JP2011219380A - Anti vegf-d antibody, and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti VEGF-D antibody and a pharmaceutical composition comprising the same as an effective component.SOLUTION: The present inventors have succeeded in obtaining an anti VEGF-D antibody binding to VEGF-D in a system wherein a cell expressing VEGF-D is transplanted, and exhibiting lymphangiogenesis inhibiting activity and/or lymph-node metastasis inhibiting activity in vivo, and in humanizing the antibody while maintaining the activity thereof. The antibody has a reduced risk of immunogenicity in humans and is useful as an agent of curing cancer.

Description

  The present invention relates to an anti-VEGF-D antibody and a pharmaceutical composition comprising an anti-VEGF-D antibody as an active ingredient.

  Vascular endothelial growth factor (hereinafter referred to as VEGF) is a protein factor purified and isolated as a substance that enhances the proliferation of vascular endothelial cells and the permeability of blood vessels (Biochem. Biophys. Res Commun., 161: 851-858 (1989)). Thereafter, VEGF-B (Non-patent document 2) and VEGF-C (Non-patent document 3) are isolated, and one VEGF family is formed. Recently, VEGF-D was found as the fourth factor of the VEGF family having significant homology with VEGF-C (Patent Document 1). VEGF-C and VEGF-D bind to one of the receptors, VEGF receptor 3 (hereinafter referred to as VEGFR-3), activate the receptor, and are involved in promoting lymphangiogenesis. These factors also bind to VEGFR-2 and are involved in promoting angiogenesis (Non-Patent Documents 4 to 8). Furthermore, it has been reported that VEGF-C and VEGF-D are highly expressed in cancer tissues and are involved in the promotion of lymphatic vessel proliferation and lymph node metastasis around and / or inside the cancer tissues (Non-Patent Documents 9- 13).

  Recently, it has been reported that an anti-VEGF-D antibody that inhibits the binding between VEGF-D and its receptor has been obtained (Patent Documents 2 and 3, Non-Patent Document 14). Furthermore, it has been reported that one of these obtained antibodies, VD1, inhibits the binding of VEGF-D and its receptor and suppresses lymph node metastasis (Non-patent Document 9).

  Prior art documents of the present invention are shown below.

WO98 / 002543 WO2000 / 037025 WO2005 / 087177

Biochem. Biophys. Res. Commun., 161: 851-858 (1989) Proc. Natl. Acad. Sci. USA 93, 2576-2581 (1996) Proc. Natl. Acad. Sci. USA 93, 1988-1992 (1996) TRENDS in Immunology Vol.25 No.7 387-394 (2004) Dev. Biol. 188, 96-109 (1997) EMBO J. 20, 1223-1231 (2001) FASEB J. 16, 1041-1049 (2002) Circ. Res. 92, 1098-1106 (2003) Nat. Med. 7, 192-198 (2001) Cancer Res. 61, 1786-1790 (2001) Nat. Med. 7, 186-191 (2001) Int. J. Cancer 98, 946-951 (2002) Nat. Med. 13, 1458-1466 (2007) Eur. J. Biochem. 267, 2505-2515 (2000)

  An object of the present invention is to provide an anti-VEGF-D antibody and a pharmaceutical composition containing such an antibody as an active ingredient.

The present inventors succeeded in obtaining an anti-VEGF-D antibody that binds to VEGF-D and has lymphangiogenesis inhibitory activity in vivo in a system in which cells expressing VEGF-D are transplanted.
In addition, the present inventors succeeded in humanizing the antibody while maintaining the activity of the anti-VEGF-D antibody obtained above.
These antibodies reduce the risk of immunogenicity in humans and are useful as cancer therapeutic agents.

That is, the present invention relates to an anti-VEGF-D antibody and a pharmaceutical composition containing an anti-VEGF-D antibody as an active ingredient, and more specifically includes the following inventions [1] to [12].
[1] The anti-VEGF-D antibody according to any one of the following (1) to (5);
(1) An antibody having a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 3. (VE29H chain CDR),
(2) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 6. (VE29 light chain CDR),
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) The antibody according to any one of (1) to (3), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (3) An antibody having an activity equivalent to that of the antibody according to
(5) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (4) binds.
[2] The anti-VEGF-D antibody according to any one of (1) to (8) below:
(1) A heavy chain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 7, CDR2 having the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 15, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 9 An antibody having a variable region (VE199 H chain CDR),
(2) an antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 17, CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 and CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 (VE199L chain CDR),
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) FR1 in which the FR of the heavy chain variable region of the antibody according to any one of (1) and (3) has the amino acid sequence described in SEQ ID NO: 10 or SEQ ID NO: 11, described in SEQ ID NO: 12 An antibody having a heavy chain variable region that is FR2 having an amino acid sequence, FR3 having an amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14, and FR4 having an amino acid sequence set forth in SEQ ID NO: 16 (VE199 humanized H Before and after chain FR modification),
(5) FR1 in which the FR of the light chain variable region of the antibody according to any one of (2) and (3) has the amino acid sequence set forth in SEQ ID NO: 20, and FR2 having the amino acid sequence set forth in SEQ ID NO: 21 FR3 having the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, an antibody having a light chain variable region that is FR4 having the amino acid sequence set forth in SEQ ID NO: 24 (before VE199 humanized L chain FR modification) After modification),
(6) an antibody having the heavy chain variable region of (4) and the light chain variable region of (5),
(7) The antibody according to any one of (1) to (6), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having an activity equivalent to that of the antibody according to
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.
[3] The anti-VEGF-D antibody according to any one of (1) to (8) below:
(1) CDR1 having the amino acid sequence described in SEQ ID NO: 25 or SEQ ID NO: 43, CDR2 having the amino acid sequence described in SEQ ID NO: 26, SEQ ID NO: 42 or SEQ ID NO: 44, described in SEQ ID NO: 27 An antibody having a heavy chain variable region comprising CDR3 having the amino acid sequence of (VE48 H chain CDR),
(2) a light chain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 41, CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 35 An antibody having a variable region (VE48 L chain CDR),
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) FR1 in which the FR of the heavy chain variable region of the antibody according to any one of (1) and (3) has the amino acid sequence set forth in SEQ ID NO: 28, FR2 having the amino acid sequence set forth in SEQ ID NO: 29 An antibody having a heavy chain variable region which is FR3 having the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31, FR4 having the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 45 (VE48 humanized HA Before and after chain FR modification),
(5) FR1 in which the FR of the light chain variable region of the antibody according to any one of (2) and (3) has the amino acid sequence set forth in SEQ ID NO: 36, FR2 having the amino acid sequence set forth in SEQ ID NO: 37 FR3 having the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39, an antibody having a light chain variable region that is FR4 having the amino acid sequence set forth in SEQ ID NO: 40 (before VE48 humanized L chain FR modification) After modification),
(6) an antibody having the heavy chain variable region of (4) and the light chain variable region of (5),
(7) The antibody according to any one of (1) to (6), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having an activity equivalent to that of the antibody according to
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.
[4] The antibody variable region or antibody according to any one of (1) to (5) below:
(1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 46 (VE199HA),
(2) Light chain variable region having the amino acid sequence set forth in SEQ ID NO: 47 (VE199LA)
(3) an antibody (VE199HALA) comprising the heavy chain variable region of (1) and the light chain variable region of (2),
(4) The antibody according to any one of (1) to (3), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (3) An antibody having an activity equivalent to that of the antibody according to
(5) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (4) binds.
[5] The antibody variable region or antibody according to any one of (1) to (13) below:
(1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 48 (VE48HA),
(2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 49 (VE48HH),
(3) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 50 (VE48HI),
(4) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 51 (VE48HJ),
(5) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 52 (VE48HK),
(6) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53 (VE48H16),
(7) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 54 (VE48H17),
(8) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 55 (VE48H18),
(9) a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 56 (VE48L6),
(10) a light chain variable region having the amino acid sequence of SEQ ID NO: 57 (VE48L14),
(11) An antibody comprising the heavy chain variable region according to any one of (1) to (8) and the light chain variable region of (9) or (10),
(12) The antibody according to any one of (1) to (11), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (11) An antibody having an activity equivalent to that of the antibody according to
(13) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (12) binds.
[6] The anti-VEGF-D antibody according to any one of [1] to [5], which is a humanized antibody.
[7] A pharmaceutical composition comprising the antibody according to any one of [1] to [6].
[8] The pharmaceutical composition according to [7], which is a cancer therapeutic agent.
[9] The pharmaceutical composition according to [7], which is a lymphangiogenesis inhibitor.
[10] The pharmaceutical composition according to [7], which is a tumor growth inhibitor.
[11] The pharmaceutical composition according to [7], which is a metastasis inhibitor.
[12] The pharmaceutical composition according to [7], which is a lymph node metastasis inhibitor.

  The anti-VEGF-D antibody of the present invention exhibits excellent VEGF-D binding inhibitory activity against VEGFR-3. The inhibitory activity inhibits the activity due to the binding of VEGF-D, and can be used as an excellent anticancer agent. In particular, it has an excellent inhibitory effect on lymphangiogenesis, lymph node metastasis and / or tumor growth.

It is a figure which shows the result of having measured the specific binding with respect to human VEGF-D of the anti-VEGF-D mouse antibody (mVE29, mVE48, and mVE199) obtained by immunizing human VEGF-D or cynomolgus monkey VEGF-D. It is a figure which shows the result of having measured the specific binding with respect to human VEGF-C of the anti-VEGF-D mouse antibody (mVE29, mVE48, and mVE199) obtained by immunizing human VEGF-D or cynomolgus monkey VEGF-D. It is a figure which shows the result of having measured the binding inhibition of human VEGF-D with respect to the human VEGFR-3 expression cell by an anti-VEGF-D chimera antibody (chimera VE29, chimera VE48, and chimera VE199). It is a figure which shows the result of having measured the binding inhibition of cynomolgus monkey VEGF-D with respect to cynomolgus monkey VEGFR-3 expression CHO cell by an anti-VEGF-D chimera antibody (chimera VE48 and chimera VE199). It is a figure which shows the result of having measured the proliferation inhibitory activity of the human lymphatic-endothelial-cell by anti-VEGF-D chimera antibody (VD1, chimera VE48, and chimera VE199). In vivo lymphangiogenesis in hD_1 cell-transplanted mice with anti-VEGF-D chimeric antibodies (chimeric VE48 10 mg / kg, 2 mg / kg and chimeric VE199 10 mg / kg, 2 mg / kg) and control (saline) It is the figure (A) and the photograph (BF) which show the evaluation result of a model. It is a figure which shows the result of having measured the lymph node metastasis inhibitory activity in vivo by an anti- VEGF-D chimeric antibody (chimeric VE48 25 mg / kg and chimeric VE48 10 mg / kg) and a control (saline). FIG. 6 is a diagram comparing VHGF-D binding inhibitory activities of a chimeric VE48 antibody and a humanized antibody VE199 antibody (199HA / 199LA). FIG. 6 is a diagram comparing VHGF-D binding inhibitory activities of a chimeric VE48 antibody and a humanized antibody VE48 antibody (48HA / 48L6). FIG. 3 is a graph comparing VHGF-D binding inhibitory activities of a chimeric VE48 antibody and a humanized antibody VE48 antibody (48HA / 48L14). It is a figure comparing the VEGF-D binding inhibitory activities of the chimeric VE48 antibody and the humanized antibody VE48 antibodies (48HH / 48L6 and 48HI / 48L6). FIG. 6 is a graph comparing the VEGF-D binding inhibitory activities of the chimeric VE48 antibody and the humanized antibody VE48 antibodies (48HJ / 48L6 and 48HK / 48L6). It is a figure comparing the VEGF-D binding inhibitory activities of the chimeric VE48 antibody and the humanized antibody VE48 antibodies (48H16 / 48L6 and 48H17 / 48L6). FIG. 3 is a graph comparing the VEGF-D binding inhibitory activities of a chimeric VE48 antibody and a humanized antibody VE48 antibody (48H18 / 48L6).

