 19 evaluation of Forming efficiency of bispecific antibody (IgG1 type) having modified CH3 interface

Wild type IgG1, KiH, w1, w2, and w3 were analyzed by cation exchange chromatography (IEX) to evaluate BiAb formation efficiency. Under the following conditions of cation exchange chromatography analysis, peak area ratios of homodimer a-Homo of the humanized a69 antibody, heterodimer BiAb of the humanized a69 antibody and humanized B26 antibody, and homodimer B-Homo of the humanized B26 antibody were calculated.

Column: ProPac WCX-10, 4X 250mm, (Dionex)

Mobile phase: a: 10mmol/L NaH₂PO₄/Na₂HPO₄，pH6.25

B：10mmol/L NaH₂PO₄/Na₂HPO₄，500mmol/L NaCl，pH6.25

Flow rate: 1.0 mL/min

And (3) detection: 220nm

The formation ratios of A-Homo, BiAb and B-Homo of wild type IgG1, KiH, w1, w2 and w3 are shown in FIG. 32. As with IGg4 type, the efficiency of formation of the target BiAb was greatly improved compared to the wild type. Like IgG4 type, high BiAb formation efficiency of 90% or more was obtained by using 4 site modifications less than KiH, and the risk of antigenicity was considered to be smaller. This example shows that: the method of modifying the amino acids at positions 356, 357, 370, 399, 409 and 439 of the H chain in the CH3 interface is applicable not only to the subclass IgG4 of the antibody constant region but also to IgG1, and can be applied to whole IgG antibodies.

Industrial applicability

In the method of the present invention, only a few amino acids need to be substituted to adjust the association without changing the structure and function (activity) of the original polypeptide, and the practicability is very high. The influence on the antigenicity is also small.

By using the method of the present invention, bispecific antibodies that actually retain activity can be efficiently obtained.

Sequence listing

<110> China and foreign pharmaceutical company

<120> method for producing polypeptide by regulating association

<130>C1-A0415Y1P

<150>JP 2005-101105

<151>2005-03-31

<150>JP 2005-378266

<151>2005-12-28

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Cys Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>12

<211>1572

<212>DNA

<213>Homo sapiens

<400>12

atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag 60

gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120

tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gcagaggcct 180

ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240

gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 300

caactgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 360

gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 420

ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctcca 480

ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 540

ctcctgcata gtaatggcaa cacttacttg tattggttcc tgcagaagcc agggcagtct 600

ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 660

ggcagtggat caggcacaga ttttacactg aaaatcagca gagtggaggc tgaggatgtt 720

ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 780

ctggaaatca aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840

gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 900

tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gcagaggcct 960

ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 1020

gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 1080

caactgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 1140

gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 1200

ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctcca 1260

ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 1320

ctcctgcata gtaatggcaa cacttacttg tattggttcc tgcagaagcc agggcagtct 1380

ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 1440

ggcagtggat caggcacaga ttttacactg aaaatcagca gagtggaggc tgaggatgtt 1500

ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 1560

ctggaaatca aa 1572

<210>13

<211>118

<212>PRT

<213>Homo sapiens

<400>13

Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Ser

20 25 30

Trp Met Asn Trp Val Arg Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr Ile Tyr Asn Gly Lys Phe

