JP5139517B2 - Anti-NR10 antibody and use thereof - Google Patents

Description

  The present invention relates to an anti-NR10 antibody and a pharmaceutical composition containing the anti-NR10 antibody.

  Numerous cytokines are known as humoral factors involved in the proliferation and differentiation of various cells or the activation of the functions of differentiated and mature cells. In living bodies, cells stimulated with cytokines produce other cytokines, and a network of a plurality of cytokines is formed. Biological homeostasis is maintained on a delicate balance by the network coordinating with each other. Many immunoinflammatory diseases are thought to be caused by the breakdown of these cytokine networks, and anti-cytokine therapy using monoclonal antibodies has attracted attention. For example, anti-TNF antibodies and anti-IL-6 receptor antibodies have been highly effective clinically. On the other hand, however, there are many cases in which the compensatory pathway works in actual pathological conditions, and if only one cytokine such as IL-4 is blocked, a therapeutic effect cannot be obtained and it fails.

  The present inventors have succeeded in isolating a novel cytokine receptor NR10 having high homology to IL-6 signaling receptor gp130 (Patent Document 1). NR10 forms a heterodimer with the oncostatin M receptor (OSMR) and functions as a receptor for IL-31 (Non-patent Document 1). Regarding IL-31, it has been reported that transgenic mice overexpressing IL-31 spontaneously develop pruritic dermatitis (Patent Document 2).

  Antibodies that bind to NR10 and inhibit the binding of IL-31 and NR10 are thought to be effective in the treatment of inflammatory diseases, but in order to use anti-NR10 antibodies clinically, antibodies with low immunogenicity are required. It is said. Further, in order to exert a high therapeutic effect, an antibody having a high binding activity or neutralizing activity to NR10 is desired.

Prior art documents of the present invention are shown below.
WO00 / 75314 WO03 / 060090 IL-31 is associated with cutaneous lymphocyte antigen-positive skin homing T cells in patients with atopic dermatitis., J Allergy Clin Immunol. 2006 Feb; 117 (2): 418-25.

  The present invention has been made in view of the above background, and an object of the present invention is to provide an anti-NR10 antibody and a pharmaceutical composition containing the anti-NR10 antibody.

  The present inventors have intensively studied to solve the above problems. The present inventors have succeeded in obtaining an anti-NR10 antibody having effective neutralizing activity against NR10. Furthermore, the present inventors succeeded in humanizing an antibody while maintaining the activity of the antibody. Furthermore, the inventors have succeeded in producing antibodies with improved pharmacokinetics, enhanced antigen binding activity, improved stability and / or reduced risk of immunogenicity. The antibody is useful as a therapeutic agent for inflammatory diseases.

  The present invention relates to an anti-NR10 antibody and a pharmaceutical composition containing the anti-NR10 antibody, and more specifically includes the following inventions [1] to [15].

[1] An antibody that recognizes domain 1 of NR10.
[2] The antibody according to [1], which has neutralizing activity.
[3] The antibody according to [1] or [2], which is a humanized antibody.
[4] The anti-NR10 antibody according to any one of (1) to (8) below:
(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 the heavy chain variable region described in SEQ ID NO: 4;
(3) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 5, CDR2 having the amino acid sequence set forth in SEQ ID NO: 6, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 7. ,
(4) an antibody having the light chain variable region set forth in SEQ ID NO: 8;
(5) an antibody having the heavy chain variable region of (1) and the light chain variable region of (3),
(6) an antibody having the heavy chain variable region of (2) and the light chain variable region of (4),
(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 described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.
[5] The anti-NR10 antibody according to any one of the following (1) to (8);
(1) An antibody having a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 10, and CDR3 having the amino acid sequence of SEQ ID NO: 11. ,
(2) an antibody having the heavy chain variable region set forth in SEQ ID NO: 12;
(3) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 13, CDR2 having the amino acid sequence set forth in SEQ ID NO: 14 and CDR3 having the amino acid sequence set forth in SEQ ID NO: 15 ,
(4) an antibody having the light chain variable region set forth in SEQ ID NO: 16;
(5) an antibody having the heavy chain variable region of (1) and the light chain variable region of (3),
(6) an antibody having the heavy chain variable region of (2) and the light chain variable region of (4),
(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 described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.
[6] The anti-NR10 antibody according to any one of (1) to (8) below:
(1) An antibody having a heavy 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. ,
(2) an antibody having the heavy chain variable region set forth in SEQ ID NO: 20;
(3) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 21, CDR2 having the amino acid sequence set forth in SEQ ID NO: 22, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 23 ,
(4) an antibody having the light chain variable region set forth in SEQ ID NO: 24,
(5) an antibody having the heavy chain variable region of (1) and the light chain variable region of (3),
(6) an antibody having the heavy chain variable region of (2) and the light chain variable region of (4),
(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 described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.
[7] The anti-NR10 antibody according to any one of (1) to (8) below:
(1) An antibody having a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 25, CDR2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 27 ,
(2) an antibody having the heavy chain variable region set forth in SEQ ID NO: 28,
(3) An antibody having a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 29, CDR2 having the amino acid sequence set forth in SEQ ID NO: 30 and CDR3 having the amino acid sequence set forth in SEQ ID NO: 31 ,
(4) an antibody having the light chain variable region set forth in SEQ ID NO: 32,
(5) an antibody having the heavy chain variable region of (1) and the light chain variable region of (3),
(6) an antibody having the heavy chain variable region of (2) and the light chain variable region of (4),
(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 described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds.
[8] The antibody variable region or antibody according to any one of (1) to (20) below;
(1) heavy chain variable region (H17) comprising CDR1 of SEQ ID NO: 196, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 11;
(2) a heavy chain variable region (H19) comprising CDR1 of SEQ ID NO: 176, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 11;
(3) heavy chain variable region (H28, H42) comprising CDR1 of SEQ ID NO: 196, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184,
(4) heavy chain variable region (H30, H44) comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184,
(5) A heavy chain variable region comprising CDR1 of SEQ ID NO: 176, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184. (H34, H46),
(6) heavy chain variable region (H57, H78) comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 198, CDR3 of SEQ ID NO: 184,
(7) A heavy chain variable region comprising CDR1 of SEQ ID NO: 176, CDR2 of SEQ ID NO: 198, CDR3 of SEQ ID NO: 184. (H71, H92),
(8) heavy chain variable region (H97, H98) comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 199, CDR3 of SEQ ID NO: 184,
(9) a light chain variable region (L11) comprising CDR1 of SEQ ID NO: 200, CDR2 of SEQ ID NO: 170, CDR3 of SEQ ID NO: 193,
(10) CDR1 described in SEQ ID NO: 201, CDR2 described in SEQ ID NO: 170, light chain variable region comprising CDR3 described in SEQ ID NO: 193 (L12),
(11) a light chain variable region (L17) comprising CDR1 of SEQ ID NO: 202, CDR2 of SEQ ID NO: 170, CDR3 of SEQ ID NO: 193,
(12) a light chain variable region (L50) comprising CDR1 of SEQ ID NO: 203, CDR2 of SEQ ID NO: 170, CDR3 of SEQ ID NO: 193,
(13) An antibody comprising the heavy chain variable region of (3) and the light chain variable region of (11),
(14) An antibody comprising the heavy chain variable region of (4) and the light chain variable region of (11),
(15) An antibody comprising the heavy chain variable region of (5) and the light chain variable region of (11),
(16) An antibody comprising the heavy chain variable region of (6) and the light chain variable region of (11),
(17) An antibody comprising the heavy chain variable region of (7) and the light chain variable region of (11),
(18) an antibody comprising the heavy chain variable region of (8) and the light chain variable region of (12),
(19) The antibody according to any one of (13) to (18), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (13) to (18) An antibody having an activity equivalent to that of the antibody described in 1.
(20) An antibody that binds to the same epitope as that to which the antibody according to any one of (13) to (18) binds.
[9] The antibody variable region or antibody according to any one of (1) to (32) below:
(1) a heavy chain variable region (H17) having the amino acid sequence of SEQ ID NO: 204,
(2) a heavy chain variable region (H19) having the amino acid sequence set forth in SEQ ID NO: 205,
(3) a heavy chain variable region (H28) having the amino acid sequence of SEQ ID NO: 206,
(4) heavy chain variable region (H30) having the amino acid sequence set forth in SEQ ID NO: 207,
(5) a heavy chain variable region (H34) having the amino acid sequence set forth in SEQ ID NO: 208,
(6) a heavy chain variable region (H42) having the amino acid sequence of SEQ ID NO: 209,
(7) heavy chain variable region (H44) having the amino acid sequence set forth in SEQ ID NO: 210;
(8) a heavy chain variable region (H46) having the amino acid sequence of SEQ ID NO: 211,
(9) a heavy chain variable region (H57) having the amino acid sequence set forth in SEQ ID NO: 212;
(10) a heavy chain variable region (H71) having the amino acid sequence of SEQ ID NO: 213,
(11) a heavy chain variable region (H78) having the amino acid sequence of SEQ ID NO: 214,
(12) a heavy chain variable region (H92) having the amino acid sequence of SEQ ID NO: 215,
(13) a heavy chain variable region (H97) having the amino acid sequence of SEQ ID NO: 216,
(14) a heavy chain variable region (H98) having the amino acid sequence of SEQ ID NO: 217,
(15) a light chain variable region (L11) having the amino acid sequence of SEQ ID NO: 218,
(16) a light chain variable region (L12) having the amino acid sequence of SEQ ID NO: 219,
(17) a light chain variable region (L17) having the amino acid sequence of SEQ ID NO: 220,
(18) a light chain variable region (L50) having the amino acid sequence of SEQ ID NO: 221;
(19) An antibody (H28L17) comprising the heavy chain variable region of (3) and the light chain variable region of (17),
(20) an antibody (H30L17) comprising the heavy chain variable region of (4) and the light chain variable region of (17),
(21) an antibody (H34L17) comprising the heavy chain variable region of (5) and the light chain variable region of (17),
(22) an antibody (H42L17) comprising the heavy chain variable region of (6) and the light chain variable region of (17),
(23) an antibody (H44L17) comprising the heavy chain variable region of (7) and the light chain variable region of (17),
(24) An antibody (H46L17) comprising the heavy chain variable region of (8) and the light chain variable region of (17),
(25) an antibody (H57L17) comprising the heavy chain variable region of (9) and the light chain variable region of (17),
(26) an antibody (H71L17) comprising the heavy chain variable region of (10) and the light chain variable region of (17),
(27) an antibody (H78L17) comprising the heavy chain variable region of (11) and the light chain variable region of (17),
(28) an antibody (H92L17) comprising the heavy chain variable region of (12) and the light chain variable region of (17),
(29) an antibody (H97L50) comprising the heavy chain variable region of (13) and the light chain variable region of (18),
(30) an antibody (H98L50) comprising the heavy chain variable region of (14) and the light chain variable region of (18),
(31) The antibody according to any one of (19) to (30), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (19) to (30) An antibody having an activity equivalent to that of the antibody described in 1.
(32) An antibody that binds to the same epitope as the epitope to which the antibody according to any one of (19) to (30) binds.
[10] The anti-NR10 antibody according to any one of [4] to [9], which is a humanized antibody.
[11] The antibody heavy chain, antibody light chain, or antibody of any one of (1) to (32) below:
(1) a heavy chain (H17) having the amino acid sequence set forth in SEQ ID NO: 222;
(2) a heavy chain (H19) having the amino acid sequence of SEQ ID NO: 223,
(3) a heavy chain (H28) having the amino acid sequence set forth in SEQ ID NO: 224,
(4) a heavy chain (H30) having the amino acid sequence set forth in SEQ ID NO: 225,
(5) a heavy chain (H34) having the amino acid sequence set forth in SEQ ID NO: 226,
(6) a heavy chain (H42) having the amino acid sequence set forth in SEQ ID NO: 227,
(7) a heavy chain (H44) having the amino acid sequence of SEQ ID NO: 228,
(8) a heavy chain (H46) having the amino acid sequence set forth in SEQ ID NO: 229,
(9) a heavy chain (H57) having the amino acid sequence set forth in SEQ ID NO: 230;
(10) a heavy chain (H71) having the amino acid sequence set forth in SEQ ID NO: 231;
(11) a heavy chain (H78) having the amino acid sequence of SEQ ID NO: 232,
(12) a heavy chain (H92) having the amino acid sequence of SEQ ID NO: 233,
(13) a heavy chain (H97) having the amino acid sequence of SEQ ID NO: 234,
(14) a heavy chain (H98) having the amino acid sequence of SEQ ID NO: 235,
(15) a light chain (L11) having the amino acid sequence of SEQ ID NO: 236,
(16) a light chain (L12) having the amino acid sequence of SEQ ID NO: 237,
(17) a light chain (L17) having the amino acid sequence of SEQ ID NO: 238,
(18) a light chain (L50) having the amino acid sequence of SEQ ID NO: 239,
(19) an antibody (H28L17) comprising the heavy chain of (3) and the light chain of (17),
(20) an antibody (H30L17) comprising the heavy chain of (4) and the light chain of (17),
(21) an antibody (H34L17) comprising the heavy chain of (5) and the light chain of (17),
(22) an antibody (H42L17) comprising the heavy chain of (6) and the light chain of (17),
(23) an antibody (H44L17) comprising the heavy chain of (7) and the light chain of (17),
(24) an antibody (H46L17) comprising the heavy chain of (8) and the light chain of (17),
(25) an antibody (H57L17) comprising the heavy chain of (9) and the light chain of (17),
(26) an antibody (H71L17) comprising a heavy chain of (10) and a light chain of (17),
(27) an antibody (H78L17) comprising the heavy chain of (11) and the light chain of (17),
(28) an antibody (H92L17) comprising a heavy chain of (12) and a light chain of (17),
(29) an antibody (H97L50) comprising the heavy chain of (13) and the light chain of (18),
(30) an antibody (H98L50) comprising the heavy chain of (14) and the light chain of (18),
(31) The antibody according to any one of (19) to (30), wherein one or more amino acids are substituted, deleted, added and / or inserted, and any one of (19) to (30) An antibody having an activity equivalent to that of the antibody described in 1.
(32) An antibody that binds to the same epitope as the epitope to which the antibody according to any one of (19) to (30) binds.
[12] A pharmaceutical composition comprising the antibody according to any one of [1] to [11].
[13] The pharmaceutical composition according to [12], which is a therapeutic agent for inflammatory diseases.
[14] A method for treating or preventing an inflammatory disease, comprising a step of administering the antibody according to any one of [1] to [11].
[15] Use of the antibody according to any one of [1] to [11] in the manufacture of a therapeutic agent for inflammatory diseases.