VEGF-D
VEGF-D is a member of the VEGF family and has been found in WO98 / 002543 for the first time as a novel protein factor involved in human angiogenesis and lymphangiogenesis. The factor is processed into a mature homodimer after forming a homodimer. The mature homodimer binds to both VEGFR-2 and VEGFR-3 (J. Biol. Chem. 274: 32127-32136, 1999). The factor functions as a ligand for VEGFR-2 and VEGFR-3, and is known to be involved in metastasis, particularly lymph node metastasis, through the process of promoting angiogenesis and lymphangiogenesis (Nat. Med. 13, 1459, 2007).

  The origin of VEGF-D of the present invention is not particularly limited, and includes VEGF-D derived from humans, mice, monkeys and other mammals, preferably VEGF-D derived from humans, mice or monkeys, Particularly preferred is VEGF-D derived from human.

Antibody (sequence)
As a preferred embodiment of the anti-VEGF-D antibody of the present invention, any of the following anti-VEGF-D antibodies can be mentioned.

(A) VE29
(1) An antibody having a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 3. (VE29H chain CDR),
(2) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 6. (VE29 light chain CDR),
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) The antibody according to any one of (1) to (3), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (3) An antibody having an activity equivalent to that of the antibody according to
(5) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (4) binds.

(B) VE199
(1) A heavy chain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 7, CDR2 having the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 15, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 9 An antibody having a variable region (VE199 H chain CDR),
(2) an antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 17, CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 and CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 (VE199L chain CDR),
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) FR1 in which the FR of the heavy chain variable region of the antibody according to any one of (1) and (3) has the amino acid sequence described in SEQ ID NO: 10 or SEQ ID NO: 11, described in SEQ ID NO: 12 An antibody having a heavy chain variable region that is FR2 having an amino acid sequence, FR3 having an amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14, and FR4 having an amino acid sequence set forth in SEQ ID NO: 16 (VE199 humanized H Before and after chain FR modification),
(5) FR1 in which the FR of the light chain variable region of the antibody according to any one of (2) and (3) has the amino acid sequence set forth in SEQ ID NO: 20, and FR2 having the amino acid sequence set forth in SEQ ID NO: 21 FR3 having the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, an antibody having a light chain variable region that is FR4 having the amino acid sequence set forth in SEQ ID NO: 24 (before VE199 humanized L chain FR modification) After modification),
(6) an antibody having the heavy chain variable region of (4) and the light chain variable region of (5),
(7) The antibody according to any one of (1) to (6), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having an activity equivalent to that of the antibody according to
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.

(C) VE48
(1) CDR1 having the amino acid sequence described in SEQ ID NO: 25 or SEQ ID NO: 43, CDR2 having the amino acid sequence described in SEQ ID NO: 26, SEQ ID NO: 42 or SEQ ID NO: 44, described in SEQ ID NO: 27 An antibody having a heavy chain variable region comprising CDR3 having the amino acid sequence of (VE48 H chain CDR),
(2) a light chain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 41, CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 35 An antibody having a variable region (VE48 L chain CDR),
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) FR1 in which the FR of the heavy chain variable region of the antibody according to any one of (1) and (3) has the amino acid sequence set forth in SEQ ID NO: 28, FR2 having the amino acid sequence set forth in SEQ ID NO: 29 An antibody having a heavy chain variable region which is FR3 having the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31, FR4 having the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 45 (VE48 humanized HA Before and after chain FR modification),
(5) FR1 in which the FR of the light chain variable region of the antibody according to any one of (2) and (3) has the amino acid sequence set forth in SEQ ID NO: 36, FR2 having the amino acid sequence set forth in SEQ ID NO: 37 FR3 having the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39, an antibody having a light chain variable region that is FR4 having the amino acid sequence set forth in SEQ ID NO: 40 (before VE48 humanized L chain FR modification) After modification),
(6) an antibody having the heavy chain variable region of (4) and the light chain variable region of (5),
(7) The antibody according to any one of (1) to (6), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having an activity equivalent to that of the antibody according to
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.

Specific examples of the substitution, deletion, addition and / or insertion of one or more amino acids described above are not particularly limited, and the following modifications can be mentioned.
Substitution of 9th A to another amino acid in heavy chain FR1 of SEQ ID NO: 10. Although the amino acid after substitution is not specifically limited, P can be mentioned as a preferable example.
Substitution of the fourth I in the heavy chain FR3 of SEQ ID NO: 13 with another amino acid. Although the amino acid after substitution is not particularly limited, L can be mentioned as a preferred example.
Replacement of the 6th A with another amino acid in the heavy chain FR3 of SEQ ID NO: 13. Although the amino acid after substitution is not particularly limited, V can be mentioned as a preferred example.
Substitution of 31st A to another amino acid in heavy chain FR3 of SEQ ID NO: 13. Although the amino acid after substitution is not particularly limited, I can be mentioned as a preferred example.
Substitution of the 16th R to another amino acid in the heavy chain CDR2 of SEQ ID NO: 8. Although the amino acid after substitution is not particularly limited, Q can be mentioned as a preferred example.
Substitution of 31st Y to other amino acid in light chain FR3 of SEQ ID NO: 22. Although the amino acid after substitution is not specifically limited, F can be mentioned as a preferable example.
Substitution of R at position 32 with another amino acid in heavy chain FR3 of SEQ ID NO: 30. Although the amino acid after substitution is not particularly limited, Q can be mentioned as a preferred example.
Substitution of 31st Y to other amino acid in light chain FR3 of SEQ ID NO: 38. Although the amino acid after substitution is not specifically limited, F can be mentioned as a preferable example.
Substitution of N at 9th other amino acid in light chain CDR1 of SEQ ID NO: 33. Although the amino acid after substitution is not particularly limited, I can be mentioned as a preferred example.
Substitution of N at the 10th position in the light chain CDR1 of SEQ ID NO: 33. The amino acid after substitution is not particularly limited, but preferred examples include K.
Substitution of N at the 5th N in the heavy chain CDR2 of SEQ ID NO: 26. Although the amino acid after substitution is not particularly limited, E can be mentioned as a preferred example.
Substitution of the first T with another amino acid in the heavy chain CDR1 of SEQ ID NO: 25. Although the amino acid after substitution is not specifically limited, D can be mentioned as a preferable example.
Substitution of 15th R and 16th S to other amino acids in heavy chain CDR2 of SEQ ID NO: 26. The amino acid after substitution is not particularly limited, but preferred examples include substitution of Q at the 15th R and D at the 16th S.
Substitution of the third Q to another amino acid in heavy chain FR4 of SEQ ID NO: 32. Although the amino acid after substitution is not particularly limited, E can be mentioned as a preferred example.

  The above-mentioned substitution may be performed alone or a plurality of substitutions may be combined. Further, the above-described substitution may be combined with substitutions other than those described above. These substitutions can improve the pharmacokinetics (retention in plasma) of the antibody, enhance the binding activity to the antigen, improve the stability, and / or reduce the risk of immunogenicity.

  Specific examples of the variable region combining the above substitutions in the present invention include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 or a light chain variable region having the amino acid sequence of SEQ ID NO: 47. Furthermore, as an example of an antibody combining the above-mentioned substitutions, an antibody comprising a heavy chain variable region having any one of the amino acid sequences of SEQ ID NO: 48 to 55 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56 or 57 Can be mentioned.

Furthermore, the following antibodies can be mentioned as specific examples of antibodies in which the above substitutions are combined.
(i) an antibody comprising a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 47
(ii) an antibody comprising the heavy chain variable region of any of SEQ ID NOs: 48 to 55 and the light chain variable region of SEQ ID NO: 56 or SEQ ID NO: 57

  Although any framework region (FR) may be used in the above-described antibody, it is preferable to use human-derived FR. In the above-described antibody, any constant region may be used as the constant region, but a human-derived constant region is preferably used. The amino acid sequence of the FR or constant region used in the antibody of the present invention may be the original FR or constant region amino acid sequence from which it is derived, or may be substituted, deleted, added and / or substituted for one or more amino acids. Alternatively, different amino acid sequences may be used by insertion or the like.

  In the present invention, “activity equivalent to antibody” means that binding activity and / or neutralizing activity to VEGF-D (eg, human VEGF-D) is equivalent, or antitumor activity is equivalent. Means. Antitumor activity includes, for example, an activity that decreases tumor cell weight or volume, an activity that suppresses increase in tumor cell weight or volume, an activity that decreases intratumoral lymphatic vessels, an activity that decreases intravascular blood vessels, Activity to normalize abnormal lymphatic vessels, activity to normalize abnormal blood vessels in the tumor, activity to normalize tumor vascular permeability, activity to normalize tumor lymphatic permeability, suppress lymph node metastasis of tumor cells Activity, activity to suppress distant metastasis of tumor cells, activity to promote reduction of tumor cell weight or volume by other drugs, activity to suppress individual death by tumor cells, and the like.

  In the present invention, “equivalent” does not necessarily have the same level of activity, and the activity may be enhanced, or the activity may be decreased as long as it has activity. Examples of antibodies with decreased activity include antibodies having 30% or more activity, preferably 50% or more activity, more preferably 80% or more activity compared to the original antibody.

  The above-described antibody has one or more amino acids in the amino acid sequence of the variable region (CDR sequence and / or FR sequence) as long as it has binding activity and / or neutralizing activity against VEGF-D or has antitumor activity. Substitutions, deletions, additions and / or insertions may be made. In order to prepare an amino acid sequence of an antibody having one or more amino acid substitutions, deletions, additions and / or insertions in the amino acid sequence and having binding activity, neutralizing activity and / or antitumor activity against VEGF-D As a method well known to those skilled in the art, a method for introducing a mutation into a protein is known. For example, those skilled in the art will understand site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction.Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Oligonucleotide- directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492) To the amino acid sequence of an antibody having binding activity and / or neutralizing activity against VEGF-D or anti-tumor activity. By appropriately introducing a mutation, a mutant functionally equivalent to an antibody having binding activity and / or neutralizing activity against VEGF-D or antitumor activity can be prepared.

  Thus, in the variable region, one or a plurality of amino acids are mutated, and an antibody having binding and / or neutralizing activity against VEGF-D or antitumor activity is also included in the antibody of the present invention.

  When altering amino acid residues, it is desirable to mutate to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), and aromatic-containing sides Amino acids having a chain (H, F, Y, W) can be mentioned (the parentheses indicate single letter symbols of amino acids). Amino acid substitutions within each of these groups are referred to as conservative substitutions. It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with other amino acids of one or more amino acid residues to a certain amino acid sequence maintains its biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81: 5662-6; Zoller, MJ and Smith, M., Nucleic Acids Res. (1982) 10: 6487-500; Wang , A. et al., Science (1984) 224: 1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6409-13). Such variants are at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 80% of the amino acid sequence of the variable region of the present invention (eg, CDR sequence, FR sequence, entire variable region). It has 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity. In this specification, the sequence identity refers to the amino acid sequence of the original heavy chain variable region or light chain variable region after aligning the sequences as necessary so that the sequence identity is maximized, and introducing gaps as appropriate. Is defined as the proportion of residues identical to The identity of amino acid sequences can be determined by the method described below.

  Further, in the amino acid sequence of the variable region (CDR sequence and / or FR sequence), one or more amino acids are substituted, deleted, added and / or inserted, and binding activity and / or neutralizing activity against VEGF-D Alternatively, the amino acid sequence of a variable region having antitumor activity can be obtained from a nucleic acid that hybridizes under stringent conditions to a nucleic acid comprising a base sequence encoding the amino acid sequence of the variable region. Stringent hybridization conditions for isolating a nucleic acid that hybridizes under stringent conditions to a nucleic acid consisting of a base sequence encoding the amino acid sequence of the variable region include 6 M urea, 0.4% SDS, 0.5 x Examples of the conditions include SSC, 37 ° C., or hybridization conditions with the same stringency. Isolation of nucleic acids with higher homology can be expected by using conditions with higher stringency, for example, 6 M urea, 0.4% SDS, 0.1 × SSC, 42 ° C. The sequence of the isolated nucleic acid can be determined by a known method described later. The homology of the isolated nucleic acid is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) in the entire base sequence. And the like).