50 55 60

Arg Val Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210>14

<211>112

<212>PRT

<213>Homo sapiens

<400>14

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His

85 90 95

Ile Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>15

<211>1572

<212>DNA

<213> Artificial sequence

<220>

<223> artificially synthesized sequence

<400>15

atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag 60

gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120

tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag ggagaggcct 180

ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240

gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 300

caactgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 360

gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 420

ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctcca 480

ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 540

ctcctgcata gtaatggcaa cacttacttg tattggttcc tggagaagcc agggcagtct 600

ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 660

ggcagtggat caggcacaga ttttacactg aaaatcagca gagtggaggc tgaggatgtt 720

ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 780

ctggaaatca aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840

gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 900

tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gaagaggcct 960

ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 1020

gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 1080

caactgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 1140

gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 1200

ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctcca 1260

ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 1320

ctcctgcata gtaatggcaa cacttacttg tattggttcc tgaagaagcc agggcagtct 1380

ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 1440

ggcagtggat caggcacaga ttttacactg aaaatcagca gagtggaggc tgaggatgtt 1500

ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 1560

ctggaaatca aa 1572

<210>16

<211>524

<212>PRT

<213> Artificial sequence

<220>

<223> artificially synthesized sequence

<400>16

Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly

1 5 10 15

Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Ser Trp Met Asn Trp Val Arg Glu Arg Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr Ile Tyr Asn

65 70 75 80

Gly Lys Phe Arg Val Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

145 150 155 160

Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg

165 170 175

Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp

180 185 190

Phe Leu Glu Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val

225 230 235 240

Gly Val Tyr Tyr Cys Met Gln His Ile Glu Tyr Pro Phe Thr Phe Gly

245 250 255

Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Pro

275 280 285

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

290 295 300

Gly Tyr Thr Phe Thr Asn Ser Trp Met Asn Trp Val Arg Lys Arg Pro

305 310 315 320

Gly Lys Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu

325 330 335

Thr Ile Tyr Asn Gly Lys Phe Arg Val Arg Val Thr Ile Thr Ala Asp

340 345 350

Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu

355 360 365

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe

370 375 380

Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

405 410 415

Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser

420 425 430

Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr

435 440 445

Tyr Leu Tyr Trp Phe Leu Lys Lys Pro Gly Gln Ser Pro Gln Leu Leu

450 455 460

Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

465 470 475 480

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

485 490 495

Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Ile Glu Tyr Pro

500 505 510

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

515 520

<210>17

<211>1572

<212>DNA

<213> Artificial sequence

<220>

<223> artificially synthesized sequence

<400>17

atggactgga cctggaggtt cctctttgtg gtggcagcag ctacaggtgt ccagtcccag 60

gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120

tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag ggagaggcct 180

ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 240

gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 300

caactgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 360

gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 420

ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctcca 480

ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 540

ctcctgcata gtaatggcaa cacttacttg tattggttcc tgaagaagcc agggcagtct 600

ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 660

ggcagtggat caggcacaga ttttacactg aaaatcagca gagtggaggc tgaggatgtt 720

ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 780

ctggaaatca aaggaggtgg tggatcgggt ggtggtggtt cgggaggcgg tggatcgcag 840

gtgcagctgg tgcagtctgg acctgaggtg aagaagcctg gggcctcagt gaaggtctcc 900

tgcaaggctt ctggatacac cttcaccaac tcctggatga actgggtgag gaagaggcct 960

ggaaagggtc ttgagtggat tggacggatt tatcctggag atggagaaac tatctacaat 1020

gggaaattca gggtcagagt cacgattacc gcggacgaat ccacgagcac agcctacatg 1080

caactgagca gcctgagatc tgaggacacg gccgtgtatt actgtgcgag aggctatgat 1140

gattactcgt ttgcttactg gggccaggga accacggtca ccgtctcttc aggtggtggt 1200

ggatccggag gtggtggatc gggtggtgga ggatcggata ttgtgatgac tcagtctcca 1260

ctctccctgc ccgtcacccc tggagagccg gcctccatct cctgcaggtc tagtaagagt 1320

ctcctgcata gtaatggcaa cacttacttg tattggttcc tggagaagcc agggcagtct 1380

ccacagctcc tgatctatcg gatgtccaac cttgcctcag gggtccctga caggttcagt 1440

ggcagtggat caggcacaga ttttacactg aaaatcagca gagtggaggc tgaggatgtt 1500

ggggtttatt actgcatgca acatatagaa tatcctttta cgttcggcca agggaccaaa 1560

ctggaaatca aa 1572

<210>18

<211>524

<212>PRT

<213> Artificial sequence

<220>

<223> artificially synthesized sequence

<400>18

Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Gly

1 5 10 15

Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Ser Trp Met Asn Trp Val Arg Glu Arg Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr Ile Tyr Asn