It is a figure which shows the amino acid sequence of the heavy chain variable region of mouse antibodies NS18, NS22, NS23, NS33. It is a figure which shows the amino acid sequence of the light chain variable region of mouse antibody NS18, NS22, NS23, NS33. It is a graph which shows about inhibition of the hybridoma culture supernatant with respect to hNR10 / hOSMR / BaF3 cell proliferation. It is a graph which shows about inhibition of the hybridoma culture supernatant with respect to cynNR10 / cynOSMR / BaF3 cell proliferation. It is a graph shown about activity evaluation (BaF) of chimera NS22. It is a graph shown about activity evaluation (DU-145) of chimera NS22. It is a graph which shows about IL-31 competitive activity evaluation of chimera NS22. It is a graph which shows about NR10 binding competition activity of an anti-NR10 antibody. It is a graph which shows about IL-31 competitive activity of humanized NS22 (H0L0). It is the figure which evaluated the influence on the heterogeneity which the constant region of humanized anti-NR10 antibody H0L0 exerted by cation exchange chromatography. It is a graph which shows the IL-31 competitive activity evaluation of the variant which can reduce the isoelectric point of a variable region, without significantly reducing the binding of humanized anti-NR10 antibody to NR10. It is the figure which evaluated the influence on the heterogeneity which the constant region of an anti- IL-6 receptor antibody exerts by the cation exchange chromatography. It is the figure which evaluated the influence on the denaturation peak which the constant region of an anti- IL-6 receptor antibody has on DSC. In the anti-IL-6 receptor antibody, it is the figure which evaluated the influence on the heterogeneity which the novel constant region M14 exerts by cation exchange chromatography. In anti-IL-6 receptor antibody, it is the figure which evaluated the influence on the heterogeneity which the novel constant region M58 exerts by cation exchange chromatography. In anti-IL-6 receptor antibody, it is the figure which evaluated the influence on the denaturation peak which the novel constant region M58 exerts on by DSC. It is a figure which shows the evaluation result of the plasma retention in the human FcRn transgenic mouse of huPM1-IgG1 and huPM1-M58. It is a graph which shows the biological activity using BaF / NR10 about each antibody. It is the figure which analyzed the thermal acceleration goods (dotted line) and unaccelerated goods (solid line) of each obtained modified antibody by the cation exchange chromatography, and compared the decomposition product production | generation before and behind thermal acceleration. The arrow indicates the basic component peak position where a change was observed. It is a graph shown about activity evaluation (BaF) of each modification. It is a graph shown about activity evaluation (BaF) of Ha401La402 and H0L0. It is a graph shown about activity evaluation (BaF) of H17L11 and H0L0. It is a graph shown about activity evaluation (BaF) of H19L12 and H0L0. It is a graph which shows the biological activity using BaF / NR10 about H0L12 and H0L17. It is a graph shown about activity evaluation (BaF) of each modification. FIG. 26 is a continuation diagram of FIG. 25-1. FIG. 2 is a schematic diagram of human / mouse wild type and chimeric NR10-ECD. It is the photograph which detected the binding domain using the Western blot. A is a photograph showing the result of detection with a humanized anti-human NR10 antibody, B is a photograph showing the result of detection with a mouse anti-human NR10 antibody, and C is a photograph showing the result of detection with an anti-Myc antibody. For some anti-human NR10 antibodies, binding antigens were detected only at hhh, hhm, and hmm, and binding could not be confirmed for mmm, mmh, and mhm. It is a figure which shows the amino acid sequence of each modification of H0 (sequence number: 50). FIG. 29 is a continuation diagram of FIG. 28-1. FIG. 29 is a continuation diagram of FIG. 28-2. It is a figure which shows the amino acid sequence of each modification of L0 (sequence number: 52). It is a figure of a continuation of FIG.

[Mode for Carrying Out the Invention]
NR10
NR10 is a protein that forms a heterodimer with the oncostatin M receptor (OSMR) and functions as a receptor for IL-31. Also known as names such as glm-r (J Biol Chem 277, 16831-6, 2002), GPL (J Biol Chem 278, 49850-9, 2003), IL31RA (Nat Immunol 5, 752-60, 2004) Thus, the NR10 of the present invention includes proteins called by such names.

  The origin of the NR10 of the present invention (also referred to as IL31RA, GPL or glm-r) is not particularly limited, and includes NR10 derived from humans, mice, monkeys and other mammals, but preferably derived from humans, mice or monkeys NR10, particularly preferably human-derived NR10.

  As human-derived NR10, a plurality of splicing variants are known (WO00 / 075314). Among the splicing variants, NR10.1 consists of 662 amino acids and has a transmembrane region. NR10.2 is a soluble receptor-like protein that does not have a transmembrane region consisting of a 252 amino acid sequence. On the other hand, NR10.3 and IL31RAv3 are known as NR10 splicing variants that function as a transmembrane receptor protein. The human NR10 in the present invention is not particularly limited as long as it forms a heterodimer with the oncostatin M receptor (OSMR) and functions as a receptor for IL-31, but preferred NR10 is NR10. 3 (also called ILRAv4 (Nat Immunol 5, 752-60, 2004)) and IL31RAv3. NR10.3 (IL31RAv4) consists of 662 amino acids (WO00 / 075314, Nat Immunol 5, 752-60, 2004), and IL31RAv3 consists of 732 amino acids (GenBank ACCESSION No: NM_139017). The amino acid sequence of IL31RAv4 is shown in SEQ ID NO: 79, and the amino acid sequence of IL31RAv3 is shown in SEQ ID NO: 80. On the other hand, examples of mouse-derived NR10 include a protein consisting of the amino acid sequence set forth in SEQ ID NO: 81. Moreover, as NR10 derived from cynomolgus monkey, for example, a protein having the amino acid sequence set forth in SEQ ID NO: 66 can be mentioned.

Antibody (sequence)
As a preferred embodiment of the anti-NR10 antibody of the present invention, the anti-NR10 antibody described in any of (1) to (8) in the following (A) to (D) can be mentioned.

(A) NS18
(1) CDR1 having the amino acid sequence described in SEQ ID NO: 1 (HCDR1), CDR2 having the amino acid sequence described in SEQ ID NO: 2 (HCDR2), CDR3 having the amino acid sequence described in SEQ ID NO: 3 (HCDR3) Having a heavy chain variable region comprising
(2) An antibody having the heavy chain variable region described in SEQ ID NO: 4 (VH)
(3) CDR1 having the amino acid sequence described in SEQ ID NO: 5 (LCDR1), CDR2 having the amino acid sequence described in SEQ ID NO: 6 (LCDR2), CDR3 having the amino acid sequence described in SEQ ID NO: 7 (LCDR3) Having a light chain variable region comprising
(4) An antibody having the light chain variable region set forth in SEQ ID NO: 8 (VL)
(5) An antibody having the heavy chain variable region of (1) and the light chain variable region of (3)
(6) An antibody having the heavy chain variable region of (2) and the light chain variable region of (4)
(7) The antibody according to any one of (1) to (6), wherein one or a plurality of amino acids are substituted, deleted, added and / or inserted, and any one of (1) to (6) An antibody having the same activity as the antibody described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds

(B) NS22
(1) CDR1 having the amino acid sequence of SEQ ID NO: 9 (HCDR1), CDR2 having the amino acid sequence of SEQ ID NO: 10 (HCDR2), CDR3 having the amino acid sequence of SEQ ID NO: 11 (HCDR3) Having a heavy chain variable region comprising
(2) An antibody having the heavy chain variable region described in SEQ ID NO: 12 (VH)
(3) CDR1 having the amino acid sequence described in SEQ ID NO: 13 (LCDR1), CDR2 having the amino acid sequence described in SEQ ID NO: 14 (LCDR2), CDR3 having the amino acid sequence described in SEQ ID NO: 15 (LCDR3) Having a light chain variable region comprising
(4) Antibody having a light chain variable region set forth in SEQ ID NO: 16 (VL)
(5) An antibody having the heavy chain variable region of (1) and the light chain variable region of (3)
(6) An antibody having the heavy chain variable region of (2) and the light chain variable region of (4)
(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 the same activity as the antibody described in 1.
(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 the third Ile to another amino acid in the heavy chain CDR1 of SEQ ID NO: 9. Although the amino acid after substitution is not particularly limited, Val can be mentioned as a preferred example.