  In place of the above-described method using the hybridization technique, a gene amplification method using a primer synthesized based on nucleotide sequence information encoding the amino acid sequence of the variable region, for example, a polymerase chain reaction (PCR) method is used. It is also possible to isolate a nucleic acid that hybridizes with a nucleic acid comprising a base sequence encoding the amino acid sequence of the region under stringent conditions.

The identity of the base sequence and amino acid sequence can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7) by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al., J. Mol. Biol. (1990) 215: 403-10). When analyzing a base sequence by BLASTN based on BLAST, parameters are set to score = 100 and wordlength = 12, for example. When the amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (refer to NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) website; http://www.ncbi.nlm.nih.gov ).

  The present invention also provides an antibody that binds to the same epitope as that to which the antibody binds.

  Whether an antibody recognizes the same epitope as another antibody can be confirmed by competition for the epitopes of both. Competition between antibodies can be evaluated by competitive binding assays, such as enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer assay (FRET), and fluorescence microassay technology (FMAT (registered trademark)). Is mentioned. The amount of the antibody bound to the antigen is indirectly correlated with the binding ability of a candidate competitive antibody (test antibody) that competes for binding to the same epitope. That is, the greater the amount and affinity of the test antibody for the same epitope, the lower the binding amount of the antibody to the antigen and the more the binding amount of the test antibody to the antigen. Specifically, the antibody labeled with an appropriate label and the antibody to be evaluated are simultaneously added to the antigen, and the bound antibody is detected using the label. The amount of the antibody bound to the antigen can be easily measured by labeling the antibody in advance. This labeling is not particularly limited, but a labeling method corresponding to the technique is selected. Specific examples of the labeling method include fluorescent labeling, radiolabeling, enzyme labeling and the like.

  For example, the fluorescently labeled antibody and the unlabeled antibody or test antibody are simultaneously added to beads immobilized with VEGF-D, and the labeled antibody is detected by a fluorescence micromeasurement technique.

The “antibody recognizing the same epitope” as used herein refers to the concentration of the antibody to be tested that is 50% lower than that of the labeled antibody due to the binding of the unlabeled antibody (IC ₅₀ ). Reduce the binding amount of the labeled antibody by at least 50%, usually 100 times, preferably 80 times, more preferably 50 times, more preferably 30 times, more preferably 10 times higher than the IC ₅₀ of the labeled antibody. It is an antibody that can.

  An antibody that binds to an epitope to which the above-described antibody binds is particularly useful in that it has a high binding inhibitory activity of VEGF-D to VEGFR-3. Further, an antibody that binds to an epitope to which the antibody described in (A) above binds binds not only to VEGF-D but also to VEGF-C, and has high binding inhibitory activity of VEGF-C to VEGFR-3. Like VEGF-D, VEGF-C binds to VEGFR-3 and has angiogenesis, lymphangiogenesis, and lymph node metastasis. Therefore, it is expected to have an excellent cancer treatment effect that is efficient in cancer treatment.

  Although the above-mentioned antibody is not particularly limited, it is preferably a humanized antibody.

  The present invention further provides a gene encoding the above-described anti-VEGF-D antibody. The gene of the present invention may be any gene, for example, DNA or RNA.

Antibody (humanized)
One preferred embodiment of the antibody in the present invention is a humanized antibody that binds to VEGF-D. Humanized antibodies can be produced using methods known to those skilled in the art.

  The variable region of an antibody is usually composed of three complementarity determining regions (CDRs) sandwiched between four frames (FR). CDRs are regions that substantially determine the binding specificity of an antibody. The amino acid sequence of CDR is rich in diversity. On the other hand, the amino acid sequence constituting FR often shows high homology among antibodies having different binding specificities. Therefore, it is generally said that the binding specificity of one antibody can be transplanted to another antibody by CDR grafting.

  A humanized antibody is also referred to as a reshaped human antibody, which is a non-human mammal, for example, a mouse antibody CDR grafted to the complementarity determining region of a human antibody, and its general gene Recombination techniques are also known (see European Patent Application Publication No. EP 125023, WO 96/02576).

  Specifically, for example, when the CDR is derived from a mouse antibody, a DNA sequence designed so as to link the CDR of the mouse antibody and the framework region (FR) of the human antibody, The oligonucleotide is synthesized by PCR using several oligonucleotides prepared so as to have an overlapping portion in the terminal region (see the method described in WO 98/13388). The obtained DNA is obtained by ligation with DNA encoding a human antibody constant region, and then incorporated into an expression vector, which is introduced into a host and produced (European Patent Application Publication Number EP 239400, International Patent Application Publication Number). WO 96/02576).

  As the framework region of the human antibody to be linked to the CDR, a region in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, the amino acid of the framework region in the variable region of the antibody may be substituted, deleted, added, and / or inserted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site. Good. For example, amino acid sequence mutations can be introduced into FRs by applying the PCR method used for transplantation of mouse CDRs into human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to the FR. A nucleotide sequence mutation is introduced into the FR synthesized by such a primer. A mutant FR sequence having a desired property can be selected by measuring and evaluating the antigen-binding activity of a mutant antibody substituted with an amino acid by the above method (Sato, K. et al., Cancer Res. (1993) 53 , 851-856).

  For humanized antibody, the C region is that of a human antibody.For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, Cε are used for the H chain, and Cκ, Cλ are used for the L chain. be able to.

  In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production. Examples of the modified human antibody C region include the C region described later. Human antibodies used for humanization may be human antibodies of any isotype such as IgG, IgM, IgA, IgE, IgD, but IgG is preferably used in the present invention. IgG1, IgG2, IgG3, IgG4, etc. can be used as IgG.

  In addition, after producing a humanized antibody, amino acids in the variable region (eg, CDR, FR) or constant region may be substituted, deleted, added and / or inserted with other amino acids. The humanized antibody also includes a humanized antibody having such amino acid substitution.

  In the humanization of antibodies, it is usually difficult to humanize while maintaining the binding activity, neutralizing activity, or antitumor activity of the derived antibody, but in the present invention, the derived mouse A humanized antibody having binding activity and / or neutralizing activity equivalent to that of an antibody or antitumor activity was successfully obtained. Since humanized antibodies have reduced immunogenicity in the human body, they are useful when administered to humans for therapeutic purposes.

Furthermore, the present invention provides a vector containing a gene encoding the antibody of the present invention.
The present invention further provides a host cell transformed with the above-described vector.
The present invention further relates to a method for producing the variable region of the antibody of the present invention, the heavy chain of the antibody of the present invention, the light chain of the antibody of the present invention, or the antibody of the present invention, comprising the step of culturing the above-mentioned host cell.

Antibody (neutralizing activity)
The present invention further provides an anti-VEGF-D antibody having neutralizing activity.
In the present invention, the neutralizing activity against VEGF-D is an activity that inhibits the binding between VEGF-D and its receptor, VEGFR-3, and preferably an activity that suppresses physiological activity based on VEGFR-3.

  Selection of an antibody having VEGF-D neutralizing activity can be performed, for example, by confirming inhibition of VEGF-D binding when a candidate antibody is added to a VEGFR-3 expressing cell line. An antibody that inhibits the binding of VEGF-D is judged to be an antibody having neutralizing activity against VEGF-D. Further, for example, when a candidate antibody is added to a cell line that proliferates in a VEGF-D-dependent manner, it can also be confirmed by confirming its growth inhibitory effect. The antibody that suppresses the proliferation of the cell is judged to be an antibody having neutralizing activity against VEGF-D.

Antibody (general)
The antibody of the present invention is not limited in its origin, and may be an antibody derived from any animal such as a human antibody, a mouse antibody, or a rat antibody. Further, it may be a recombinant antibody such as a chimeric antibody or a humanized antibody. As mentioned above, humanized antibodies can be mentioned as preferred antibodies of the present invention.

  A chimeric antibody is an antibody comprising a heavy chain and light chain variable region of a mouse antibody other than a human, for example, a mouse antibody heavy chain and a light chain constant region. Chimeric antibodies can be produced using known methods. For example, an antibody gene can be cloned from a hybridoma, incorporated into an appropriate vector, and introduced into a host (for example, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA of the variable region (V region) of the antibody is synthesized from the hybridoma mRNA using reverse transcriptase. If DNA encoding the V region of the target antibody can be obtained, it is ligated with DNA encoding the desired human antibody constant region (C region) and incorporated into an expression vector. Alternatively, DNA encoding the V region of the antibody may be incorporated into an expression vector containing DNA of the human antibody C region. It is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells can be transformed with this expression vector to express the chimeric antibody.

  A method for obtaining a human antibody is also known. For example, human lymphocytes are sensitized with a desired antigen or a cell that expresses the desired antigen in vitro, and the sensitized lymphocyte is fused with a human myeloma cell such as U266 to have a desired human antibody having an antigen-binding activity (See Japanese Patent Publication No. 1-59878). Further, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (International Patent Application Publication Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

  Furthermore, a technique for obtaining a human antibody by a panning method using a human antibody phage library is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, an appropriate expression vector having the sequence can be prepared and a human antibody can be obtained. These methods are well known and can be referred to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, and the like. .

  The antibody of the present invention is represented not only by a bivalent antibody represented by IgG but also by a monovalent antibody or IgM as long as it has binding activity and / or neutralizing activity to VEGF-D or antitumor activity. Also included are multivalent antibodies, or Bispecific antibodies that can bind to different antigens. The multivalent antibodies of the present invention include multivalent antibodies that all have the same antigen-binding site, or multivalent antibodies that have some or all different antigen-binding sites. The antibody of the present invention is not limited to the full length molecule of the antibody, and may be a low molecular weight antibody or a modified product thereof as long as it binds to the VEGF-D protein.

  The antibody in the present invention may be a low molecular weight antibody. A low molecular weight antibody is an antibody comprising an antibody fragment in which a part of a full-length antibody (whole antibody such as whole IgG) is deleted, and has a binding activity and / or neutralizing activity to VEGF-D, or an antitumor activity As long as it has, it will not specifically limit. In the present invention, the low molecular weight antibody is not particularly limited as long as it includes a part of the full-length antibody, but preferably includes a heavy chain variable region (VH) or a light chain variable region (VL), and particularly preferably VH and VL. It is a low molecular weight antibody containing both. Another preferred example of the low molecular weight antibody of the present invention is a low molecular weight antibody containing the CDR of the antibody. The CDR included in the low molecular weight antibody may include all six CDRs of the antibody or may include a part of the CDRs.

  The low molecular weight antibody in the present invention preferably has a smaller molecular weight than the full-length antibody. However, for example, it may form a multimer such as a dimer, trimer or tetramer, and the molecular weight is larger than that of the full-length antibody. There is also.

  Specific examples of antibody fragments include, for example, Fab, Fab ′, F (ab ′) 2, and Fv. Specific examples of the low molecular weight antibody include, for example, Fab, Fab ′, F (ab ′) 2, Fv, scFv (single chain Fv), Diabody, sc (Fv) 2 (single chain (Fv) 2) And so on. Multimers (for example, dimers, trimers, tetramers, polymers, etc.) of these antibodies are also included in the low molecular weight antibody of the present invention.

  Antibody fragments can be obtained, for example, by treating an antibody with an enzyme to produce antibody fragments. Known enzymes that produce antibody fragments include, for example, papain, pepsin, and plasmin. Alternatively, genes encoding these antibody fragments can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (eg, Co, MS et al., J. Immunol. (1994) 152). , 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Lamoyi, E. , Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669, Bird, RE et al., TIBTECH (1991) 9, 132-137. ).

The digestive enzyme cleaves a specific position of the antibody fragment to give an antibody fragment having a specific structure as follows. If a genetic engineering technique is used for such an enzymatically obtained antibody fragment, any part of the antibody can be deleted.
The antibody fragments obtained when the above digestive enzymes are used are as follows.
Papain digestion: F (ab) 2 or Fab
Pepsin digestion: F (ab ') 2 or Fab'
Plasmin digestion: Facb

  The low molecular weight antibody in the present invention can include an antibody fragment lacking any region as long as it has binding activity and / or neutralizing activity to VEGF-D or antitumor activity.