65 70 75 80

Gly Lys Phe Arg Val Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe Ala Tyr Trp Gly

115 120 125

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

145 150 155 160

Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg

165 170 175

Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr Trp

180 185 190

Phe Leu Lys Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met

195 200 205

Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val

225 230 235 240

Gly Val Tyr Tyr Cys Met Gln His Ile Glu Tyr Pro Phe Thr Phe Gly

245 250 255

Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly

260 265 270

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Pro

275 280 285

Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

290 295 300

Gly Tyr Thr Phe Thr Asn Ser Trp Met Asn Trp Val Arg Lys Arg Pro

305 310 315 320

Gly Lys Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu

325 330 335

Thr Ile Tyr Asn Gly Lys Phe Arg Val Arg Val Thr Ile Thr Ala Asp

340 345 350

Glu Ser Thr Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Arg Ser Glu

355 360 365

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Asp Asp Tyr Ser Phe

370 375 380

Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

405 410 415

Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser

420 425 430

Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr

435 440 445

Tyr Leu Tyr Trp Phe Leu Glu Lys Pro Gly Gln Ser Pro Gln Leu Leu

450 455 460

Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

465 470 475 480

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu

485 490 495

Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Ile Glu Tyr Pro

500 505 510

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

515 520

<210>19

<211>411

<212>DNA

<213>Homo sapiens

<400>19

atggactgga cctggagaat cctctttttg gtggcagcag ccaaaggtgc ccactccgag 60

gtccagcttg tgcagtctgg ggctgaggtg gtgaagcctg ggtcctcagt gaaggtttcc 120

tgcacggcct ctggatacac cttcagtgac tactatatgc actgggtgcg ccaggccccc 180

ggagaagggc ttgagtggat gggatacatt aatcctagca gtggttatac taagtacaat 240

cggaagttca gggacagagt caccattacc gcggacaaat ccacgagcac agcctacatg 300

gagctgagca gcctgagatc tgaagacacg gctgtgtatt actgtgcgag agggggtctc 360

ggttactacc ttgactactg gggcgagggc accacggtca ccgtctcctc a 411

<210>20

<211>137

<212>PRT

<213>Homo sapiens

<400>20

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Lys Gly

1 5 10 15

Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys

20 25 30

Pro Gly Ser Ser Val Lys Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe

35 40 45

Ser Asp Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Glu Gly Leu

50 55 60

Glu Trp Met Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn

65 70 75 80

Arg Lys Phe Arg Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Gly Leu Gly Tyr Tyr Leu Asp Tyr Trp Gly

115 120 125

Glu Gly Thr Thr Val Thr Val Ser Ser

130 135

<210>21

<211>414

<212>DNA

<213>Homo sapiens

<400>21

atggactgga cctggagcat ccttttcttg gtggcagcag caacaggtgc ccactccgag 60

gtgcagctgg tgcagtctgg agctcaggtg aagaagccgg gggcctcagt gaaggtctcc 120

tgcaaggcct ctggctacac gttttccgac aacaacatgg actgggtgcg acaggcccct 180

ggaaaagggc ttgagtggat gggagatatt aatactaaaa gtggtggttc tatctacaac 240

cagaagttca agggcagagt catcatgacc atagacaaat ccacgggcac agcctacatg 300

gaattgagga gcctgagatc agacgacacg gccatatatt actgtgcgag gaggaggagc 360

tacggctact actttgacta ctggggccgg ggaaccctgg tcaccgtctc ctca 414

<210>22

<211>138

<212>PRT

<213>Homo sapiens

<400>22

Met Asp Trp Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Gln Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Ser Asp Asn Asn Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Met Gly Asp Ile Asn Thr Lys Ser Gly Gly Ser Ile Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Ile Met Thr Ile Asp Lys Ser Thr Gly