  Substitution of the fourth Met to another amino acid in the heavy chain CDR1 of SEQ ID NO: 9. The amino acid after substitution is not particularly limited, but preferred examples include Ile.

  Substitution of the fourth Met to another amino acid in the heavy chain CDR1 of SEQ ID NO: 9. The amino acid after substitution is not particularly limited, but preferred examples include Leu.

  Substitution of the third Ile to another amino acid in the heavy chain CDR1 of SEQ ID NO: 9. The amino acid after substitution is not particularly limited, but preferred examples include Ala.

  Substitution of the first Leu to another amino acid in the heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but preferred examples include Glu.

  Substitution of the third Asn to another amino acid in the heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of the 13th Gln to another amino acid in the heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of 14th Lys to another amino acid in heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but preferred examples include Gln.

  Substitution of 16th Lys to another amino acid in heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but preferred examples include Gln.

  Substitution of Gly at position 17 in the heavy chain CDR2 of SEQ ID NO: 10 with another amino acid. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of 16th Lys and 17th Gly to other amino acids in heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but preferred examples include substitution of 16th Lys with Gln and 17th Gly with Asp.

  Replacement of 14th Lys, 16th Lys and 17th Gly with other amino acids in heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but preferred examples include substitution of Gln at the 14th Lys, Gln at the 16th Lys, and Asp at the 17th Gly.

  Replacement of 13th Gln, 14th Lys, 16th Lys and 17th Gly with other amino acids in heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, and preferred examples include substitution of 13th Gln Asp, 14th Lys Gln, 16th Lys Gln, and 17th Gly Asp.

  Substitution of the 10th Ser to another amino acid in the heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of the 13th Gln to another amino acid in the heavy chain CDR2 of SEQ ID NO: 10. The amino acid after substitution is not particularly limited, but Pro can be mentioned as a preferred example.

  Substitution of the third Tyr to another amino acid in the heavy chain CDR3 of SEQ ID NO: 11. The amino acid after substitution is not particularly limited, but preferred examples include Leu.

  Substitution of the 10th Met to another amino acid in the heavy chain CDR3 of SEQ ID NO: 11. The amino acid after substitution is not particularly limited, but preferred examples include Leu.

  Substitution of 11th Asp to another amino acid in heavy chain CDR3 of SEQ ID NO: 11. The amino acid after substitution is not particularly limited, but preferred examples include Glu.

  Substitution of the 12th Tyr to another amino acid in the heavy chain CDR3 of SEQ ID NO: 11. The amino acid after substitution is not particularly limited, but preferred examples include Thr and Ser.

  In the heavy chain CDR3 of SEQ ID NO: 11, substitution of the 10th Met, 11th Asp and 12th Tyr with another amino acid. The amino acid after substitution is not particularly limited, but preferred examples include substitution of Leu at the 10th Met, Glu at the 11th Asp, and Thr at the 12th Tyr.

  Substitution of 11th Asp and 12th Tyr to other amino acids in heavy chain CDR3 of SEQ ID NO: 11. The amino acid after substitution is not particularly limited, but preferred examples include substitution of 11th Asp with Glu and 12th Tyr with Thr.

  In the heavy chain CDR3 of SEQ ID NO: 11, substitution of the 3rd Tyr, 11th Asp and 12th Tyr with another amino acid. The amino acid after substitution is not particularly limited, and preferred examples include substitution of Leu at the 3rd Tyr, Glu at the 11th Asp, and Thr or Ser at the 12th Tyr.

  Substitution of the first Arg to another amino acid in the light chain CDR1 of SEQ ID NO: 13. The amino acid after substitution is not particularly limited, but preferred examples include Gln.

  Substitution of 5th Asn to another amino acid in light chain CDR1 of SEQ ID NO: 13. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of the first Arg and the fifth Asn to other amino acids in the light chain CDR1 of SEQ ID NO: 13. The amino acid after the substitution is not particularly limited, but preferred examples include substitution of the first Arg to Gln and the fifth Asn to Asp.

  Substitution of the 8th Ser to another amino acid in the light chain CDR1 of SEQ ID NO: 13. The amino acid after substitution is not particularly limited, but preferred examples include Arg.

  Substitution of the 10th Leu to another amino acid in the light chain CDR1 of SEQ ID NO: 13. Although the amino acid after substitution is not particularly limited, Val can be mentioned as a preferred example.

  Substitution of the 8th Ser and 10th Leu to other amino acids in the light chain CDR1 of SEQ ID NO: 13. The amino acid after substitution is not particularly limited, and preferred examples include substitution of Arg at the 8th Ser and Val at the 10th Leu.

  Replacement of the second Thr with another amino acid in the light chain CDR1 of SEQ ID NO: 13. The amino acid after substitution is not particularly limited, but preferred examples include Ala or Ser.

  Replacement of the first Asn with another amino acid in the light chain CDR2 of SEQ ID NO: 14. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Replacement of the third Lys with another amino acid in the light chain CDR2 of SEQ ID NO: 14. The amino acid after substitution is not particularly limited, but preferred examples include Gln.

Substitution of the 5th Leu to another amino acid in the light chain CDR2 of SEQ ID NO: 14. The amino acid after substitution is not particularly limited, but preferred examples include Glu.

  Replacement of 7th Lys with another amino acid in light chain CDR2 of SEQ ID NO: 14. The amino acid after substitution is not particularly limited, but preferred examples include Gln or Asp.

  Substitution of 3rd Lys, 5th Leu and 7th Lys to other amino acids in light chain CDR2 of SEQ ID NO: 14. The amino acid after the substitution is not particularly limited, but preferred examples include substitution of the third Lys to Gln, the fifth Leu to Glu, and the seventh Lys to Gln.

  Substitution of the 5th Glu to another amino acid in the light chain CDR3 of SEQ ID NO: 15. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of the sixth Ser in the light chain CDR3 of SEQ ID NO: 15 with another amino acid. The amino acid after substitution is not particularly limited, but a preferred example is Asp.

  Substitution of 9th Thr to another amino acid in light chain CDR3 of SEQ ID NO: 15. The amino acid after substitution is not particularly limited, but preferred examples include Phe.

  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-described substitutions in the present invention include a heavy chain variable region having the amino acid sequence of SEQ ID NO: 167 or a light chain variable region having the amino acid sequence of SEQ ID NO: 168. Furthermore, as an example of an antibody combining the above-mentioned substitution, an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 167 and a light chain variable region having the amino acid sequence of SEQ ID NO: 168 can be mentioned.

Specific examples of the heavy chain variable region or light chain variable region in which the above substitutions are combined include the following variable regions.
(a) A heavy chain variable region comprising CDR1 of SEQ ID NO: 196, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 11. (H17)
(b) heavy chain variable region (H19) comprising CDR1 of SEQ ID NO: 176, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 11
(c) A heavy chain variable region comprising CDR1 of SEQ ID NO: 196, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184. (H28, H42)
(d) A heavy chain variable region comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184. (H30, H44)
(e) A heavy chain variable region comprising CDR1 described in SEQ ID NO: 176, CDR2 described in SEQ ID NO: 197, and CDR3 described in SEQ ID NO: 184. (H34, H46)
(f) A heavy chain variable region comprising CDR1 set forth in SEQ ID NO: 9, CDR2 set forth in SEQ ID NO: 198, CDR3 set forth in SEQ ID NO: 184. (H57, H78)
(g) A heavy chain variable region comprising CDR1 described in SEQ ID NO: 176, CDR2 described in SEQ ID NO: 198, and CDR3 described in SEQ ID NO: 184. (H71, H92)
(h) A heavy chain variable region comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 199, CDR3 of SEQ ID NO: 184. (H97, H98)
(i) A light chain variable region comprising CDR1 described in SEQ ID NO: 200, CDR2 described in SEQ ID NO: 170, CDR3 described in SEQ ID NO: 193. (L11)
(j) A light chain variable region comprising CDR1 of SEQ ID NO: 201, CDR2 of SEQ ID NO: 170, CDR3 of SEQ ID NO: 193. (L12)
(k) A light chain variable region comprising CDR1 of SEQ ID NO: 202, CDR2 of SEQ ID NO: 170, CDR3 of SEQ ID NO: 193. (L17)
(l) A light chain variable region comprising CDR1 described in SEQ ID NO: 203, CDR2 described in SEQ ID NO: 170, CDR3 described in SEQ ID NO: 193. (L50)

Furthermore, the following antibodies can be mentioned as specific examples of antibodies in which the above substitutions are combined.
(i) an antibody comprising the heavy chain variable region of (c) and the light chain variable region of (k)
(ii) an antibody comprising the heavy chain variable region of (d) and the light chain variable region of (k)
(iii) an antibody comprising the heavy chain variable region of (e) and the light chain variable region of (k)
(iv) an antibody comprising the heavy chain variable region of (f) and the light chain variable region of (k)
(v) an antibody comprising the heavy chain variable region of (g) and the light chain variable region of (k)
(vi) an antibody comprising the heavy chain variable region of (h) and the light chain variable region of (l)

(C) NS23
(1) CDR1 having the amino acid sequence of SEQ ID NO: 17 (HCDR1), CDR2 having the amino acid sequence of SEQ ID NO: 18 (HCDR2), CDR3 having the amino acid sequence of SEQ ID NO: 19 (HCDR3) Having a heavy chain variable region comprising
(2) An antibody having the heavy chain variable region described in SEQ ID NO: 20 (VH)
(3) CDR1 having the amino acid sequence described in SEQ ID NO: 21 (LCDR1), CDR2 having the amino acid sequence described in SEQ ID NO: 22 (LCDR2), CDR3 having the amino acid sequence described in SEQ ID NO: 23 (LCDR3) Having a light chain variable region comprising
(4) Antibody having the light chain variable region set forth in SEQ ID NO: 24 (VL)
(5) An antibody having the heavy chain variable region of (1) and the light chain variable region of (3)
(6) An antibody having the heavy chain variable region of (2) and the light chain variable region of (4)
(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 the same activity as the antibody described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds

(D) NS33
(1) CDR1 having the amino acid sequence described in SEQ ID NO: 25 (HCDR1), CDR2 having the amino acid sequence described in SEQ ID NO: 26 (HCDR2), CDR3 having the amino acid sequence described in SEQ ID NO: 27 (HCDR3) Having a heavy chain variable region comprising
(2) An antibody having the heavy chain variable region described in SEQ ID NO: 28 (VH)
(3) CDR1 having the amino acid sequence described in SEQ ID NO: 29 (LCDR1), CDR2 having the amino acid sequence described in SEQ ID NO: 30 (LCDR2), CDR3 having the amino acid sequence described in SEQ ID NO: 31 (LCDR3) Having a light chain variable region comprising
(4) Antibody having the light chain variable region set forth in SEQ ID NO: 32 (VL)
(5) An antibody having the heavy chain variable region of (1) and the light chain variable region of (3)
(6) An antibody having the heavy chain variable region of (2) and the light chain variable region of (4)
(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 the same activity as the antibody described in 1.
(8) An antibody that binds to the same epitope as that to which the antibody according to any one of (1) to (7) binds

  In the antibody described in (1) or (3) above, any framework region (FR) may be used, but human-derived FR is preferably used. In the antibodies described in (1) to (8) above, 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.