  Diabodies refer to bivalent antibody fragments constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO93 / 11161 etc.). Diabodies are dimers composed of two polypeptide chains. Usually, in the polypeptide chain constituting the dimer, VL and VH are connected by a linker in the same chain. The linker in the diabody is generally so short that VL and VH cannot bind to each other. Specifically, the amino acid residues constituting the linker are, for example, about 5 residues. Therefore, VL and VH encoded on the same polypeptide chain cannot form a single chain variable region fragment but form a dimer with another single chain variable region fragment. As a result, the diabody has two antigen binding sites.

  The scFv antibody is an antibody in which a heavy chain variable region ([VH]) and a light chain variable region ([VL]) are combined with a linker or the like to form a single chain polypeptide (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883, Plickthun “The Pharmacology of Monoclonal Antibodies” Vol. 113, Resenburg and Moore, Springer Verlag, New York, pp.269-315, (1994)). The H chain V region and L chain V region in scFv may be derived from any of the antibodies described herein. There is no restriction | limiting in particular in the peptide linker which connects V area | region. For example, any single chain peptide consisting of about 3 to 25 residues can be used as a linker. Specifically, for example, a peptide linker described later can be used.

The V regions of both strands can be linked by, for example, the PCR method as described above. In order to link the V regions by the PCR method, first of all, the DNA encoding the desired partial amino acid sequence is used as a template.
DNA sequence encoding antibody H chain or H chain V region, and DNA sequence encoding antibody L chain or L chain V region

By the PCR method using a pair of primers having sequences corresponding to the sequences at both ends of the DNA to be amplified, DNAs encoding the V regions of the H chain and the L chain are respectively amplified. Next, DNA encoding a peptide linker portion is prepared. DNA encoding a peptide linker can also be synthesized using PCR. At this time, a base sequence that can be linked to the amplification product of each V region synthesized separately is added to the 5 ′ side of the primer to be used. Then
[H chain V region DNA]-[peptide linker DNA]-[L chain V region DNA]
PCR reaction is carried out using each of the DNA and primers for assembly PCR.

  The primer for assembly PCR consists of a combination of a primer that anneals to the 5 ′ side of [H chain V region DNA] and a primer that anneals to the 3 ′ side of [L chain V region DNA]. That is, the assembly PCR primer is a primer set that can amplify DNA encoding the full-length sequence of scFv to be synthesized. On the other hand, a base sequence that can be linked to each V region DNA is added to [peptide linker DNA]. As a result, these DNAs are ligated, and the full length of scFv is finally produced as an amplification product by the primers for assembly PCR. Once a DNA encoding scFv is prepared, an expression vector containing them and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. Further, the scFv can be obtained by culturing the resulting recombinant cells and expressing the DNA encoding the scFv.

The order of the heavy chain variable region (VH) and the light chain variable region (VL) to be bound is not particularly limited, and may be arranged in any order. For example, the following arrangements can be given: .
[VH] Linker [VL]
[VL] Linker [VH]

  sc (Fv) 2 is a low molecular weight antibody in which two VHs and two VLs are combined with a linker or the like to form a single chain (Hudson et al, J Immunol. Methods 1999; 231: 177-189). sc (Fv) 2 can be prepared, for example, by linking scFv with a linker.

Two VHs and two VLs are arranged in the order of VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]) starting from the N-terminal side of the single-chain polypeptide. However, the order of the two VHs and the two VLs is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangements can also be mentioned.
[VL] Linker [VH] Linker [VH] Linker [VL]
[VH] Linker [VL] Linker [VL] Linker [VH]
[VH] Linker [VH] Linker [VL] Linker [VL]
[VL] Linker [VL] Linker [VH] Linker [VH]
[VL] Linker [VH] Linker [VL] Linker [VH]

  The amino acid sequence of the heavy chain variable region or light chain variable region in the small molecule antibody may be substituted, deleted, added and / or inserted. Furthermore, when the heavy chain variable region and the light chain variable region are associated, a part may be deleted or another polypeptide may be added as long as it has antigen binding activity. The variable region may be chimerized or humanized.

  In the present invention, the linker that binds the variable region of the antibody may be any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker such as the linker disclosed in Protein Engineering, 9 (3), 299-305, 1996. Can be used.

  A preferred linker in the present invention is a peptide linker. The length of the peptide linker is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose, but is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, Particularly preferred is 12 to 18 amino acids (for example, 15 amino acids).

Examples of the amino acid sequence of the peptide linker include the following sequences.
Ser
Gly ・ Ser
Gly ・ Gly ・ Ser
Ser ・ Gly ・ Gly
Gly, Gly, Gly, Ser (SEQ ID NO: 84)
Ser, Gly, Gly, Gly (SEQ ID NO: 85)
Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 86)
Ser, Gly, Gly, Gly, Gly (SEQ ID NO: 87)
Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 88)
Ser, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 89)
Gly, Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 90)
Ser, Gly, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 91)
(Gly, Gly, Gly, Gly, Ser) n (the amino acid sequence in parentheses is described as SEQ ID NO: 86)
(Ser, Gly, Gly, Gly, Gly) n (The amino acid sequence in parentheses is described as SEQ ID NO: 87)
[N is an integer of 1 or more].

  The amino acid sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose. For example, n which determines the length of the above peptide linker is usually 1 to 5, preferably 1 to 3, more preferably 1 or 2.

  Synthetic compound linkers (chemical cross-linking agents) are commonly used for cross-linking peptides such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) Suberate (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene Glycol bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimideoxycarbonyloxy ) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl ] And the like sulfone (sulfo-BSOCOES), These crosslinking agents are commercially available.

  When linking four antibody variable regions, usually three linkers are required. A plurality of linkers may be the same or different linkers may be used.

  The antibody of the present invention also includes an antibody in which one or more amino acid residues are added to the amino acid sequence of the antibody of the present invention. Also included are fusion proteins in which these antibodies are fused with other peptides or proteins. In the method for producing a fusion protein, a polynucleotide encoding an antibody of the present invention and a polynucleotide encoding another peptide or polypeptide are linked so that the frames coincide with each other, introduced into an expression vector, and expressed in a host. Any technique known to those skilled in the art can be used. Other peptides or polypeptides to be subjected to fusion with the antibody of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 His (histidine) residues 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen Known peptides such as fragments, lck tag, α-tubulin fragment, B-tag, and protein C fragment can be used. Examples of other polypeptides to be subjected to fusion with the antibody of the present invention include GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose). Binding protein) and the like. Preparing a fusion polypeptide by fusing a commercially available polynucleotide encoding the peptide or polypeptide with a polynucleotide encoding the antibody of the present invention and expressing the fusion polynucleotide prepared thereby. Can do.

  The antibody of the present invention may be a conjugated antibody bound to various molecules such as high molecular weight substances such as polyethylene glycol (PEG) and hyaluronic acid, radioactive substances, fluorescent substances, luminescent substances, enzymes, and toxins. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. In addition, the modification method of an antibody has already been established in this field (for example, US5057313, US5156840). The “antibody” in the present invention includes these conjugated antibodies.

  Furthermore, the antibody used in the present invention may be a bispecific antibody. Bispecific antibodies refer to antibodies that have variable regions that recognize different epitopes within the same antibody molecule. In the present invention, the bispecific antibody may be a bispecific antibody that recognizes different epitopes on the VEGF-D molecule, or one antigen binding site recognizes VEGF-D and the other antigen binding. Bispecific antibodies whose sites recognize other substances can also be used.

  Furthermore, from another viewpoint, one antigen-binding site can be a bispecific antibody that recognizes VEGF-D and the other antigen-binding site recognizes an antigen of human effector cells. Examples of the antigen to which the other antigen-binding site of the bispecific antibody comprising the antibody of the present invention that recognizes VEGF-D binds include, for example, CD2, CD3, CD16, CD19, CD20, CD25, CD28, CD33, CD30, CD44, CD44v6, CD52, VEGF, VEGFR, EGF, EGFR, EGFRvIII, HER-2 neu, HER-3, HER-4, cMET, EpCAM, IGF-1R, TRAIL-R2, Tie-1, PDGFR-alpha, NKG2D , CCR5, Gas6, Mer, Tyro3, NCAM, Transferin receptor, Folate binding protein, IL-15, IL-15R, CEA, CA125, MUC-1, Ganglioside GD3, Glypican-3, GM2, Sonic Hedgehog (Shh) Can be mentioned.

  Examples of different epitopes on the VEGF-D molecule to which the other antigen-binding site of the bispecific antibody comprising the antibody of the present invention that recognizes VEGF-D binds include IgD1, IgD2, and FND2.

  Methods for producing bispecific antibodies are known. For example, bispecific antibodies can be produced by combining two types of antibodies with different recognition antigens. The antibody to be bound may be a ½ molecule each having an H chain and an L chain, or may be a ¼ molecule consisting only of an H chain. Alternatively, bispecific antibody-producing fused cells can be prepared by fusing hybridomas that produce different monoclonal antibodies. Furthermore, bispecific antibodies can be produced by genetic engineering techniques.

  The antibody of the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence / absence of sugar chain, form, etc., depending on the antibody-producing cell, host or purification method described below. However, as long as the obtained antibody has a function equivalent to the antibody of the present invention, it is included in the present invention. For example, amino acids included in the amino acid sequences described in the present invention undergo post-translational modifications (eg, modification to pyroglutamic acid by pyroglutamylation of N-terminal glutamine is a modification well known to those skilled in the art) In some cases, even if the amino acid is post-translationally modified as such, it is naturally included in the amino acid sequence described in the present invention. In addition, when the antibody of the present invention is expressed in prokaryotic cells such as E. coli, a methionine residue is added to the N-terminus of the original antibody amino acid sequence. The antibody of the present invention also includes such an antibody. Furthermore, post-translational modifications to sites other than known post-translational modifications are also included in the present invention as long as they have a function equivalent to that of the antibody of the present invention.

Production of Antibody The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Monoclonal antibodies having binding and / or neutralizing activity against VEGF-D or antitumor activity can be obtained by known methods using, for example, VEGF-D derived from mammals such as humans and mice, or fragmented peptides thereof as an immunogen. After preparing an anti-VEGF-D monoclonal antibody, by obtaining an antibody having VEGF-D binding activity and / or neutralizing activity or antitumor activity from the obtained anti-VEGF-D monoclonal antibody I can do it. That is, a desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen and immunized according to a normal immunization method. The anti-VEGF-D monoclonal antibody can be produced by fusing the obtained immune cells with known parental cells by a normal cell fusion method and screening monoclonal antibody-producing cells (hybridomas) by a normal screening method. Is possible. Examples of animals to be immunized include mammals such as mice, rats, rabbits, sheep, monkeys, goats, donkeys, cows, horses, and pigs. The antigen can be prepared using a known VEGF-D gene sequence according to a known method such as W0 98/002543.

  The hybridoma can be produced, for example, according to the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). When the immunogenicity of the antigen is low, immunization may be performed by binding to an immunogenic macromolecule such as albumin.

  As an embodiment of the antibody having binding activity and / or neutralizing activity or antitumor activity to VEGF-D of the present invention, a monoclonal antibody having binding activity and / or neutralizing activity to human VEGF-D or antitumor activity Is mentioned. As an immunogen for producing a monoclonal antibody having binding activity and / or neutralization activity or antitumor activity against human VEGF-D, binding activity and / or neutralization activity against human VEGF-D, or antitumor activity The antibody is not particularly limited as long as an antibody having can be prepared. Alternatively, a VEGF-D fragment peptide or a product obtained by adding an artificial mutation to the natural VEGF-D sequence under the same conditions may be used as the immunogen. Mature human VEGF-D is one of the preferred immunogens for producing antibodies having binding activity and / or neutralizing activity or antitumor activity for VEGF-D of the present invention.

  Further, the measurement of the binding activity and / or neutralizing activity of the antibody to VEGF-D, or the antitumor activity can be performed, for example, by the method described in the Examples.