85 90 95

Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Ile

100 105 110

Tyr Tyr Cys Ala Arg Arg Arg Ser Tyr Gly Tyr Tyr Phe Asp Tyr Trp

115 120 125

Gly Arg Gly Thr Leu Val Thr Val Ser Ser

130 135

<210>23

<211>384

<212>DNA

<213>Homo sapiens

<400>23

atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc 60

agatgtgaca tcgtgatgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 120

gtcaccatca cttgcaaggc cagtcagaat gtggggactg ctgtagcctg gtatcagcag 180

aaaccaggga aagcccctaa gctcctgatc tattcggcat cctaccgggc cagtggggtc 240

ccatcaaggt tcagtggcag tcgatatggg acagatttca ctctcaccat ctcaagcttg 300

caacctgaag atttagcaac ttactactgt cagcaatata gcaactatat cacgttcggc 360

caagggacca aggtggagat caaa 384

<210>24

<211>128

<212>PRT

<213>Homo sapiens

<400>24

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser

35 40 45

Gln Asn Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Ala Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Ser Asn Tyr Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

115 120 125

<210>25

<211>327

<212>PRT

<213>Homo sapiens

<400>25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>26

<211>327

<212>PRT

<213>Homo sapiens

<400>26

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>27

<211>327

<212>PRT

<213>Homo sapiens

<400>27

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>28

<211>327

<212>PRT

<213>Homo sapiens

<400>28

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>29

<211>327

<212>PRT

<213>Homo sapiens

<400>29

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Lys Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>30

<211>327

<212>PRT

<213>Homo sapiens

<400>30

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>31

<211>327

<212>PRT

<213>Homo sapiens

<400>31

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>32

<211>327

<212>PRT

<213>Homo sapiens

<400>32

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>33

<211>327

<212>PRT

<213>Homo sapiens

<400>33

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Lys Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>34

<211>327

<212>PRT

<213>Homo sapiens

<400>34

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>35

<211>327

<212>PRT

<213>Homo sapiens

<400>35

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>36

<211>327

<212>PRT

<213>Homo sapiens

<400>36

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>37

<211>327

<212>PRT

<213>Homo sapiens

<400>37

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Lys Lys Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>38

<211>327

<212>PRT

<213>Homo sapiens

<400>38

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>39

<211>327

<212>PRT

<213>Homo sapiens

<400>39

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>40

<211>327

<212>PRT

<213>Homo sapiens

<400>40

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>41

<211>327

<212>PRT

<213>Homo sapiens

<400>41

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Lys Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>42

<211>327

<212>PRT

<213>Homo sapiens

<400>42

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>43

<211>327

<212>PRT

<213>Homo sapiens

<400>43

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Lys Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>44

<211>327

<212>PRT

<213>Homo sapiens

<400>44

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>45

<211>327

<212>PRT

<213>Homo sapiens

<400>45

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Lys Lys Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>46

<211>327

<212>PRT

<213>Homo sapiens

<400>46

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Glu Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>47

<211>327

<212>PRT

<213>Homo sapiens

<400>47

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Lys Lys Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>48

<211>330

<212>PRT

<213>Homo sapiens

<400>48

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>49

<211>330

<212>PRT

<213>Homo sapiens

<400>49

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>50

<211>330

<212>PRT

<213>Homo sapiens

<400>50

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp As nSer Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>51

<211>330

<212>PRT

<213>Homo sapiens

<400>51

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>52

<211>330

<212>PRT

<213>Homo sapiens

<400>52

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Lys

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>53

<211>330

<212>PRT

<213>Homo sapiens

<400>53

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Asp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>54

<211>330

<212>PRT

<213>Homo sapiens

<400>54

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Lys Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>55

<211>330

<212>PRT

<213>Homo sapiens

<400>55

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Glu Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Glu Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>56