  The above-mentioned NS18 heavy chain amino acid sequence is shown in SEQ ID NO: 34, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 33. The amino acid sequence of the light chain is shown in SEQ ID NO: 36, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 35.

  The amino acid sequence of the heavy chain of NS22 is shown in SEQ ID NO: 38, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 37. The amino acid sequence of the light chain is shown in SEQ ID NO: 40, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 39.

  The heavy chain amino acid sequence of NS23 is shown in SEQ ID NO: 42, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 41. The amino acid sequence of the light chain is shown in SEQ ID NO: 44, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 43.

  The heavy chain amino acid sequence of NS33 is shown in SEQ ID NO: 46, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 45. The amino acid sequence of the light chain is shown in SEQ ID NO: 48, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 47.

  In the present invention, the “activity equivalent to the antibody described in any one of (1) to (6)” means that the binding activity and / or neutralizing activity to NR10 (eg, human NR10) is equivalent. To do. 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 antibody according to any one of the above (1) to (6) has one or more amino acid sequences in the variable region (CDR sequence and / or FR sequence) as long as it has binding activity and / or neutralizing activity against NR10. Of amino acids may be substituted, deleted, added and / or inserted. One skilled in the art for preparing 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 and / or neutralizing activity against NR10. As a known method, a method of 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 appropriately introduce mutations in the amino acid sequence of an antibody having binding activity and / or neutralizing activity against NR10. Accordingly, it is possible to prepare antibodies functionally equivalent variants with binding activity and / or neutralizing activity against NR10. Thus, an antibody having one or more amino acids mutated in the variable region and having binding activity and / or neutralizing activity for NR10 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. Defined as the percentage of residues that are identical to the residues. The identity of amino acid sequences can be determined by the method described below.

  In addition, 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 have binding activity and / or neutralizing activity against NR10. The amino acid sequence of the variable region can also 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 nucleic acids that hybridize under stringent conditions to nucleic acids consisting of the base sequence encoding the variable region amino acid sequence include 6M urea, 0.4% SDS, 0.5 x SSC. , 37 ° C., or hybridization conditions with stringency equivalent thereto. Isolation of nucleic acids with higher homology can be expected by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC, and 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 publicly known (refer to the BLAST (Basic Local Alignment Search Tool) website of NCBI (National Center for Biotechnology Information); 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 according to any one of (1) to (7) 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 a competitive binding assay. Examples of the means include ELISA, fluorescence energy transfer measurement (FRET), and fluorescence micromeasurement technique (FMAT (registered trademark)). 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 animal cells expressing NR10, 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.

  The antibody that binds to the epitope to which the antibody according to any one of the above (1) to (7) binds is useful in that it has a particularly high neutralizing activity.

  The antibody described in any of (1) to (8) above is not particularly limited, but is preferably a humanized antibody.

  Furthermore, the present invention provides a gene encoding the anti-NR10 antibody according to any one of (1) to (8) in the above (A) to (D). 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 NR10. 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 a portion overlapping with the terminal region (see the method described in WO98 / 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. etal., 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. The amino acid sequence of Cκ is shown in SEQ ID NO: 58, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 57. The amino acid sequence of Cγ1 is shown in SEQ ID NO: 60, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 59. The amino acid sequence of Cγ2 is shown in SEQ ID NO: 62, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 61. The amino acid sequence of Cγ4 is shown in SEQ ID NO: 64, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 63. 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.

  The origin of CDR in the humanized antibody is not particularly limited and may be derived from any animal. For example, sequences of mouse antibody, rat antibody, rabbit antibody, camel antibody and the like can be used, but the CDR sequence of mouse antibody is preferable.

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

In the present invention, preferred examples of the humanized anti-NR10 antibody include the antibodies described in any of the following (a) to (e).
(a) a humanized antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 (H0-VH)
(b) a humanized antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 112 (H1-VH)
(c) a humanized antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 52 (L0-VL)
(d) a humanized antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 (H0-VH) and a light chain variable region having the amino acid sequence of SEQ ID NO: 52 (L0-VL)
(e) a human antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 112 and a light chain variable region having the amino acid sequence of SEQ ID NO: 52

  In addition, the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 50 (H0-VH), the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 112, and the sequence described in SEQ ID NO: 52 (L0-VL) In the light chain variable region having the amino acid sequence, one or more amino acids may be substituted, deleted, added and / or inserted. Amino acid substitutions, deletions, additions and / or insertions may be made in the CDR or FR, or in both the CDR and FR.

Accordingly, other preferred embodiments of the humanized anti-NR10 antibody in the present invention include, for example, the antibodies described in any of (f) to (j) below.
(f) an antibody comprising a heavy chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 50 (H0-VH)
(g) an antibody comprising a heavy chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 112 (H1-VH)
(h) an antibody comprising a light chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 52 (L0-VL)
(i) a heavy chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 50 (H0-VH); and SEQ ID NO: 52 (L0 -VL) antibody comprising a light chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence
(j) a heavy chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 112 (H1-VH); and SEQ ID NO: 52 (L0 -VL) antibody comprising a light chain variable region having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence

  Although not particularly limited, the antibody described in any of (f) to (j) preferably has the same activity as the antibody described in any of (a) to (e).

The amino acid substitution, deletion, addition and / or insertion is not particularly limited, but as a specific example, for example, the amino acid substitution described above can be performed.
More specifically, for example, the following amino acid substitutions can be mentioned.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR1 (SEQ ID NO: 9) with the third Ile to Val (SEQ ID NO: 173). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR1 having the amino acid sequence of SEQ ID NO: 9 is substituted with CDR1 having the amino acid sequence of SEQ ID NO: 173. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR1 (SEQ ID NO: 9) with the 4th Met to Ile (SEQ ID NO: 174). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR1 having the amino acid sequence of SEQ ID NO: 9 is substituted with CDR1 having the amino acid sequence of SEQ ID NO: 174. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, replacement of CDR1 (SEQ ID NO: 9) with the fourth Met to Leu (SEQ ID NO: 175). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR1 having the amino acid sequence of SEQ ID NO: 9 is substituted with CDR1 having the amino acid sequence of SEQ ID NO: 175. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR1 (SEQ ID NO: 9) with the third Ile to Ala (SEQ ID NO: 176). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR1 having the amino acid sequence of SEQ ID NO: 9 is substituted with CDR1 having the amino acid sequence of SEQ ID NO: 176. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with the first Leu to Glu (SEQ ID NO: 113). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 113. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with Asp at the third Asn (SEQ ID NO: 114). Accordingly, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 114. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with the 13th Gln to Asp (SEQ ID NO: 115). Accordingly, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 115. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with the 14th Lys to Gln (SEQ ID NO: 116). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 116. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) at the 16th Lys with Gln (SEQ ID NO: 117). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 117. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with 17th Gly to Asp (SEQ ID NO: 118). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 118. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, replacement of CDR2 (SEQ ID NO: 10) with 16th Lys in Gln and replacement of 17th Gly with Asp (SEQ ID NO: 119). Accordingly, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 119. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with 14th Lys to Gln, substitution of 16th Lys with Gln, and 17th Gly Asp (SEQ ID NO: 167). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 171. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR2 (SEQ ID NO: 10) with 13th Gln to Asp, substitution of 14th Lys with Gln, 16th Lys with Gln And 17th Gly to Asp (SEQ ID NO: 172). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 172. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 (SEQ ID NO: 10) is replaced with Asp at the 10th Ser (SEQ ID NO: 177). Accordingly, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 177. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, replacement of CDR2 (SEQ ID NO: 10) with 13th Gln to Pro (SEQ ID NO: 178). Accordingly, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR2 having the amino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acid sequence of SEQ ID NO: 178. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, replacement of CDR3 (SEQ ID NO: 11) with the third Tyr to Leu (SEQ ID NO: 179). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is replaced with CDR3 having the amino acid sequence of SEQ ID NO: 179. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR3 (SEQ ID NO: 11) at the 10th Met with Leu (SEQ ID NO: 180). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is replaced with CDR3 having the amino acid sequence of SEQ ID NO: 180. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR3 (SEQ ID NO: 11) with the 11th Asp to Glu (SEQ ID NO: 181). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is substituted with CDR3 having the amino acid sequence of SEQ ID NO: 181. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR3 (SEQ ID NO: 11) with 12th Tyr to Thr (SEQ ID NO: 182). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is replaced with CDR3 having the amino acid sequence of SEQ ID NO: 182. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR3 (SEQ ID NO: 11) with 12th Tyr to Ser (SEQ ID NO: 183). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is replaced with CDR3 having the amino acid sequence of SEQ ID NO: 183. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 (SEQ ID NO: 11) is substituted with 10th Met to Leu, 11th Asp to Glu, and 12th Tyr Thr. (SEQ ID NO: 184). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is substituted with CDR3 having the amino acid sequence of SEQ ID NO: 184. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, replacement of CDR3 (SEQ ID NO: 11) with 11th Asp to Glu and replacement of 12th Tyr with Thr (SEQ ID NO: 185). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is replaced with CDR3 having the amino acid sequence of SEQ ID NO: 185. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR3 (SEQ ID NO: 11) with 3rd Tyr to Leu, substitution of 11th Asp with Glu, and Thr of 12th Tyr (SEQ ID NO: 186). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is replaced with CDR3 having the amino acid sequence of SEQ ID NO: 186. Provides a heavy chain variable region.