On the other hand, monoclonal antibodies can also be obtained by DNA immunization. DNA immunization refers to immunization by administering a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in an immunized animal, and expressing the immunizing antigen in the body of the immunized animal. It is a method of giving stimulation. Compared to general immunization methods that administer protein antigens, DNA immunization can be expected to have the following advantages.
-Maintains the structure of membrane proteins and can provide immune stimulation-No need to purify immune antigens

However, on the other hand, in DNA immunization, it is difficult to combine with immune stimulation means such as an adjuvant.
To obtain a monoclonal antibody by DNA immunization, first, DNA encoding VEGF-D is administered to an immunized animal. DNA encoding VEGF-D can be synthesized by a known method such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to an immunized animal. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be used. As a method of administering the vector to a living body, a generally used method can be used. For example, DNA immunization can be performed by driving gold particles adsorbed with an expression vector into cells with a gene gun. Boosting with VEGF-D or VEGF-D expressing cells after DNA immunization is a preferred method for obtaining monoclonal antibodies.

  After the mammal is immunized in this way and the increase in the desired amount of antibody in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. As preferable immune cells, spleen cells can be used.

  Mammalian myeloma cells are used as the cells fused with the above immune cells. The myeloma cell is preferably provided with an appropriate selection marker for screening. A selectable marker refers to a trait that can (or cannot) survive under certain culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells having HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells are unable to synthesize DNA in HAT-selective media and die, but when fused with normal cells, they can continue to synthesize DNA using the salvage circuit of normal cells. Above all, it will proliferate.

  HGPRT-deficient and TK-deficient cells can be selected in media containing 6 thioguanine, 8 azaguanine (hereinafter abbreviated as 8AG), or 5 'bromodeoxyuridine, respectively. Normal cells die because they incorporate these pyrimidine analogs into the DNA, but cells deficient in these enzymes cannot survive these pyrimidine analogs and can survive in selective media. In addition, a selectable marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) with a neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

  Basically, immune cells and myeloma cells according to known methods, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46) And cell fusion.

  More specifically, for example, cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like can be used. Further, an auxiliary agent such as dimethyl sulfoxide can be added as desired in order to increase the fusion efficiency.

  The usage ratio of immune cells and myeloma cells can be set arbitrarily. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture solution used for cell fusion, for example, RPMI1640 culture solution suitable for growth of myeloma cell line, MEM culture solution, and other normal culture solutions used for this type of cell culture can be used. In addition, serum supplements such as fetal calf serum (FCS) can be added to the culture medium.

  In cell fusion, a predetermined amount of immune cells and myeloma cells are mixed well in a culture solution, and a target PEG (hybridoma) is formed by mixing a PEG solution preheated to about 37 ° C. In the cell fusion method, for example, PEG having an average molecular weight of about 1000 to 6000 can be usually added at a concentration of 30 to 60% (w / v). Subsequently, cell fusion agents and the like that are undesirable for the growth of hybridomas are removed by sequentially adding the appropriate culture medium listed above, and then centrifuging to remove the supernatant.

  The hybridoma thus obtained can be selected by using a selective culture solution corresponding to the selection marker possessed by the myeloma used for cell fusion. For example, cells having HGPRT or TK deficiency can be selected by culturing in a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have succeeded in cell fusion with normal cells can be selectively proliferated in the HAT culture solution. The culture using the HAT culture solution is continued for a time sufficient for cells other than the target hybridoma (non-fusion cells) to die. Specifically, in general, the target hybridoma can be selected by culturing for several days to several weeks. Subsequently, by carrying out the usual limiting dilution method, screening and single cloning of the hybridoma producing the target antibody can be performed.

  Screening and single cloning of the target antibody can be preferably performed by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as beads made of polystyrene or the like, or a commercially available 96-well microtiter plate, and reacted with the culture supernatant of the hybridoma. Next, after washing the carrier, a secondary antibody labeled with an enzyme is reacted. If the culture supernatant contains an antibody of interest that reacts with the sensitizing antigen, the secondary antibody binds to the carrier via this antibody. By detecting the secondary antibody that finally binds to the carrier, it can be determined whether the antibody of interest is present in the culture supernatant. It becomes possible to clone a hybridoma producing a desired antibody having the ability to bind to an antigen by a limiting dilution method or the like.

  The base sequence and amino acid sequence encoding the anti-VEGF-D antibody obtained by the above-described method can be obtained by methods known to those skilled in the art.

  Based on the sequence of the obtained anti-VEGF-D antibody, it is possible to produce an anti-VEGF-D antibody using gene recombination techniques known to those skilled in the art. Specifically, a polynucleotide encoding an antibody is constructed based on the sequence of an antibody recognizing VEGF-D, introduced into an expression vector, and then expressed in an appropriate host cell (for example, Co , MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

  Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vector, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned. An expression vector is particularly useful when a vector is used for the purpose of producing the antibody of the present invention. As an expression vector, for example, for the purpose of expression in E. coli, in addition to the above characteristics that the vector is amplified in E. coli, the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, etc. In some cases, promoters that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al. , Science (1988) 240, 1041-1043), or having a T7 promoter or the like. Examples of such vectors include pGEX-5X-1 (manufactured by Pharmacia), “QIAexpress system” (manufactured by Qiagen), pEGFP, or pET (in this case, BL21 in which the host expresses T7 RNA polymerase). Are preferred).

  The vector may contain a signal sequence for antibody secretion. As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used in the case of production in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli, for example, vectors for producing the antibody of the present invention include mammalian-derived expression vectors (for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990, 18 (17) p5322), pEF, pCDM8), expression vectors derived from insect cells (eg “Bac-to-BAC baculovairus expression system” (Gibco BRL), pBacPAK8), plant-derived expression vectors (eg pMH1, pMH2), animal viruses Derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01), Examples include expression vectors derived from Bacillus subtilis (for example, pPL608, pKTH50).

  When intended for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

  Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , PSV2-dhfr (“Molecular Cloning 2nd edition” Cold Spring Harbor Laboratory Press, (1989), etc.) is introduced and amplified by methotrexate (MTX), and for the purpose of transient expression of genes In this case, there is a method of transforming with a vector having SV40 replication origin (such as pcD) using COS cells having a gene expressing SV40 T antigen on the chromosome. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, for gene copy number amplification in host cell systems, the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.

  The antibody of the present invention thus obtained can be isolated from the inside of the host cell or outside the cell (such as a medium) and purified as a substantially pure and homogeneous antibody. Separation and purification of antibodies may be carried out using separation and purification methods used in normal antibody purification, and are not limited in any way. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, When combined, antibodies can be separated and purified.

  Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel). R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Examples of the column used for affinity chromatography include a protein A column and a protein G column. Examples of the column using protein A include Hyper D, POROS, Sepharose FF (GE Amersham Biosciences). The present invention also encompasses antibodies highly purified using these purification methods.

  Measurement of the binding activity of the obtained antibody to VEGF-D can be performed by methods known to those skilled in the art. For example, ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method can be used as a method for measuring the antigen binding activity of an antibody. For example, when an enzyme immunoassay is used, a sample containing an antibody, for example, a culture supernatant of an antibody-producing cell or a purified antibody is added to a plate coated with an antigen. It is possible to evaluate the antigen binding activity by adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating the plate, washing, adding an enzyme substrate such as p-nitrophenyl phosphate and measuring the absorbance. it can.

The pharmaceutical composition and the present invention also provide a pharmaceutical composition containing the above-described antibody as an active ingredient. Furthermore, this invention provides the therapeutic agent of the cancer which uses the above-mentioned antibody as an active ingredient.

  The pharmaceutical composition of the present invention can be further used as a lymphangiogenesis inhibitor, a metastasis inhibitor, a lymph node metastasis inhibitor, or a tumor growth inhibitor. The lymphangiogenesis inhibitor or lymph node metastasis inhibitor neutralizes VEGF-D and inhibits VEGFR-3 signaling that promotes lymphangiogenesis and lymph node metastasis.

  The decrease in the expression level of VEGF-D may reduce the amount of VEGF-D that already exists, such as by degradation of VEGF-D, or it may be newly expressed by suppressing the expression of VEGF-D. The amount of -D may be reduced.

  The lymphangiogenesis inhibitor or lymph node metastasis inhibitor containing the anti-VEGF-D antibody of the present invention can also be expressed as a method of neutralizing VEGF-D using an anti-VEGF-D antibody. Further, the lymphangiogenesis inhibitor or lymph node metastasis inhibitor containing the anti-VEGF-D antibody of the present invention is also expressed as the use of an anti-VEGF-D antibody for the production of a lymphangiogenesis inhibitor or lymph node metastasis inhibitor. it can.

  The anti-VEGF-D antibody of the present invention can be expected to have effects such as lymphangiogenesis inhibitory effect, lymph node metastasis inhibitory effect, and tumor growth inhibitory effect by inhibiting VEGFR-3 signaling.

  The anti-VEGF-D antibody of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A). Further, if necessary, a pharmaceutically acceptable carrier and / or additive may be included. For example, surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (phosphoric acid, citric acid, other organic acids) Etc.), chelating agents (EDTA, etc.), suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity promoters, flavoring agents, and the like. However, the chemical | medical agent of this invention is not restrict | limited to these, The other normal carrier may be included suitably. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts. Further, it may contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine and lysine. In the case of an aqueous solution for injection, the anti-VEGF-D antibody is dissolved in an isotonic solution containing, for example, physiological saline, glucose or other adjuvants. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and further suitable solubilizers such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), A nonionic surfactant (polysorbate 80, HCO-50) may be used in combination.

  If necessary, anti-VEGF-D antibody can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nano Particles, nanocapsules, etc.) (see Remington's Pharmaceutical Science 16th edition &, Oslo Ed. (1980), etc.). Furthermore, a method of making a drug a sustained-release drug is also known and can be applied to an anti-VEGF-D antibody (Langer et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer Chem. Tech. (1982) 12: 98-105; U.S. Pat.No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers (1983) 22: 547-56; EP 133,988 ).

  The pharmaceutical composition of the present invention can be administered either orally or parenterally, but is preferably administered parenterally. Specifically, it is administered to a patient by injection and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection or subcutaneous injection. Local injection, particularly intramuscular injection, may be performed at or around the site where lymphangiogenesis, lymph node metastasis or tumor growth is to be suppressed. The administration method can be appropriately selected depending on the age and symptoms of the patient. The dose can be selected, for example, within the range of 0.0001 mg to 100 mg of active ingredient per kg of body weight per time. Alternatively, for example, when administered to a human patient, the active ingredient per patient can be selected within the range of 0.001 to 1000 mg / kg body weight, and the dose per dose is, for example, 0.01% of the antibody of the present invention. An amount of about ˜50 mg / kg · body · weight is preferably included. However, the pharmaceutical composition of the present invention is not limited to these doses.

  It should be noted that 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 hybridomas using human VEGF-D and cynomolgus monkey VEGF-D immunized mice
Balb / c mice (female, 6 weeks old at the start of immunization, Charles River, Japan) and MRL mice were immunized with human VEGF-D (Reference Example 3) or cynomolgus VEGF-D (Reference Example 3) as follows. When the first immunization was defined as day 0, 100 μg human VEGF-D was subcutaneously administered on day 0 together with Freund's complete adjuvant (Difco). On day 14, subcutaneously administer 50 μg cynomolgus VEGF-D with incomplete Freund's adjuvant (Difco), then subcutaneously administer 50 μg human or cynomolgus VEGF-D with Freund's incomplete adjuvant alternately 3 or 4 times every other week did. One week after the last dose of VEGF-D (Day 42 or Day 49), human VEGF-D was administered intravenously as a boost, and 3 days later, mouse spleen cells and mouse myeloma cells P3X63Ag8U.1 (P3U1 and ATCC CRL-1597) was cell-fused according to a conventional method using PEG1500 (Roche Diagnostics). The fused cells, that is, hybridomas, were cultured in RPMI1640 medium containing 10% FBS (hereinafter referred to as 10% FBS / RPMI1640).

  On the next day after the fusion, (1) the fused cells were suspended in a semi-fluid medium (StemCells) to carry out selective culture of the hybridoma and colonize the hybridoma.