<211>330

<212>PRT

<213>Homo sapiens

<400>56

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Lys

225 230 235 240

Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>57

<211>119

<212>PRT

<213>Mus musculus

<400>57

Met Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly

1 5 10 15

Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp

20 25 30

Tyr Tyr Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

35 40 45

Leu Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Arg Lys

50 55 60

Phe Arg Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala

65 70 75 80

Tyr Met Gln Leu Thr Ser Leu Thr Tyr Glu Asp Ser Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Gly Asn Gly Tyr Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210>58

<211>120

<212>PRT

<213>Mus musculus

<400>58

Met Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

1 5 10 15

Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp

20 25 30

Asn Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Asp Ile Asn Thr Lys Ser Gly Gly Ser Ile Tyr Asn Gln Lys

50 55 60

Phe Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala

65 70 75 80

Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Arg Arg Ser Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>59

<211>107

<212>PRT

<213>Homo sapiens

<400>59

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Leu Cys Gln Gln Tyr Ser Asn Tyr Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg

100 105

Claims

1. A method for producing a mutant polypeptide having a mutation in an amino acid residue forming an internal interface of the polypeptide to regulate the association of the polypeptide, comprising:

(a) in a polypeptide capable of forming 2 or more conformers, a nucleic acid encoding amino acid residues that form an interface within the polypeptide is modified from the original nucleic acid to inhibit association within the polypeptide that forms 1 or more conformers,

wherein a mutation of an amino acid residue is introduced into the interface so that 2 or more amino acid residues forming the interface are charged with the same kind of charge, and the introduced amino acid residue is selected from glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) and histidine (H);

(b) culturing a host cell to express the nucleic acid; and

(c) recovering the polypeptide from the host cell culture,

(iv) the mutation is introduced into one or more positions selected from the following (i) to (iii),

(i) amino acid residues contained in the CH3 region of the heavy chain, which are 356 to 439 according to EU numbering;

(ii) amino acid residues contained in the CH3 region of the heavy chain, which are located at positions 357 and 370 according to EU numbering; and

(iii) heavy chain CH3 region contains amino acid residues at positions 399 and 409 according to EU numbering,

the polypeptide is an antibody comprising 2 heavy chain CH3 regions derived from a human IgG-type antibody.

2. A method for producing a heteromultimer having a mutation in an amino acid residue forming an interface between polypeptides to regulate the association of the heteromultimer, the method comprising:

(a) in a heteromultimer capable of forming 2 or more multimers, a nucleic acid encoding amino acid residues which form an interface between polypeptides forming 1 or more multimers is modified from the original nucleic acid to inhibit association between polypeptides forming 1 or more multimers,

(b) culturing a host cell to express the nucleic acid; and

(c) recovering the heteromultimer from the host cell culture,

the heteromultimer is a bispecific antibody in which the CH3 region is derived from a human IgG type antibody.

3. A mutant polypeptide or heteromultimer produced by the method of claim 1 or 2.

4. A polypeptide mutant comprising a modification of amino acid residues forming an interface within the original polypeptide to inhibit association within the polypeptide,

(1) wherein the modification of the amino acid residues forming the interface of the polypeptide is a mutation introducing an amino acid residue into the interface so that 2 or more amino acid residues forming the interface have the same charge, and the introduced amino acid residue is selected from glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) and histidine (H); or

(2) Wherein the modification of the amino acid residues forming the interface of the polypeptide is a mutation introducing an amino acid residue into the interface so that the amino acid residues forming the hydrophobic core present in the interface are charged amino acid residues having the same charge, and

the introduced amino acid residue is selected from glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) and histidine (H),

the mutation of (1) or (2) is introduced into one or more positions selected from the following (i) to (iii),

the polypeptide mutant is an antibody comprising 2 heavy chain CH3 regions derived from a human IgG type antibody.

5. Heteromultimer comprising modification of amino acid residues forming an interface between original polypeptides to inhibit association between the polypeptides,

6. The polypeptide mutant of claim 4, wherein the original polypeptide is capable of forming 2 or more conformers.

7. The heteromultimer of claim 5, wherein the original polypeptide is capable of forming 2 or more multimers.

8. A composition comprising the polypeptide mutant of claim 4 or the heteromultimer of claim 5 and a pharmaceutically acceptable carrier.