  In the heavy chain variable region of SEQ ID NO: 50 or SEQ ID NO: 112, substitution of CDR3 (SEQ ID NO: 11) with the third Tyr to Leu, substitution of the 11th Asp with Glu, and Ser with the 12th Tyr (SEQ ID NO: 187). Therefore, in the present invention, in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112, CDR3 having the amino acid sequence of SEQ ID NO: 11 is substituted with CDR3 having the amino acid sequence of SEQ ID NO: 187. Provides a heavy chain variable region.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR1 (SEQ ID NO: 13) with the first Arg to Gln (SEQ ID NO: 121). Therefore, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 121. provide.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR1 (SEQ ID NO: 13) with Asp at the 5th Asn (SEQ ID NO: 122). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 122. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR1 (SEQ ID NO: 13) at the 8th Ser with Arg (SEQ ID NO: 188). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 188. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR1 (SEQ ID NO: 13) at the 10th Leu with Val (SEQ ID NO: 189). Therefore, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 189. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR1 (SEQ ID NO: 13) at the 8th Ser to Arg and substitution of the 10th Leu to Val (SEQ ID NO: 190). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 190. provide.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR1 (SEQ ID NO: 13) with the second Thr of Ala (SEQ ID NO: 191). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted with CDR1 having the amino acid sequence of SEQ ID NO: 191. provide.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR1 (SEQ ID NO: 13) with the second Thr to Ser (SEQ ID NO: 192). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 192. provide.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR2 (SEQ ID NO: 14) with Asp at the first Asn (SEQ ID NO: 123). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR2 having the amino acid sequence of SEQ ID NO: 14 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 123. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR2 (SEQ ID NO: 14) with the third Lys to Gln (SEQ ID NO: 124). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR2 having the amino acid sequence of SEQ ID NO: 14 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 124. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR2 (SEQ ID NO: 14) at the fifth Leu with Glu (SEQ ID NO: 125). Therefore, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR2 having the amino acid sequence of SEQ ID NO: 14 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 125. provide.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR2 (SEQ ID NO: 14) with the seventh Lys to Gln (SEQ ID NO: 126). Accordingly, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR2 having the amino acid sequence of SEQ ID NO: 14 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 126. provide.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR2 (SEQ ID NO: 14) with 7th Lys to Asp (SEQ ID NO: 127). Therefore, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR2 having the amino acid sequence of SEQ ID NO: 14 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 127. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR1 (SEQ ID NO: 13) with the first Arg to Gln and substitution of the fifth Asn with Asp (SEQ ID NO: 169). Therefore, the present invention relates to a light chain variable region having the amino acid sequence of SEQ ID NO: 52, a light chain variable region in which CDR1 having the amino acid sequence of SEQ ID NO: 13 is replaced with CDR1 having the amino acid sequence of SEQ ID NO: 169. provide.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR2 (SEQ ID NO: 14) with third Lys to Gln, substitution of fifth Leu with Glu, and substitution of seventh Lys with Gln (sequence) Number: 170). Accordingly, the present invention provides a light chain variable region in which the CDR2 having the amino acid sequence of SEQ ID NO: 14 is replaced with the CDR2 having the amino acid sequence of SEQ ID NO: 170 in the light chain variable region of SEQ ID NO: 52.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR3 (SEQ ID NO: 15) at the fifth Glu with Asp (SEQ ID NO: 193). Accordingly, the present invention provides a light chain variable region in which the CDR3 having the amino acid sequence of SEQ ID NO: 15 is replaced with the CDR3 having the amino acid sequence of SEQ ID NO: 193 in the light chain variable region of SEQ ID NO: 52.

  In the light chain variable region of SEQ ID NO: 52, replacement of CDR3 (SEQ ID NO: 15) with the 6th Ser of Asp (SEQ ID NO: 194). Accordingly, the present invention provides a light chain variable region in which the CDR3 having the amino acid sequence of SEQ ID NO: 15 is replaced with the CDR3 having the amino acid sequence of SEQ ID NO: 194 in the light chain variable region of SEQ ID NO: 52.

  In the light chain variable region of SEQ ID NO: 52, substitution of CDR3 (SEQ ID NO: 15) at the 9th Thr with Phe (SEQ ID NO: 195). Accordingly, the present invention provides a light chain variable region in which the CDR3 having the amino acid sequence of SEQ ID NO: 15 is replaced with the CDR3 having the amino acid sequence of SEQ ID NO: 195 in the light chain variable region of SEQ ID NO: 52.

  Furthermore, examples of substitution other than the above-mentioned amino acid substitution include substitution of the third Arg with another amino acid in the heavy chain FR2 having the amino acid sequence of SEQ ID NO: 97. The amino acid after substitution is not particularly limited, but preferred examples include Gln. Furthermore, when the 3rd Arg of SEQ ID NO: 97 is substituted with Gln, the 5th Ala can be substituted with Ser to make FR2 a human sequence. In the amino acid sequence of SEQ ID NO: 97, the amino acid sequence in which the third Arg is substituted with Gln and the fifth Ala with Ser is shown in SEQ ID NO: 120. Therefore, in the present invention, FR2 having the amino acid sequence of SEQ ID NO: 97 is substituted with FR2 having the amino acid sequence of SEQ ID NO: 120 in the heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 112. Provides a heavy chain variable region.

  The above amino acid substitutions may be used alone or in combination with other amino acid substitutions described above. Moreover, you may combine with amino acid substitution other than the above-mentioned.

  Specific examples of the antibody in which the above substitution has been made include an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 167, an antibody comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 168, sequence And an antibody comprising a heavy chain variable region having the amino acid sequence of No. 167 and a light chain variable region having the amino acid sequence of SEQ ID No. 168.

In addition, specific examples of the heavy chain variable region subjected to the above-described substitution include the following heavy chain variable regions.
(1) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 204 (H17)
(2) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 205 (H19)
(3) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 206 (H28)
(4) Heavy chain variable region (H30) having the amino acid sequence set forth in SEQ ID NO: 207
(5) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 208 (H34)
(6) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 209 (H42)
(7) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 210 (H44)
(8) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 211 (H46)
(9) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 212 (H57)
(10) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 213 (H71)
(11) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 214 (H78)
(12) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 215 (H92)
(13) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 216 (H97)
(14) Heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 217 (H98)

Moreover, the following light chain variable regions can be mentioned as specific examples of the light chain variable regions subjected to the above substitution.
(15) Light chain variable region (L11) having the amino acid sequence of SEQ ID NO: 218
(16) Light chain variable region (L12) having the amino acid sequence set forth in SEQ ID NO: 219
(17) Light chain variable region having the amino acid sequence set forth in SEQ ID NO: 220 (L17)
(18) Light chain variable region (L50) having the amino acid sequence of SEQ ID NO: 221

Furthermore, the following antibodies can be mentioned as specific examples of antibodies comprising the above-mentioned heavy chain variable region and light chain variable region.
(19) An antibody (H28L17) comprising the heavy chain variable region of (3) and the light chain variable region of (17)
(20) An antibody comprising the heavy chain variable region of (4) and the light chain variable region of (17) (H30L17)
(21) An antibody (H34L17) comprising the heavy chain variable region of (5) and the light chain variable region of (17)
(22) An antibody comprising the heavy chain variable region of (6) and the light chain variable region of (17) (H42L17)
(23) An antibody comprising the heavy chain variable region of (7) and the light chain variable region of (17) (H44L17)
(24) An antibody comprising the heavy chain variable region of (8) and the light chain variable region of (17) (H46L17)
(25) An antibody (H57L17) comprising the heavy chain variable region of (9) and the light chain variable region of (17)
(26) An antibody comprising the heavy chain variable region of (10) and the light chain variable region of (17) (H71L17)
(27) An antibody comprising the heavy chain variable region of (11) and the light chain variable region of (17) (H78L17)
(28) An antibody (H92L17) comprising the heavy chain variable region of (12) and the light chain variable region of (17)
(29) An antibody comprising the heavy chain variable region of (13) and the light chain variable region of (18) (H97L50)
(30) An antibody (H98L50) comprising the heavy chain variable region of (14) and the light chain variable region of (18)

  The constant region used in the humanized antibody of the present invention may be any constant region derived from a human antibody. Preferable examples of constant regions derived from human antibodies include constant regions derived from IgG1 or IgG2. In addition, a constant region in which one or more amino acids are substituted, deleted, added and / or inserted in a constant region derived from a human antibody may be used.

The constant region in which one or more amino acids are substituted, deleted, added and / or inserted in the constant region derived from a human antibody is not particularly limited, and examples thereof include the following constant regions.
Constant region (M58) having the amino acid sequence set forth in SEQ ID NO: 128
Constant region (M14) having the amino acid sequence set forth in SEQ ID NO: 129
Constant region (SKSC) having the amino acid sequence set forth in SEQ ID NO: 62

Specific examples of the heavy chain or antibody using the above constant region include the following heavy chains or antibodies.
(1) A heavy chain comprising a variable region having the amino acid sequence of SEQ ID NO: 167 and a constant region having the amino acid sequence of SEQ ID NO: 128
(2) In the heavy chain of (1), CDR2 having the amino acid sequence of SEQ ID NO: 171 is replaced with CDR2 having the amino acid sequence of SEQ ID NO: 172
(3) An antibody comprising a heavy chain of (1) and a light chain having the amino acid sequence of SEQ ID NO: 152
(4) An antibody comprising a heavy chain of (2) and a light chain having the amino acid sequence of SEQ ID NO: 152

As a more specific example of the humanized anti-NR10 antibody of the present invention, for example, the antibody described in any of (k) to (o) below can be mentioned.
(k) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 54 (H0-VH + constant region)
(l) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 130 (H1-VH + constant region)
(m) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 56 (L0-VL + constant region)
(n) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 54 (H0-VH + constant region) and a light chain having the amino acid sequence of SEQ ID NO: 56 (L0-VL + constant region)
(o) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 130 (H1-VH + constant region) and a light chain having the amino acid sequence of SEQ ID NO: 56 (L0-VL + constant region)

  The heavy chain having the amino acid sequence set forth in SEQ ID NO: 54 (H0-VH + constant region) and the light chain having the amino acid sequence set forth in SEQ ID NO: 56 (L0-VL + constant region) are one or more amino acids. May be substituted, deleted, added and / or inserted. Amino acid substitutions, deletions, additions and / or insertions may be made in the variable region or constant region, or in both the variable region and constant region.

Therefore, the present invention provides the antibody described in any of (p) to (t) below.
(p) an antibody comprising a heavy chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 54 (H0-VH + constant region)
(q) an antibody comprising a heavy chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 130 (H1-VH + constant region)
(r) an antibody comprising a light chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 56 (L0-VL + constant region)
(s) a heavy chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 54 (H0-VH + constant region); and SEQ ID NO: 56 (L0 -VL + constant region) antibody comprising a light chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted
(t) a heavy chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 130 (H1-VH + constant region); and SEQ ID NO: 56 (L0 -VL + constant region) antibody comprising a light chain having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted

  Although not particularly limited, the antibody described in any of (p) to (t) preferably has the same activity as the antibody described in any of (k) to (o).

  The amino acid substitution, deletion, addition and / or insertion is not particularly limited, but as a specific example, for example, the amino acid substitution described above can be performed.

  The base sequence encoding the amino acid sequence (SEQ ID NO: 50) of the above-mentioned humanized heavy chain variable region is shown in SEQ ID NO: 49, and the base sequence encoding the amino acid sequence of the humanized light chain variable region (SEQ ID NO: 52). Is SEQ ID NO: 51, the base sequence encoding the humanized heavy chain amino acid sequence (SEQ ID NO: 54) is SEQ ID NO: 53, and the humanized light chain amino acid sequence (SEQ ID NO: 56) is the base sequence. Is shown in SEQ ID NO: 55.