  On day 9 or 10 after fusion, colonies of hybridomas were picked up and HAT selection medium (10% FBS / RPMI1640, 2 vol% HAT 50x concentrate (Dainippon Pharmaceutical), 5 vol% BM-Condimed H1 (Roche Diagnostics) ) Was inoculated in a 96-well plate with 1 colony per well. After culturing for 3 to 4 days, the culture supernatant of each well was collected, and the mouse IgG concentration in the culture supernatant was measured. Production of antibody that specifically binds to human VEGF-D or human VEGF-C by ELISA (Reference Example 4) in which human VEGF-D or human VEGF-C is immobilized on the culture supernatant in which mouse IgG was confirmed Clones to be selected were selected (FIGS. 1 and 2).

[Example 2] Preparation of chimeric antibody Total RNA was extracted from hybridoma cells using RNeasy Mini Kits (QIAGEN), and cDNA was synthesized using SMART RACE cDNA Amplification Kit (BD Biosciences). Using the prepared cDNA, the antibody variable region gene was inserted into a cloning vector by PCR. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions. The determined heavy chain variable region and light chain variable region of the VE29, VE48, and VE199 mouse antibodies are shown in Table 1 (VE29, 48, 199 mouse antibody heavy chain variable region) and Table 2 (VE29, 48, 199 mouse antibody L Chain variable region). CDR and FR were determined according to Kabat numbering.

The correspondence between each sequence in Tables 1 and 2 and SEQ ID NO is shown below.
H chain variable region of VE29 mouse antibody (SEQ ID NO: 60)
VE29 mouse antibody L chain variable region (SEQ ID NO: 61)
H chain variable region of VE48 mouse antibody (SEQ ID NO: 64)
VE48 mouse antibody L chain variable region (SEQ ID NO: 65)
VE199 mouse antibody heavy chain variable region (SEQ ID NO: 62)
VE199 mouse antibody L chain variable region (SEQ ID NO: 63)

  Expression of the chimeric antibody H chain in which the mouse antibody H chain variable region and human antibody IgG1 chain constant region are combined, and the chimeric antibody L chain gene in which the mouse antibody L chain variable region and human antibody Kappa chain constant region are combined, expressed in animal cells Incorporated into the vector. VE29, VE48, and VE199 chimeric antibodies were expressed and purified using the prepared expression vector (Reference Example 7).

[Example 3] Evaluation of in vitro activity of chimeric antibody For the chimeric anti-VEGF-D antibody prepared in Example 2, human VEGFR-3 expressing CHO cells were evaluated by the VEGF-D binding inhibition evaluation system for VEGFR-3 (Reference Example 5). It was confirmed that the binding of human VEGF-D to (Reference Example 2) was inhibited (FIG. 3) and that the binding of cynomolgus VEGF-D to cynomolgus monkey VEGFR-3 expressing CHO cells (Reference Example 2) was inhibited ( FIG. 4).

  Further, using this chimeric antibody, inhibition of proliferation of human lymphatic endothelial cells proliferating in a VEGF-D-dependent manner was evaluated (Reference Example 6). For comparison, the VD1 antibody, which is a mouse IgG1 antibody whose variable region has the amino acid sequence described in Fig. 1 of WO2005 / 087177, was also evaluated at the same time. As a result, it was confirmed that the VE48 chimeric antibody and the VE199 chimeric antibody showed stronger lymphatic endothelial cell growth inhibitory activity than VD1 (FIG. 5).

[Example 4] In vivo activity evaluation of chimeric antibody (inhibition of lymphangiogenesis)
Production of hD_1 cells
A VEGF-D expression plasmid (geneticin resistance) was introduced into HEK293 cells (ATCC) by electroporation. Genetic cells (Invitrogen) were selected at 500 μg / mL from the introduced cells, and hD_1 cells were prepared by a single cell cloning method.
The hD_1 cells were maintained and passaged in a medium supplemented with 10% FBS, 0.1 mM NEAA (GIBCO), 1 mM Sodium Pyruvate (GIBCO), and 500 μg / mL geneticin in E-MEM (SIGMA).

Preparation of administered antibody The chimeric anti-VEGF-D antibody prepared in Example 2 was used on the day of administration using physiological saline at 1 mg / mL (10 mg / kg administration group) and 0.2 mg / mL (2 mg / kg), respectively. (Administration group) to prepare an administration sample.

In vivo lymphangiogenesis model using hD_1 cell transplanted mice
hD_1 cells were prepared in HBSS (GIBCO / Invitrogen) at 4 × 10 ⁸ cells / mL, and 50 μL (2 × 10 ⁷ cells / mouse) was transplanted subcutaneously into the abdomen of a skid mouse (Claire Japan).
Tumor volume was calculated by the following formula, and antibody administration was started when the average tumor volume reached about 100 mm ³ .

Tumor volume = major axis x minor axis x minor axis / 2

  The anti-VEGF-D antibody was administered twice a week for 2 weeks, and the administration sample prepared in (2) above was administered at 10 mL / kg from the tail vein. As a negative control, physiological saline was similarly administered twice a week for 2 weeks at 10 mL / kg from the tail vein. All groups were performed with 3 animals per group.

Evaluation of in vivo lymphangiogenesis model by hD_1 cell-transplanted mice Each anti-VEGF-D antibody was evaluated for in vivo lymphangiogenesis by the following immunostaining method.

Immunostaining
(i) The tumor mass excised from the mouse was embedded in OCT compound (Tissuetek), a frozen section was prepared, and then sliced into thin sections.
(ii) Rabbit anti-mouse Lyve-1 antibody (RELIATech GmbH) was used as a primary antibody and biotinylated labeled anti-rabbit IgG antibody (Vector Laboratories) was used as a secondary antibody for staining of mouse lymphatic vessels.
(iii) Hematoxylin was used for cell nucleus staining.
(iv) The staining of (ii) and (iii) was performed using a fully automatic immunostaining system (VENTANA).
(v) Lymphatic vessel quantification was performed using image analysis software Image Pro Plus (Media Cybernetics), and the stained part of the image of each specimen was measured as the number of pixels and expressed as an average value.

As a result, as shown in FIG. 6, it was confirmed that VE48 and VE199 showed strong in vivo lymphangiogenesis inhibitory activity at both concentrations of 10 mg / kg and 2 mg / kg.
Further, regarding lymphatic vessel formation at each antibody concentration, a significant difference test was performed between physiological saline and the antibody administration group. As a result, a significant difference was observed in lymphatic vessel formation at any antibody concentration.

[Example 5] Evaluation of in vivo activity of chimeric antibody (anti-metastasis of cancer cells)
A VEGF-D expression plasmid (Geneticin resistance) was introduced into mouse melanoma B16F10 cells (Medical Cell Resource Center, Institute of Aging Medicine, Tohoku University) by electroporation. Genetic cells (Invitrogen) were selected at 500 μg / mL from the introduced cells, and B16F10_VEGFD # 4 cells were prepared by a single cell cloning method. B16F10_VEGFD # 4 cells were maintained and subcultured in RPMI-1640 (SIGMA) supplemented with 10% FBS and 500 μg / mL geneticin.

Prepare and administer anti-VEGF-D antibody to 2.5 mg / mL (25 mg / kg administration group) and 1 mg / mL (10 mg / kg administration group) using physiological saline on the day of administration A sample was used.
B16F10_VEGFD # 4 cells were prepared in HBSS (Invitrogen) at 2 × 10 ⁷ cells / mL, and 50 μL (1 × 10 ⁶ cells / mouse) was transplanted subcutaneously into the abdomen of nude mice (Charles River Japan). Immediately after transplantation of B16F10_VEGFD # 4 cells, the anti-VEGF-D antibody was administered twice a week for 2 weeks, and the administration sample prepared above was administered at 10 mL / kg from the tail vein. As a negative control, physiological saline was similarly administered twice a week for 2 weeks at 10 mL / kg from the tail vein. All groups were conducted with 10 animals per group.

  For the evaluation of lymph node metastasis of each anti-VEGF-D antibody, lymph nodes on the same side as cell transplantation were taken out and evaluated by lymph node weight. As a result, as shown in FIG. 7, it was revealed that VE48 exhibits strong lymph node metastasis inhibitory activity at both 25 mg / kg and 10 mg / kg concentrations. Further, the lymph node weight at each antibody concentration was subjected to a significant difference test between the physiological saline and the antibody administration group. As a result, a significant difference was observed in the lymph node weight at any antibody concentration. The SAS preclinical package (SAS Institute Inc.) was used for statistical analysis.

[Example 6] Humanization of VE199 antibody
6-1. Selection of each framework arrangement
In order to humanize the VE199 antibody, the variable region sequence of the VE199 antibody was compared with the human germline sequence. Among them, FR sequences serving as templates for humanization are summarized in Table 3. As a humanized variable region, the H chain was a sequence composed of FR1 (2), FR2, FR3 (2) and FR4 described in Table 3 as a 199HA variable region (SEQ ID NO: 46). For the L chain, a sequence composed of FR1, FR2, FR3 (2), and FR4 was used as a 199LA variable region (SEQ ID NO: 47). The CDR and FR were determined according to Kabat numbering.

  In the sequence of H chain FR1, it has been reported that a series of residues including the 9th Kabat numbering has a great influence on the conformation of FR1 (Jung et al., J Mol Biol. 2001 Jun 8; 309 (3): 701-16.). The residue of VE199 is proline (P) (SEQ ID NO: 62), but the germline sequence selected for humanization is the residue shown in FR1 (1) (SEQ ID NO: 10) of Table 3. Since the group was alanine (A), it was replaced with alanine by humanization, and a decrease in activity was expected. Therefore, for the ninth H chain, the FR1 (2) sequence was used, in which the VE199 sequence remained and proline was used (SEQ ID NO: 11). In addition, in the sequence of H chain FR3, it has been reported that residues 71 and 94 of Kabat numbering affect the three-dimensional structure of CDR (Xiang et al. J Mol Biol. 1995 Oct 27; 253 (3): 385-90.) And residues 69 and 71 are reported to form an upper core and participate in structural stabilization (Ewert et al., Methods. 2004 Oct; 34 (2): 184 -99). The residue of VE199 is 69 for leucine (L), 71 for valine (V), and 93 for isoleucine (I) (SEQ ID NO: 62). As shown in FR3 (1) (SEQ ID NO: 13) of No. 3, the residue is No. 69 isoleucine (I), No. 71 is alanine (A), No. 93 is alanine (A). A decrease in activity was expected due to substitution for each residue. Therefore, the FR3 (2) sequence in which the VE199 sequence remained was used for the 69th, 71st, and 93rd chains (SEQ ID NO: 14). Furthermore, in consideration of immunogenicity, we decided to adopt the CDR2 immediately before FR3 by replacing the 64th arginine (R) with glutamine (Q) with reference to the germline IGHV1-45 sequence (SEQ ID NO: : 15).

  In the sequence of L chain FR3, the 87th residue of Kabat numbering has been reported to affect VH / VL interface formation (Vargas-Madrazo et al., J Mol Recognit. 2003; 16 (3): 113-20 .). The residue of VE199 is phenylalanine (F) (SEQ ID NO: 63), but the germline sequence selected for humanization is the residue shown in FR3 (1) (SEQ ID NO: 22) of Table 3. Since the group was tyrosine (Y), it was replaced with tyrosine by humanization, and a decrease in activity was expected. Therefore, the FR3 (2) sequence in which the VE199 sequence was left and phenylalanine was used for the 87th L chain was used (SEQ ID NO: 23).

6-2. Preparation of humanized VE199 variable regions 199HA and 199LA To create variable regions of humanized VE199 antibody by grafting CDR region of VE199 antibody into FR region of humanized template sequence, synthetic oligo DNA was designed for H chain and L chain respectively. . Each synthetic oligo DNA was mixed and a gene encoding the variable region of humanized VE199 was prepared by assembling PCR, and the H chain was 199HA and the L chain was 199LA. Assemble PCR was performed using PirmeSTAR GXL (TaKaRa Bio), and PCR was performed according to the following conditions. The reaction mixture consisting of the attached PCR Buffer, dNTPs, PirmeSTAR GXL and 15 pmol synthetic oligo DNA was heated at 94 ° C for 5 minutes, then at 94 ° C for 2 minutes, at 55 ° C for 2 minutes, and at 68 ° C. After eight PCR reaction cycles consisting of 2 minutes, 5 pmol each of a primer with a restriction enzyme site and Kozak sequence added to the 5 'end of the variable region and a primer with a restriction enzyme site added to the 3' end A PCR reaction cycle consisting of 10 seconds at 94 ° C., 5 seconds at 55 ° C. and 1 minute at 68 ° C. was performed 30 times to obtain an amplified fragment. The obtained amplified fragment was cloned using an animal cell expression vector and ligated to the constant region.