9. A nucleic acid encoding the polypeptide mutant of claim 4 or the heteromultimer of claim 5.

10. A host cell comprising the nucleic acid of claim 9.

11. A method for producing a mutant polypeptide of claim 4 or a heteromultimer of claim 5, which comprises: a step of culturing the host cell of claim 10; and recovering the polypeptide from the cell culture.

12. A method of modulating polypeptide association, the method comprising modifying amino acid residues forming an interface within an original polypeptide to inhibit association within the polypeptide,

(1) wherein the modification is to introduce mutation of amino acid residue into the interface, to make 2 or more amino acid residues forming the interface have the same charge, and to inhibit association of polypeptides forming 1 or more conformers among polypeptides capable of forming 2 or more conformers, and

the introduced amino acid residue is selected from glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) and histidine (H); or

(2) Wherein the modification is to introduce mutation of amino acid residue into the interface so that the amino acid residue forming the hydrophobic core existing in the interface becomes a charged amino acid residue having the same charge, and

the polypeptide association is an association of polypeptides comprising antibodies derived from the 2 heavy chain CH3 regions of human IgG.

13. A method of modulating the association of heteromultimers, the method comprising modifying amino acid residues forming an interface between original polypeptides to inhibit the association between the polypeptides,

(1) wherein the modification is to introduce mutation of amino acid residues into the interface, to make 2 or more amino acid residues forming the interface have the same charge, and in the heteromultimer capable of forming 2 or more multimers, to inhibit association between polypeptides forming 1 or more multimers, and

wherein the heteromultimer is a bispecific antibody in which the CH3 region is derived from a human IgG type antibody.

14. An antibody, which comprises 2 human heavy chain CH3 regions, wherein amino acid residues in the 1 st heavy chain CH3 region, which are selected from one to three groups of amino acid residues shown in the following (1) to (3), have the same charge:

(2) amino acid residues contained in the CH3 region of the heavy chain, which are located at positions 357 and 370 according to EU numbering; and

(3) heavy chain CH3 region contains amino acid residues at positions 399 and 409 according to EU numbering,

wherein the amino acid residue having the same charge in the 1 st heavy chain CH3 region is selected from the group consisting of the amino acid residues contained in any one of the following (a) or (b):

(a) glutamic acid (E) and aspartic acid (D); or

(b) Lysine (K), arginine (R) and histidine (H),

one to three groups of amino acid residues in the 2 nd heavy chain CH3 region are:

(i) selected from the group consisting of amino acid residues represented by (1) to (3);

(ii) the amino acid residues shown in (1) to (3) corresponding to the 1 st heavy chain CH3 region; and

(iii) with a charge opposite to that of the corresponding amino acid residue in the heavy chain 1 CH3 region,

wherein the amino acid residue in the 2 nd heavy chain CH3 region is selected from the group consisting of the amino acid residues contained in any one of the following (a) or (b):

(a) glutamic acid (E) and aspartic acid (D); or

(b) Lysine (K), arginine (R) and histidine (H).

15. The antibody of claim 14, wherein said 1 st heavy chain CH3 region and 2 nd heavy chain CH3 region are cross-linked by disulfide bonds.

16. The antibody of claim 14, which is an antibody having 2 or more heavy chain constant regions.

17. The antibody of claim 14, which is a humanized bispecific antibody.

18. A composition comprising the antibody of claim 14 and a pharmaceutically acceptable carrier.

19. A nucleic acid encoding the antibody-constituting polypeptide of claim 14.

20. A host cell comprising the nucleic acid of claim 19.

21. A method of producing the antibody of claim 14, the method comprising: a step of culturing the host cell of claim 20; and recovering the polypeptide from the cell culture.