  Furthermore, the present invention provides an antibody that recognizes the same epitope as the epitope recognized by the antibody described in any of (a) to (t) above. The binding to the same epitope has already been described.

Furthermore, the present invention provides the antibody described in any of (u) to (w) below.
(u) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 151
(v) an antibody comprising a light chain having the amino acid sequence of SEQ ID NO: 152
(w) an antibody comprising the heavy chain of (u) and the light chain of (v)

Furthermore, the present invention provides a heavy chain, light chain, or antibody described in any of the following.
(1) Heavy chain (H17) having the amino acid sequence set forth in SEQ ID NO: 222
(2) Heavy chain (H19) having the amino acid sequence set forth in SEQ ID NO: 223
(3) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 224 (H28)
(4) Heavy chain (H30) having the amino acid sequence set forth in SEQ ID NO: 225
(5) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 226 (H34)
(6) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 227 (H42)
(7) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 228 (H44)
(8) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 229 (H46)
(9) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 230 (H57)
(10) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 231 (H71)
(11) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 232 (H78)
(12) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 233 (H92)
(13) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 234 (H97)
(14) Heavy chain having the amino acid sequence set forth in SEQ ID NO: 235 (H98)
(15) A light chain having the amino acid sequence set forth in SEQ ID NO: 236 (L11)
(16) Light chain having the amino acid sequence set forth in SEQ ID NO: 237 (L12)
(17) A light chain having the amino acid sequence of SEQ ID NO: 238 (L17)
(18) A light chain (L50) having the amino acid sequence of SEQ ID NO: 239
(19) An antibody comprising the heavy chain of (3) and the light chain of (17) (H28L17)
(20) An antibody comprising the heavy chain of (4) and the light chain of (17) (H30L17)
(21) An antibody comprising the heavy chain of (5) and the light chain of (17) (H34L17)
(22) An antibody comprising the heavy chain of (6) and the light chain of (17) (H42L17)
(23) An antibody comprising the heavy chain of (7) and the light chain of (17) (H44L17)
(24) An antibody comprising the heavy chain of (8) and the light chain of (17) (H46L17)
(25) An antibody comprising a heavy chain of (9) and a light chain of (17) (H57L17)
(26) An antibody comprising a heavy chain of (10) and a light chain of (17) (H71L17)
(27) An antibody comprising a heavy chain of (11) and a light chain of (17) (H78L17)
(28) An antibody comprising a heavy chain of (12) and a light chain of (17) (H92L17)
(29) An antibody comprising a heavy chain of (13) and a light chain of (18) (H97L50)
(30) Antibody comprising heavy chain (14) and light chain (18) (H98L50)
(31) The heavy chain according to any one of (1) to (14), wherein the heavy chain has an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted
(32) The light chain according to any one of (15) to (18), wherein one or more amino acids have an amino acid sequence substituted, deleted, added and / or inserted
(33) The antibody according to any one of (19) to (30), wherein one or more amino acids have an amino acid sequence substituted, deleted, added and / or inserted
(34) An antibody that recognizes the same epitope as the epitope recognized by the antibody according to any one of (19) to (33).

  Amino acid substitutions, deletions, additions and / or insertions are as described above. An antibody that recognizes the same epitope as that recognized by a certain antibody is as described above.

  The present invention further provides a gene encoding the variable region of the present invention, the heavy chain of the present invention, the light chain of the present invention or the antibody of the present invention.

  Furthermore, this invention provides the vector containing the above-mentioned gene.

  The present invention further provides a host cell transformed with the above-described vector.

  Furthermore, the present invention relates to a method for producing the variable region of the present invention, the heavy chain of the present invention, the light chain of the present invention or the antibody of the present invention, which comprises the step of culturing the above host cell.

  The vector, host cell, and host cell culture are as described later.

Domain Recognizing Antibody One of the preferred embodiments of the anti-NR10 antibody in the present invention is an antibody that recognizes domain 1 or an antibody that recognizes domain 2. In the present invention, domain 1 refers to the region (LPAKP to LENIA) from the 21st amino acid to the 120th amino acid in the number containing the signal peptide in the amino acid sequence of human NR10 described in SEQ ID NO: 76. . Further, in the present invention, domain 2 is a region from the 121st amino acid to the 227th amino acid (KTEPP to EEEAP) in the amino acid sequence of human NR10 described in SEQ ID NO: 76 in the state containing a signal peptide. Say.

  Such an antibody is not particularly limited, but is usually an antibody having neutralizing activity, and preferably a humanized antibody.

  An example of a preferable antibody in the present invention includes an antibody that recognizes domain 1. An antibody that recognizes domain 1 has a high neutralizing activity and is particularly useful as a pharmaceutical product.

Antibody (neutralizing activity)
The present invention further provides an anti-NR10 antibody having neutralizing activity.

  In the present invention, the neutralizing activity for NR10 is an activity that inhibits the binding between NR10 and its ligand IL-31, and preferably an activity that suppresses physiological activity based on NR10.

  Selection of an antibody having NR10 neutralizing activity can be performed, for example, by a method of observing the growth inhibitory effect of the IL-31-dependent cell line when a candidate antibody is added to the IL-31-dependent cell line. Is possible. The antibody that suppresses the growth of the IL-31-dependent cell line in the above method is judged to be an antibody having neutralizing activity against NR10.

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 is 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. .

  As long as it has binding activity and / or neutralizing activity to NR10, the antibody of the present invention is not only a bivalent antibody represented by IgG, but also a monovalent antibody, or a multivalent antibody represented by IgM, or different. Bispecific antibodies that can bind to an antigen are also included. 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 NR10 protein.

  The antibody in the present invention may be a low molecular weight antibody. The low molecular weight antibody is an antibody including an antibody fragment in which a part of a full-length antibody (whole antibody, for example, whole IgG) is deleted, and is not particularly limited as long as it has a binding activity to NR10 and / or a neutralizing activity. 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 of these antibodies (eg, dimer, trimer, tetramer, polymer) 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 to NR10 and / or neutralizing 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. Next, a PCR reaction is performed using each DNA of [H chain V region DNA]-[peptide linker DNA]-[L chain V region DNA] and a primer 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 and the light chain variable region to be combined is not particularly limited, and may be arranged in any order, and examples thereof include the following arrangements.
[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: 82)
Ser, Gly, Gly, Gly (SEQ ID NO: 83)
Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 84)
Ser, Gly, Gly, Gly, Gly (SEQ ID NO: 85)
Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 86)
Ser, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 87)
Gly, Gly, Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 88)
Ser, Gly, Gly, Gly, Gly, Gly, Gly (SEQ ID NO: 89)
(Gly, Gly, Gly, Gly, Ser (SEQ ID NO: 84)) n
(Ser, Gly, Gly, Gly, Gly (SEQ ID NO: 85)) n
[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 NR10 molecule, or one antigen binding site recognizes NR10 and the other antigen binding site is the other. Bispecific antibodies that recognize substances can also be used.

  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, 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.

Production of Antibody The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. A monoclonal antibody having binding activity and / or neutralizing activity for NR10 is prepared, for example, by preparing an anti-NR10 monoclonal antibody by a known method using NR10 derived from a mammal such as a human or a mouse or a fragment peptide thereof as an immunogen. It can be obtained by selecting an antibody having NR10 binding activity and / or neutralizing activity from the obtained anti-NR10 monoclonal antibody. 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. It is possible to produce anti-NR10 monoclonal antibodies 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 there. 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 NR10 gene sequence according to a known method such as a method using a baculovirus (WO98 / 46777 etc.).

  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.

  One embodiment of the antibody having binding activity and / or neutralizing activity for NR10 of the present invention includes a monoclonal antibody having binding activity and / or neutralizing activity for human NR10. The immunogen for producing a monoclonal antibody having binding activity and / or neutralizing activity against human NR10 is not particularly limited as long as an antibody having binding activity and / or neutralizing activity against human NR10 can be produced. For example, human NR10 is known to have a plurality of variants, but any variant may be used as an immunogen as long as an antibody having binding activity and / or neutralizing activity against human NR10 can be produced. Alternatively, under the same conditions, an NR10 fragment peptide or a natural NR10 sequence with an artificial mutation may be used as an immunogen. Human NR10.3 is one of the preferred immunogens for producing antibodies having binding activity and / or neutralizing activity for NR10 of the present invention.

  In addition, the measurement of the binding activity and / or neutralizing activity of the antibody to NR10 can be performed, for example, by the method for observing the growth inhibitory effect of the IL-31-dependent cell line 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 a stimulus. Compared to general immunization methods that administer protein antigens, DNA immunization can be expected to have the following advantages.
-The structure of the membrane protein can be maintained to provide immune stimulation-There is no need to purify the immune antigen. 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 NR10 is administered to an immunized animal. DNA encoding NR10 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 NR10-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. Alternatively, an antibody that recognizes NR10 can be produced by the method described in International Publication WO03 / 104453.

  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. In this case, substantially the same NR10 protein can be preferably used as the antigen, including those used for immunization. For example, a cell line expressing NR10, an extracellular domain of NR10, or an oligopeptide consisting of a partial amino acid sequence constituting the region can be used as an antigen.

  In addition to the above-described method for obtaining a hybridoma by immunizing an animal other than a human with an antigen, a target antibody can be obtained by sensitizing human lymphocytes with the antigen. Specifically, human lymphocytes are first sensitized with NR10 protein in vitro. The immunized lymphocytes are then fused with an appropriate fusion partner. As the fusion partner, for example, a myeloma cell derived from human and having a permanent division ability can be used (see Japanese Patent Publication No. 1-59878). The antibody obtained by this method is a human antibody having binding activity to the NR10 protein.

  The base sequence and amino acid sequence encoding the anti-NR10 antibody obtained by the above-described method and the like can be obtained by methods known to those skilled in the art. The amino acids contained in the amino acid sequences described in the present invention are modified after translation (for example, 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.

  Based on the sequence of the obtained anti-NR10 antibody, it is possible to produce an anti-NR10 antibody using gene recombination techniques known to those skilled in the art. Specifically, a polynucleotide encoding the antibody is constructed based on the sequence of the antibody recognizing NR10, 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. 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, examples of vectors for producing the antibody of the present invention include expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen)), pEF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), insect cell-derived expression vectors (for example, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (for example, pMH1, pMH2) Animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Expression Kit” (manufactured by Invitrogen), pNV11, SP-Q01), and an expression vector 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.
Accordingly, the present invention is encoded by a polypeptide of the present invention or a gene encoding a polypeptide of the present invention, comprising the step of culturing a host cell containing a vector into which a polynucleotide encoding the polypeptide of the present invention has been introduced. A method for producing a polypeptide is provided.
More specifically, a method for producing the polypeptide of the present invention comprising the following steps is provided.
(a) culturing a host cell containing a vector into which a gene encoding the polypeptide of the present invention has been introduced,
(b) A step of obtaining a polypeptide encoded by the gene.