  Here, as a heterogeneity derived from the H chain C-terminal sequence of the IgG antibody (SEQ ID NO: 58), a C-terminal amino acid deletion of a lysine residue and a C-terminal 2-amino acid deletion of both glycine and lysine Amidation of the terminal amino group has been reported (Anal Biochem. 2007 Jan 1; 360 (1): 75-83.). As a method for reducing these heterogeneities, a method of deleting two amino acids at the C-terminal of the H chain, that is, glycine at EU numbering 446 and lysine at 447 is known (WO 2009/041613). Even in the humanized VE199 antibody, it is desirable that there is no heterogeneity derived from the H chain C-terminal sequence. Therefore, the IgG1 sequence lacking the EU numbering 446th glycine and the 447th lysine of human IgG1 (SEQ ID NO: 68) was used as the constant region sequence. On the other hand, the natural human κ chain (SEQ ID NO: 59) was used as the constant region sequence for the L chain. Antibody expression and purification were carried out according to the procedure of Reference Example 7. Regarding humanized VE199 antibody, inhibition of binding of human VEGF-D to human VEGFR-3 expressing CHO cells (Reference Example 2) was examined using a VEGF-D binding inhibition evaluation system for VEGFR-3 (Reference Example 5) (FIG. 8). . Humanized VE199 antibody (H chain: VE199HA / SEQ ID NO: 69, L chain: VE199LA / SEQ ID NO: 70) is a chimeric VE48 antibody (H chain: cHVE48 / SEQ ID NO: 66, L chain: cLVE48 / SEQ ID NO: 67) It was judged that the activity was equivalent to that of the standard chimeric antibody even after humanization.

[Example 7] Humanization of VE48 antibody
7-1. Selection of each framework arrangement
In order to humanize the VE48 antibody, the variable region sequence of the VE48 antibody was compared with the human germline sequence. Among them, FR sequences serving as templates for humanization are summarized in Table 4. As a humanized variable region, the sequence consisting of FR1, FR2, FR3 (2) and FR4 described in Table 4 as the H chain was designated as the HA variable region (SEQ ID NO: 48). The L chain was a sequence consisting of FR1, FR2, FR3 (2), and FR4 as an L6 variable region (SEQ ID NO: 56). The CDR and FR were determined according to Kabat numbering.

  In the sequence of H chain FR3, the 94th residue of Kabat numbering has been reported to greatly affect the conformation of CDR3 (Morea et al. J. Mol. Biol. 1998; 275: 269-294). The residue of the VE48 variable region is glutamine (Q) (SEQ ID NO: 64), but the germline sequence selected for humanization is as shown in FR3 (1) (SEQ ID NO: 30) in Table 4, Since the residue is arginine (R), it was substituted with arginine by humanization, and a decrease in activity was expected. Therefore, for the 94th H chain, the FR3 (2) sequence was used in which the VE48 sequence remained and glutamine (Q) was used (SEQ ID NO: 31).

  In the sequence of L chain FR3, the 87th residue of Kabat numbering has been reported to affect VH / VL interface formation (Vargas-Madrazo et al., J Mol Recognit. 2003; 16 (3): 113-20 .). The residue of the VE48 variable region is phenylalanine (F) (SEQ ID NO: 65), but the germline sequence selected for humanization is as shown in FR3 (1) (SEQ ID NO: 38) of Table 4, Since the residue is tyrosine (Y), it was replaced with tyrosine by humanization, and a decrease in activity was expected. Therefore, the FR3 (2) sequence in which the VE48 sequence remained and the phenylalanine sequence was used for the 87th L chain was used (SEQ ID NO: 39).

7-2. Preparation of humanized VE48 variable regions HA and L6 In order to create a variable region of a humanized VE48 antibody in which the CDR region of the VE48 antibody was transplanted into the FR region of the humanized template sequence, a synthetic oligo DNA was designed only for the H chain. Each synthetic oligo DNA was mixed and a gene encoding the variable region of humanized VE48 was prepared by assembly PCR, and this was designated as HA (SEQ ID NO: 48). The variable region and the vector linked to the constant region were prepared in the same manner as in Example 6.

  Since the homology between the VE48 antibody and the VE199 antibody is high with respect to the L chain, a modification (No. 91) was made with only CDR3 as the VE48 antibody sequence based on the 199LA variable region (SEQ ID NO: 47) prepared in Example 6. Glycine was substituted with serine, and 96th arginine was substituted with tryptophan). The mutant was prepared by performing Assemble PCR using PCR. Specifically, first, normal- and reverse-strand oligo DNAs designed based on the amino acid sequence including the modification site were synthesized. A reverse-strand oligo DNA that binds to a normal-chain oligo DNA containing the modification site and a vector into which the gene to be modified is inserted, or a reverse-strand oligo DNA that contains the modification site and a vector into which the gene to be modified is inserted By combining the normal strand oligo DNAs to be combined and performing PCR using PrimeSTAR (TAKARA), two fragments containing the modification site were prepared on the 5 ′ end side and the 3 ′ end side. The two fragments were joined together by Assemble PCR to prepare a gene fragment of the L6 variable region sequence (SEQ ID NO: 56) in which the 91st glycine was replaced with serine and the 96th arginine was replaced with tryptophan.

  As the L chain constant region, a natural human kappa chain (SEQ ID NO: 59) was used as a constant region sequence to obtain an animal cell expression vector. Antibody expression and purification were carried out according to the procedure of Reference Example 7. For humanized VE48 antibody (H chain: VE48HA / SEQ ID NO: 71, L chain: VE48L6 / SEQ ID NO: 72), a human VEGFR-3 expression CHO was evaluated by a VEGF-D binding inhibition evaluation system for VEGFR-3 (Reference Example 5). The binding inhibition of human VEGF-D to cells (Reference Example 2) was examined (FIG. 9). The humanized VE48 antibody has no significant decrease in activity compared to the chimeric VE48 antibody (H chain: cHVE48 / SEQ ID NO: 66, L chain: cLVE48 / SEQ ID NO: 67), Judged to have equivalent activity.

[Example 8] Introduction of mutation that suppresses deamidation reaction Although the antibody used for the pharmaceutical product is a monoclonal antibody obtained from a clone derived from a single antibody-producing cell, heterogeneity exists. . It is known that such antibody heterogeneity is caused by modifications such as oxidation and deamidation, and increases upon long-term storage and exposure to stress conditions such as heat stress and light stress (reference) Literature: Heterogeneity of Monoclonal Antibodies: Journal of pharmaceutical sciences, vol. 97, No. 7, 2426-2447). However, in developing an antibody as a pharmaceutical product, the physical properties, particularly homogeneity and stability, of the protein are extremely important, and it is desired that the heterogeneity of the target substance is reduced and that it is as single as possible.

  The deamidation reaction is a reaction that occurs non-enzymatically in the side chains of asparagine (N) and glutamine (Q), and the amide present in the side chains of asparagine and glutamine is changed to a carboxylic acid. Since the deamidation reaction that occurs during storage causes the above-described heterogeneity, it is desired to be suppressed as much as possible. In addition, it has been reported that deamidation reaction is likely to occur particularly at a site where asparagine (N) and glycine (G) are adjacent (... NG ...) (Geiger et al. J. Biol. Chem. 1987). ; 262: 785-794). Since there is an adjacent sequence of asparagine (N) and glycine (G) in CDR1 of L6 (SEQ ID NO: 72), it is considered possible to suppress the deamidation reaction by amino acid substitution at this site. It was.

  Suppression of the deamidation reaction by amino acid substitution was specifically performed as follows. It was thought that the deamidation reaction could be suppressed by substituting asparagine (N) present in Kabat numbering No. 28 of L6 (SEQ ID NO: 72) with a different residue. Therefore, L14 (SEQ ID NO: 73) was prepared by replacing asparagine (N) present in Kabat numbering 28 of L6 (SEQ ID NO: 72) with lysine (K). The mutant was prepared by performing Assemble PCR using PCR. Specifically, it was carried out according to the method of Example 7. The prepared gene fragment was inserted into an animal cell expression vector, and the base sequence of the obtained expression vector was determined by a method known to those skilled in the art. Antibody production and purification were performed according to the method of Reference Example 7. Using these, HA / L14 (H chain: VE48HA / SEQ ID NO: 71, L chain: VE48L14 / SEQ ID NO: 73) was prepared, and these variants were evaluated for inhibition of VEGF-D binding to VEGFR-3. The inhibition of binding of human VEGF-D to human VEGFR-3 expressing CHO cells (Reference Example 2) was examined by the system (Reference Example 5). The results are shown in FIG. The prepared variant has no significant decrease in activity compared to chVE48 (H chain: cHVE48 / SEQ ID NO: 66, L chain: cLVE48 / SEQ ID NO: 67), and can suppress deamidation reaction. it was thought.

[Example 9] Introduction of mutation that changes isoelectric point As one of the methods for controlling the plasma half-life of an antibody, a method for controlling the surface charge of an antibody molecule by modifying an amino acid residue exposed on the surface of the antibody molecule Are known (WO2007 / 114319 and WO2009 / 041543). Specifically, it is known that the plasma half-life of an antibody can be extended by reducing the isoelectric point (pI) value of the antibody. Contrary to this, it is known that an increase in the isoelectric point of the antibody shortens the plasma half-life and improves the tissue migration of the antibody (non-patent literature: Vaisitti T, Deaglio S, Malavasi F ., Cationization of monoclonal antibodies: another step towards the "magic bullet" ?, J Biol Regul Homeost Agents. (2005) 19 (3-4), 105-12 and: Pardridge WM, Buciak J, Yang J, Wu D. Enhanced endocytosis in cultured human breast carcinoma cells and in vivo biodistribution in rats of a humanized monoclonal antibody after cationization of the protein. J Pharmacol Exp Ther. (1998) 286 (1), 548-54).

  Based on the above, the humanized VE48 antibody with an altered isoelectric point is expected to have stronger antitumor activity due to the extension of plasma half-life or improved tissue migration. Therefore, the identification of amino acid residues that can control the pharmacokinetics of an antibody by regulating the charge on the surface of the antibody molecule without affecting the binding activity of the humanized VE48 antibody to the antigen or its conformation. Went. Specifically, the isoelectric point of the variable region of HA / L6 (H chain VE48HA / SEQ ID NO: 71, L chain VE48L6 / SEQ ID NO: 72) is changed without significantly reducing the antigen binding inhibitory activity. We searched for possible mutation sites.

  Using a three-dimensional structure model of the humanized VE48 antibody, mutation sites that can change the isoelectric point of the variable region without significantly reducing the binding to VEGF-D were screened. As a result, several mutation sites were found. Modifications that lower the isoelectric point in the H chain are shown in Table 5 (modified points for lowering the isoelectric point of the H chain). Each variant was prepared and purified by the method described in Example 7.

  About the produced modified body, the binding inhibition of human VEGF-D with respect to a human VEGFR-3 expression CHO cell (reference example 2) was investigated by the VEGF-D binding inhibition evaluation system (reference example 5) with respect to VEGFR-3. As shown in FIG. 11 to FIG. 14, the antigen binding inhibitory activity of each variant is greatly reduced compared to that of chVE48 (H chain: cHVE48 / SEQ ID NO: 66, L chain: cLVE48 / SEQ ID NO: 67). Was not.

[Reference Example 1] Isolation of human VEGF-D, human VEGFR-3, cynomolgus VEGF-D, cynomolgus VEGFR-3 gene Human VEGF-D gene, human VEGFR-3 gene, cynomolgus VEGF-D gene, cynomolgus VEGFR-3 An attempt was made to isolate the gene. Primers were designed from publicly available human VEGF-D gene sequence information, etc., and the human VEGF-D gene was successfully amplified from the human lung cDNA library (Clontech) by PCR. Similarly, primers were designed from publicly available human VEGFR-3 gene sequence information and the like, and the human VEGFR-3 gene was successfully amplified from HUVEC (Normal Human Umbilical Vein Endothelial cells, Lonza) by PCR. Similarly, primers were designed based on publicly available human VEGF-D gene sequence information, etc., and the cynomolgus VEGF-D gene was successfully amplified from Monkey (Cynomolgus) Tissue cDNA: Breast (CytoMol) by PCR. Similarly, primers were designed from publicly available human VEGFR-3 gene sequence information and the like, and the cynomolgus monkey VEGFR-3 gene was successfully amplified from cynomolgus monkey spleen cDNA by PCR. For isolated human VEGF-D and cynomolgus VEGF-D, the gene sequences predicted to encode mature proteins after processing are shown in SEQ ID NOs: 74 and 76. The gene sequences of human VEGFR-3 and cynomolgus monkey VEGFR-3 are shown in SEQ ID NOs: 78 and 80. The amino acid sequences of mature human VEGF-D, mature cynomolgus monkey VEGF-D, human VEGFR-3, and cynomolgus monkey VEGFR-3 are shown in SEQ ID NOs: 75, 77, 79, and 81.

[Reference Example 2] Establishment of a VEGFR-3 expressing CHO cell line A sequence (SEQ ID NO: 82) obtained by removing the predicted region from the intracellular region of the human VEGFR-3 gene (Reference Example 1, SEQ ID NO: 78) was subjected to PCR. Amplified by the method. Similarly, a sequence (SEQ ID NO: 83) from which the intracellular region of the cynomolgus monkey VEGFR-3 gene (Reference Example 1, SEQ ID NO: 80) was removed was amplified by PCR. These were inserted into an expression vector for mammalian cells, linearized with a restriction enzyme, and then introduced into CHO cells by electroporation (BioRad Gene Pulser, 25 μF, 1.5 kV). Human VEGFR-3 expressing CHO cells and cynomolgus monkey VEGFR-3 expressing CHO cells were established by selection with drugs and FCM analysis using anti-human VEGFR-3 antibody (R & D).

[Reference Example 3] Preparation of VEGF-D
Inserted mature human VEGF-D gene (Reference Example 1, SEQ ID NO: 74) and mature cynomolgus monkey VEGF-D gene (Reference Example 1, SEQ ID NO: 76) with His tag into mammalian cell expression vectors. Then, transient expression was performed by gene transfer into FreeStyle293-F cells. The culture supernatant was applied to a HisTrap column (GE Healthcare), affinity purification eluting with imidazole was performed, and a fraction containing VEGF-D was separated. Further, the obtained fraction was subjected to gel filtration using Superdex75 (GE Healthcare) on the developing solution D-PBS to purify the active VEGF-D dimer.

[Reference Example 4] Establishment of VEGF-C and VEGF-D ELISA systems
Add 1 μg / mL VEGF-C (R & D) or 1 μg / mL VEGF-D (Reference Example 3) to Ni-coated 96-well plate (PIERCE) at 100 μL / well and fix under shaking for 1 hour at 25 ° C. Phased. After washing 5 times with 0.05% Tween20 / PBS, blocking was performed by allowing to stand at room temperature for 1 hour using 200 μL / well blocking one (Nacalai Tesque). Anti-VEGF-D antibody diluted with blocking one as a primary antibody was added at 100 μL / well, reacted at room temperature for 1 hour, and washed 3 times with 0.05% Tween20 / PBS. Further, HRP-labeled Goat anti mouse IgG antibody (Sigma) diluted with blocking one as a secondary antibody was added at 100 μL / well, reacted at room temperature for 1 hour, and then washed 5 times with 0.05% Tween20 / PBS. A chromogenic substrate (KPL) was added at 100 μL / well and reacted at room temperature, and then the reaction was stopped by adding 1% SDS at 100 μL / well. A wavelength of 405 nm was measured with a plate reader (BioRad).

[Reference Example 5] Establishment of VEGF-D binding inhibition system by competitive cell ELISA Human and cynomolgus monkey VEGFR-3-expressing CHO cells (Reference Example 2) were extracted from the flask in which they were cultured, and a-MEM / 10% FBS / 1% Penicillin -Streptomycin (Invitrogen) / 1 × HT (Invitrogen) / 500μg / mL Suspended to 1 × 10 ⁵ cell / mL in Geneticin (Invitrogen) medium and seeded in 96 well collagen plate (Beckton Dickinson) at 200μL / well It was crowded. After culturing at 37 ° C. and 5% CO ₂ for 2 days, it was used for competitive Cell ELISA. After removing the medium, anti-VEGF-D antibody diluted with 2% FBS / HBSS containing 2.5 μg / mL human or cynomolgus VEGF-D (Reference Example 3) was added. After incubation at room temperature for 1 hour, VEGF-D and anti-VEGF-D antibody were removed, and washing was performed 3 times with 200 μL 0.05% Tween20 / PBS. PolyHistidine HRP MAb (R & D) diluted 500 times as a secondary antibody was added at 100 μL / well. After incubating at room temperature for 1 hour, the secondary antibody was removed and washed 5 times with 200 μL 0.05% Tween20 / PBS. A chromogenic substrate (KPL) was added at 100 μL / well and reacted at room temperature, and then the reaction was stopped by adding 1% SDS at 100 μL / well. Wavelengths of 405 nm / 620 nm were measured with a plate reader (BioRad).

[Reference Example 6] Human lymphatic endothelial cell proliferation inhibition assay HMVEC-LLy cells (TAKARA BIO) were used as human lymphatic endothelial cells. HMVEC-LLy cells were maintained and subcultured with microvascular endothelial cell medium kit-2 (5% FBS) medium (TAKARA BIO). HMVEC-LLy cells were prepared in microvascular endothelial cell medium kit-2 medium so as to be 5 × 10 ⁴ cells / mL. 100 μL (5 × 10 ³ cells / well) was plated on a 96-well plate (Corning) and cultured in a 5% carbon dioxide incubator at 37 ° C. for 24 hours. After culture, add anti-VEGF-D antibody to assay medium (RPMI-1640 medium (SIGMA) 0.1% FBS, 500 ng / mL human VEGF-D (R & D)) to an appropriate concentration, and add 96-well plate. Microvascular endothelial cell medium kit-2 medium was added after removal. The cells were cultured for 72 hours at 37 ° C. in a 5% carbon dioxide incubator. After the culture, Cell Counting Kit-8 (Dojindo Laboratories) was added at 10 μL / well, and cultured at 37 ° C. for 3 hours in a 5% carbon dioxide incubator. After incubation, the absorbance at 450 nm was measured.

[Reference Example 7] Preparation of antibody expression vector and expression and purification of antibody The synthesis of full-length genes encoding the heavy chain and light chain base sequences of the variable regions of antibodies is known to those skilled in the art using Assemble PCR and the like. It was produced by the method. Amino acid substitution was introduced by a method known to those skilled in the art using QuikChange Site-Directed Mutagenesis Kit (Stratagene) or PCR. The obtained plasmid fragment was inserted into an animal cell expression vector to prepare an H chain expression vector and an L chain expression vector. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. The prepared plasmid was transiently introduced into a human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) or FreeStyle293 cells (Invitrogen) to express the antibody. After collecting the obtained culture supernatant, the culture supernatant was obtained through a 0.22 μm filter MILLEX®-GV (Millipore) or a 0.45 μm filter MILLEX®-GV (Millipore). The antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose ^™ Fast Flow (GE Healthcare). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

Claims (12)

The anti-VEGF-D antibody according to any one of the following (1) to (5);
(1) An antibody having a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 3. ,
(2) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 6. ,
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) The antibody according to any one of (1) to (3), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (3) An antibody having an activity equivalent to that of the antibody according to
(5) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (4) binds. The anti-VEGF-D antibody according to any one of the following (1) to (8);
(1) A heavy chain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 7, CDR2 having the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 15, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 9 An antibody having a variable region,
(2) an antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 17, CDR2 having the amino acid sequence set forth in SEQ ID NO: 18 and CDR3 having the amino acid sequence set forth in SEQ ID NO: 19 ,
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) FR1 in which the FR of the heavy chain variable region of the antibody according to any one of (1) and (3) has the amino acid sequence described in SEQ ID NO: 10 or SEQ ID NO: 11, described in SEQ ID NO: 12 An antibody having a heavy chain variable region which is FR2 having an amino acid sequence, FR3 having an amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14, and FR4 having an amino acid sequence set forth in SEQ ID NO: 16;
(5) FR1 in which the FR of the light chain variable region of the antibody according to any one of (2) and (3) has the amino acid sequence set forth in SEQ ID NO: 20, and FR2 having the amino acid sequence set forth in SEQ ID NO: 21 An antibody having a light chain variable region which is FR3 having the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, FR4 having the amino acid sequence set forth in SEQ ID NO: 24,
(6) an antibody having the heavy chain variable region of (4) and the light chain variable region of (5),
(7) The antibody according to any one of (1) to (6), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having an activity equivalent to that of the antibody according to
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds. The anti-VEGF-D antibody according to any one of the following (1) to (8);
(1) CDR1 having the amino acid sequence described in SEQ ID NO: 25 or SEQ ID NO: 43, CDR2 having the amino acid sequence described in SEQ ID NO: 26, SEQ ID NO: 42 or SEQ ID NO: 44, described in SEQ ID NO: 27 An antibody having a heavy chain variable region comprising CDR3 having the amino acid sequence of
(2) a light chain comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 41, CDR2 having the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 35 An antibody having a variable region,
(3) an antibody having the heavy chain variable region of (1) and the light chain variable region of (2),
(4) FR1 in which the FR of the heavy chain variable region of the antibody according to any one of (1) and (3) has the amino acid sequence set forth in SEQ ID NO: 28, FR2 having the amino acid sequence set forth in SEQ ID NO: 29 An antibody having a heavy chain variable region which is FR3 having the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31, FR4 having the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 45,
(5) FR1 in which the FR of the light chain variable region of the antibody according to any one of (2) and (3) has the amino acid sequence set forth in SEQ ID NO: 36, FR2 having the amino acid sequence set forth in SEQ ID NO: 37 An antibody having a light chain variable region which is FR3 having the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39, FR4 having the amino acid sequence set forth in SEQ ID NO: 40,
(6) an antibody having the heavy chain variable region of (4) and the light chain variable region of (5),
(7) The antibody according to any one of (1) to (6), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having an activity equivalent to that of the antibody according to
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds. The antibody variable region or antibody according to any one of (1) to (5) below;
(1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 46;
(2) a light chain variable region having the amino acid sequence of SEQ ID NO: 47,
(3) an antibody comprising the heavy chain variable region of (1) and the light chain variable region of (2),
(4) The antibody according to any one of (1) to (3), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (3) An antibody having an activity equivalent to that of the antibody according to
(5) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (4) binds. The antibody variable region or antibody according to any one of the following (1) to (13);
(1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 48,
(2) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49,
(3) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 50,
(4) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51,
(5) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 52,
(6) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 53,
(7) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 54,
(8) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 55,
(9) a light chain variable region having the amino acid sequence of SEQ ID NO: 56,
(10) a light chain variable region having the amino acid sequence of SEQ ID NO: 57,
(11) An antibody comprising the heavy chain variable region according to any one of (1) to (8) and the light chain variable region of (9) or (10),
(12) The antibody according to any one of (1) to (11), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (11) An antibody having an activity equivalent to that of the antibody according to
(13) An antibody that binds to the same epitope to which the antibody according to any one of (1) to (12) binds.   The anti-VEGF-D antibody according to any one of claims 1 to 5, which is a humanized antibody.   A pharmaceutical composition comprising the antibody according to claim 1.   The pharmaceutical composition according to claim 7, which is a cancer therapeutic agent.   The pharmaceutical composition according to claim 7, which is a lymphangiogenesis inhibitor.   The pharmaceutical composition according to claim 7, which is a tumor growth inhibitor.   The pharmaceutical composition according to claim 7, which is a metastasis inhibitor.   The pharmaceutical composition according to claim 7, which is a lymph node metastasis inhibitor.