  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.

  The binding activity of the obtained antibody to NR10 can be measured by methods known to those skilled in the art. For example, ELISA (enzyme-linked immunosorbent assay), 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 inflammatory disease which uses the above-mentioned antibody as an active ingredient.

  In the present invention, an inflammatory disease is a pathological condition related to cytological / histological reactions occurring in affected blood vessels and adjacent tissues due to damage or abnormal stimulation by a physical, chemical, or biological agent. This refers to a disease with findings (Stedman Medical Dictionary 5th edition, Medical Review Inc., 2005). In general, inflammatory diseases are dermatitis (atopic dermatitis, chronic dermatitis, etc.), inflammatory bowel disease (colitis, etc.), asthma, arthritis (rheumatoid arthritis, osteoarthritis, etc.), bronchitis , Th-2 autoimmune disease, systemic lupus erythematosus, myasthenia gravis, chronic GVHD, Crohn's disease, osteoarthritis spondylitis, low back pain, gout, post-traumatic inflammation, swelling relief, neuralgia, sore throat, bladder Examples include inflammation, hepatitis (nonalcoholic steatohepatitis, alcoholic hepatitis, etc.), hepatitis B, hepatitis C, arteriosclerosis, pruritus.

  Preferable examples of the inflammatory disease that is the subject of the present invention include atopic dermatitis, chronic dermatitis, rheumatism, osteoarthritis, chronic asthma, and pruritus.

  “Containing anti-NR10 antibody as an active ingredient” means that the anti-NR10 antibody is contained as at least one active ingredient, and does not limit the content. In addition, the therapeutic agent for inflammatory diseases of the present invention may contain a component that promotes treatment of other inflammatory diseases in combination with the anti-NR10 antibody described above.

  In addition, the therapeutic agent of this invention may be used for the purpose of prevention.

  The anti-NR10 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 preventive or therapeutic agent for inflammatory diseases of the present invention is not limited to these, and may contain other conventional carriers as appropriate. 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-NR10 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-NR10 antibody can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and Nanocapsule 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 the antibody NR10 antibody (Langer et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12: 98-105; US Patent 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 inflammation 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, in 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 preventive or therapeutic agent for inflammatory diseases 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 Hereinafter, the present invention will be specifically described using examples, but the present invention is not limited to these examples.

[Example 1] Hybridoma production
1.1. Preparation of human NR10 plasmid and cynomolgus monkey NR10 plasmid for DNA immunization
1.1.1. Preparation of hNR10 and cynNR10 Expression Vector Human NR10 (base sequence SEQ ID NO: 75, amino acid sequence SEQ ID NO: 76) is expressed in vector pMacII (WO2005 / 054467) that expresses protein under the control of mouse β-actin promoter. Was used as an integrated hNR10 expression vector. Similarly, a cynNR10 expression vector was constructed from cynomolgus monkey NR10 (base sequence SEQ ID NO: 65, amino acid sequence SEQ ID NO: 66).

1.1.2. Preparation of DNA cartridge
In order to use the hNR10 or cynNR10 expression vector prepared in 1.1.1 for DNA immunization of mice, each cartridge can be immunized with 1 μg of DNA using Herios Gene Gun cartridge kit (manufactured by BIO-RAD). A DNA cartridge was prepared.

1.2. Production of anti-human NR10 antibody-producing hybridoma
1.2.1. Production of hybridomas using human NR10 and cynomolgus monkey NR10 immunized mice
Ten Balb / c mice (female, 6 weeks old at the start of immunization, Nippon Charles River) were immunized with human NR10 or cynomolgus monkey NR10 as follows. As the first immunization, a DNA cartridge prepared with the hNR10 expression vector was immunized using the Herios Gene Gun system (BIO-RAD). The second immunization was performed 1 week later by administering a DNA cartridge prepared with the cynNR10 expression vector using the Herios Gene Gun system. From the third time onwards, hNR10 and cynNR10 expression vectors were alternately immunized at weekly intervals. After confirming an increase in serum antibody titer against human NR10, human NR10 protein (extracellular region) diluted in PBS (−) (Reference Example 4) was intravenously administered at 10 μg / head as final immunization. Four days after the final immunization, mouse spleen cells and mouse myeloma cells P3X63Ag8U.1 (referred to as P3U1, ATCC CRL-1597) were cell-fused according to a conventional method using PEG1500 (Roche Diagnostics). The fused cells, ie, hybridomas, were cultured in RPMI1640 medium containing 10% FBS (hereinafter referred to as 10% FBS / RPMI1640).

1.2.2. Selection of hybridomas On the day after the fusion, (1) the fused cells were suspended in a semi-fluid medium (StemCells) to perform selective culture of the hybridomas and to colonize the hybridomas.

  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. The culture supernatant in which mouse IgG was confirmed was evaluated by neutralizing activity using a human IL-31-dependent cell line (hNR10 / hOSMR / BaF3 cells; Reference Example 2), and some of them had high NR10 neutralizing activity. Clones were obtained (FIG. 3). A clone that suppresses cell growth induced by human IL-31 in a concentration-dependent manner and a cell that suppresses cell growth induced by cynomolgus monkey IL-31 (cynNR10 / cynOSMR / BaF3 cells; Reference Example 2) in a concentration-dependent manner were obtained. (FIG. 4).

[Example 2] Production of chimeric antibody
Preparation of chimeric antibody expression vector 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 10X Universal Primer A Mix attached to the SMART RACE cDNA Amplification Kit (BD Biosciences) and primers (H chain: mIgG1-rnot, L chain mIgK-rnot) set in each antibody constant region, PrimeSTAR HS DNA polymerase ( PCR was performed by TaKaRa), and the antibody variable region gene was isolated. The base sequence of each isolated DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions. Were determined. FIG. 1 shows the heavy chain variable region and FIG. 2 shows the light chain variable region of the amino acid sequences of the obtained NS18, NS22, NS23 and NS33 mouse antibodies.

  For each of the obtained H chain and L chain fragments, PCR was performed with PrimeSTAR HS DNA Polymerase (TaKaRa) using the primer combinations shown in Table 1, and the resulting amplified fragments were converted into constant regions (respectively human γ1 or It was combined with γ2, human κ) and inserted into an animal cell expression vector. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions.

Preparation of chimeric antibody Suspended human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and dish for adherent cells at a cell density of 6 × 10 ⁵ cells / mL Cultivate 10 mL each dish (diameter 10 cm, CORNING), incubate overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ), remove the medium by suction, and select CHO-S-SFMII (Invitrogen) medium. 6.9 mL was added. Add CHO-S-SFMII medium to 700 μL to the prepared plasmid DNA mixture (13.8 μg in total), add 1 μg / mL Polyethylenimine (Polysciences Inc.) 20.7 μL, mix and leave for 10 minutes at room temperature. Cells were loaded and incubated for 4-5 hours in a CO ₂ incubator (5% CO ₂ at 37 ° C.). Then, 6.9 mL of CHO-S-SFMII (Invitrogen) medium was added and cultured in a CO ₂ incubator for 3-4 days. After recovering the culture supernatant, the cells were removed by centrifugation (about 2000 g, 5 minutes, room temperature), and further passed through a 0.22 μm filter MILLEX®-GV (Millipore). Each sample was stored at 4 ° C until use. The antibody was purified from this supernatant using Protein G Sepharose (Amersham Biosciences). The purified antibody was concentrated using Amicon Ultra 15 (Millipore), and the solvent was replaced with PBS (−) containing 0.05% NaN ₃ using PD-10 Desalting columns (Amersham Biosciences). Absorbance at 280 nm was measured with an ND-1000 Spectrophotometer (NanoDrop), and the concentration was calculated by the method of Pace et al. (Protein Science (1995) 4: 2411-2423).

Evaluation of chimera NS22 activity
hIL-31 neutralizing activity was evaluated using the hNR10 / hOSMR / BaF3 cell line that grew in a hIL-31 dose-dependent manner as shown below.

hNR10 / hOSMR / BaF3 cells were prepared in RPMI 1640 medium (GIBCO) containing 10% FBS (MOREGATE) and 1% Penicillin-Streptomycin (Invitrogen) so that the concentration was 1.5 × 10 ⁵ cells / mL. A part of this was added and hIL-31 (R & D Systems) was added to 4 ng / mL (IL-31 (+), final conc .; 2 ng / mL). The remaining cell suspension was designated IL-31 (-). The purified NS22 was adjusted to 2 μg / mL with a medium, and a dilution ratio of 3, and a total of 8 series of dilutions were prepared (final conc.; ≦ 1 μg / mL). Each well of a 96-well flat bottom plate (CORNING) was seeded with 50 μL of the cell suspension and a diluted solution of chimeric NS22 (human γ1, κ), and cultured for 2 days at 37 ° C. in a 5% CO ₂ incubator. After completion of the culture, 20 μL of a mixed solution of Cell Counting Kit-8 (Dojindo) and PBS in equal amounts was added to each well, and the absorbance (450 nm / 620 nm) was measured (TECAN, SUNRISE CLASSIC). After reacting for 2 hours in a 37 ° C., 5% CO ₂ incubator, the absorbance was measured again. The neutralization activity of NS22 was expressed as an inhibition rate using a value obtained by subtracting the 0 hour value from the 2 hour value. As a result, NS22 was found to suppress cell growth induced by IL-31 stimulation in a concentration-dependent manner in the hNR10 / hOSMR / BaF3 cell line, and proved to have neutralizing activity against human IL-31 signaling. (FIG. 5).

  Using a DU145 cell line (human prostate cancer cell line) showing IL-6 production induction by IL-31 stimulation, IL-31 neutralizing activity was evaluated as shown below.

Prepare DU145 in MEM medium (Invitrogen) containing 10% FBS (MOREGATE), 2 mmol / L L-glutamine (Invitrogen), and 1 mmol / L sodium pyruvate (SIGMA) to 2.5 x 10 ⁵ cells / mL. In each well of 48 well-plate (CORNING), 200 μL was dispensed and cultured overnight at 37 ° C. under 5% CO ₂ . The purified chimeric NS22 (human γ1, κ) is diluted to MEM medium containing 10% FBS, 2 mmol / L L-glutamine and sodium pyruvate so as to be 100 μg / mL. A total of 6 dilutions were prepared, each was mixed 1: 1 with 100 ng / mL human interleukin-31 (R & D systems), and 50 μL was added to each well. The IL-6 concentration in the culture supernatant after culturing at 37 ° C. under 5% CO ₂ for 2 days was measured using DuoSet ELISA Development kit (R & D systems). NS22 neutralization activity was evaluated as an inhibition rate (%). That is, IL-6 concentration (A) in the absence of IL-31 has the maximum inhibitory activity (100% inhibition), and IL-6 concentration (B) in the absence of IL-31 and NS22 does not. As none (0% inhibition), the IL-6 concentration (C) under the addition of IL-31 and NS22 was calculated from the following formula.

                    Inhibition rate (%) = (B−C) / (B−A) × 100

  As a result, NS22 was found to suppress IL-6 production by IL-31 stimulation in a concentration-dependent manner in the DU145 cell line, and proved to have neutralizing activity against human IL-31 signaling. (FIG. 6).

Evaluation of IL-31 competitive activity of chimeric anti-NR10 antibody
Human IL-31 (R & D Systems) was labeled with FMAT Blue Monofunctional Reactive Dye (Applied Biosystems). After adding Vortex by adding 25 nmoles FMAT Blue 5.25μL dissolved in DMSO (Junsei) to hIL-31 100μL adjusted to 0.5 mg / mL with 50 mM sodium phosphate buffer (pH 8.0) And left at room temperature for 15 minutes. After stopping the introduction of FMAT Blue into hIL-31 by adding 5 μL of 1 M Tris-HCl (pH 7.4) and 1.1 μL of 10% Tween20, Superdex 75 (GE Healthcare, 17-0771-01) FMAT Blue-labeled hIL-31 and unreacted FMAT Blue were separated on 0.1% Tween20 / PBS developing solution by gel filtration used as Column.

  Using hNR10-expressing CHO cells, the IL-31 / NR10 binding inhibitory activity of the antibody was evaluated as shown below.

NS22 and NA633 (constant regions are γ1, κ, respectively) with assay buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl ₂ , 3 mM MgCl ₂ , 2% FBS, 0.01% NaN ₃ ) Dilution was further performed, and a dilution ratio of 2, and a total of 7 series of dilutions were prepared and added to a plate (96-Well FMAT Plates, Applied Biosystems) at 40 μL / well. Subsequently, FMAT Blue-labeled hIL-31 was diluted 400 times with Assay buffer and added at 20 μL / well. Finally, the cell suspension adjusted to 2.5 × 10 ⁵ / mL with Assay buffer was added at 40 μL / well (final 1 × 10 ⁴ / well). Two hours after adding the cells, fluorescence (FL1) was measured with 8200 Cellular Detection System (Applied Biosystems). As a result, NS22 was shown to inhibit hIL-31 / hNR10 binding in a dose-dependent manner, demonstrating that its activity is superior to NA633 (FIG. 7).

[Example 3] Competition of anti-NR10 antibody against NR10
NS22 antibody purified from the hybridoma culture supernatant was labeled with FMAT Blue (Applied Biosystems, 4328853). 17 μL 1 M NaHCO ₃ solution and 3.4 μL of 17 nmoles FMAT Blue dissolved in DMSO were added to 170 μL of NS22 prepared in 1 mg / ml-PBS, vortexed, and allowed to stand at room temperature for 30 minutes. After adding 8 μL 1 M Tris-HCl (pH 7.4) and 1.9 μL 1% Tween 20 to stop the reaction of introducing FMAT Blue into NS22, Superdex 75 (GE Healthcare, 17-0771-01) was added to the column. FMAT Blue-labeled NS22 (FMAT Blue-NS22) and unreacted FMAT Blue were separated on 0.01% Tween20 / PBS developing solution by gel filtration.

The inhibition of the binding of the obtained FMAT Blue-NS22 to hNR10-expressing CHO cells (Reference Example 3) by various antibodies was examined using 8200 Cellular Detection System (Applied Biosystems, 4342920). 8.8 × 10 ^-2 μg / mL FMAT Blue-NS22, 7500 cells / well of various concentrations of chimeric anti-NR10 antibody (constant regions are γ1, κ, respectively), and left in the dark for 4 hours The fluorescence signal derived from FMAT Blue bound to the cells was measured. The reaction was performed in 10 mM Hepes-KOH containing 2.5 mM CaCl ₂ , 3 mM MgCl ₂ , 140 mM NaCl, 2% FBS, 0.01% NaNO ₃ . The results are shown in FIG. As the concentrations of NS22 and NS23 antibodies increased, the fluorescence value FL1 representing the binding of FMAT Blue-NS22 to NR10 expressing cells decreased. On the other hand, there was almost no decrease in FL1 with increasing concentration of NA633 antibody (Reference Example 6) (FIG. 8).

[Example 4] Humanization of NS22 antibody
Selection of each framework sequence The variable region of mouse NS22 antibody was compared with human germline sequence. Among them, FR sequences used for humanization are summarized in Table 2. CDR and FR were determined according to Kabat numbering. As a humanized variable region, the H chain is the sequence consisting of FR1, FR2, FR3_1, FR4 listed in Table 2 as H0-VH (SEQ ID NO: 50), and the sequence consisting of FR1, FR2, FR3_2, FR4 as H1. -VH (SEQ ID NO: 112). In addition, the L chain has a sequence composed of FR1, FR2, FR3, and FR4 as L0 (SEQ ID NO: 52).

Preparation of variable region of humanized NS22 H0L0 In order to prepare a variable region of humanized NS22 in which the CDR region of NS22 was transplanted into the FR region used for humanization, a synthetic oligo DNA was designed for each of the H chain and the L chain. Each synthetic oligo DNA was mixed, and a gene encoding the variable region of humanized NS22 was prepared by assembly PCR. Assemble PCR was performed using KOD-Plus (TOYOBO), and PCR was performed according to the following conditions. The reaction mixture consisting of the attached PCR Buffer, dNTPs, MgSO ₄ , KOD-Plus and 10 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, After two PCR reaction cycles consisting of 2 minutes at 68 ° C, 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 10 pmol of each was added, and a PCR reaction cycle consisting of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 68 ° C. for 1 minute was performed 35 times to obtain amplified fragments. The obtained amplified fragment was cloned into a TOPO TA Cloning vector (TOYOBO), and the nucleotide sequence was confirmed by sequencing. The prepared variable region and constant region were combined to prepare H0-SKSC (SEQ ID NO: 54) and L0 (SEQ ID NO: 56), which were incorporated into an expression vector that enables expression of the inserted gene in animal cells. The base sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the DNA sequencer ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached instructions.

Production of variable region of humanized NS22 H1
Preparation of H1-SKSC (SEQ ID NO: 130) was performed by replacing the 73rd glutamine (E) of kabat numbering present in FR3 of H0-SKSC (SEQ ID NO: 54) with lysine (K). The mutant was prepared using a commercially available QuikChange Site-Directed Mutagenesis Kit (Stratagene), and the mutant was prepared according to the method described in the attached instructions.

Expression of antibody converted to IgG Expression of antibody was performed by the following method. HEK293H strain derived from human fetal kidney cancer cells (Invitrogen) is suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and a dish (diameter) for cells with a cell density of 5-6 × 10 ⁵ cells / mL 10 mL to each dish of 10 cm, CORNING) After incubating overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ), the medium was removed by suction and 6.9 mL of CHO-S-SFMII (Invitrogen) medium Was added. The prepared plasmid DNA mixture (13.8 μg in total) was mixed with 20.7 μL of 1 μg / mL Polyethylenimine (Polysciences Inc.) and 690 μL of CHO-S-SFMII medium and allowed to stand for 10 minutes at room temperature. Incubated in a CO ₂ incubator (5% CO ₂ at 37 ° C.) for 4-5 hours. Then, 6.9 mL of CHO-S-SFMII (Invitrogen) medium was added and cultured in a CO ₂ incubator for 3 days. After the culture supernatant was recovered, centrifuged (approximately 2000 g, 5 min, room temperature) to remove the cells, and sterilized through a further 0.22μm filter MILLEX ^(R) -GV (Millipore). Each sample was stored at 4 ° C until use.

Purification of IgG antibody 50 μL of rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the obtained culture supernatant and mixed by inverting at 4 ° C. for 4 hours or more. The solution was transferred to a 0.22 μm filter cup Ultrafree (registered trademark) -MC (Millipore), washed 3 times with 500 μL of TBS, and then suspended in 100 μL of 50 mM sodium acetate aqueous solution, pH 3.3 in rProtein A Sepharose ^™ resin. After standing for 3 minutes, the antibody was eluted. Immediately, 6.7 μL of 1.5 M Tris-HCl, pH 7.8 was added for neutralization. Elution was performed twice to obtain 200 μL of purified antibody. Measure the absorbance at 280 nm by applying 2 μL of the antibody-containing solution to the ND-1000 Spectrophotometer (NanoDrop) (Thermo Scientific NanoDrop ^TM 1000 Spectrophotometer (Thermo Scientific)) or 50 μL to the spectrophotometer DU-600 (BECKMAN) The antibody concentration was calculated by the method of Pace et al. (Protein Science (1995) 4: 2411-2423).

Measurement of IL-31 competitive activity using FMAT
Using hNR10-expressing CHO cells, the IL-31 / NR10 binding inhibitory activity of the antibodies was evaluated as shown below. NS22 chimeric antibody and NS22_H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56) were assay buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl ₂ , 3 mM MgCl ₂ , 2% FBS, 0.01% NaN ₃ , pH 7.4) is diluted to an appropriate concentration. Further, a dilution ratio of 2, and a total of 8 series of dilutions are prepared, and the plate (96-Well FMAT Plates, Applied Biosystems). Subsequently, FMAT Blue-labeled hIL-31 was diluted 400 times with Assay buffer and added at 20 μL / well. Finally, the cell suspension adjusted to 2.5 × 10 ⁵ / mL with Assay buffer was added at 40 μL / well (final 1 × 10 ⁴ / well). Two hours after adding the cells, fluorescence (FL1) was measured with 8200 Cellular Detection System (Applied Biosystems).

  As a result, humanized NS22 antibody, H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56), H1L0 (H chain H1-SKSC / SEQ ID NO: 130, L chain L0 / SEQ ID NO: : 56) showed almost the same competitive activity as the chimeric antibody as shown in FIG. 9, and thus can be said to be a H0L0, H1L0 humanized anti-IL-31 receptor antibody. Further, it is considered that both FRs used for H0L0 and H1L0 can be used as FRs during humanization.

  Therefore, it is considered that the mutation site for CDR shown in the following examples can be introduced into both H0 and H1.

[Example 5] Heterogeneity reduction effect by novel constant regions M14 and M58 in humanized anti-IL31 receptor antibody As shown in Reference Examples 7 to 9, in the huPM1 antibody which is a humanized anti-IL-6 receptor antibody, the constant region is It was confirmed that by converting from IgG2 to M14 or M58, the heterogeneity derived from the hinge region of IgG2 can be reduced without reducing stability. Thus, it was examined whether the heterogeneity of humanized anti-IL-31 receptor antibody can be reduced by converting the constant region from wild-type IgG2 to M14 or M58.

  Example 4 including IgG1 (SEQ ID NO: 60) and IgG2 (SEQ ID NO: 132) containing M14 (SEQ ID NO: 129) and M58 (SEQ ID NO: 128) prepared in Reference Examples 8 and 9 as H chains. H0-M14, H0-M58, H0-IgG1, and H0-IgG2, combined with the variable region H0 (H0-VH / SEQ ID NO: 50) of the H chain of the humanized anti-IL-31 receptor antibody prepared in step 1, as the L chain Using L0 (L0 / SEQ ID NO: 56) prepared in Example 4, H0L0-IgG1 (H chain H0-IgG1 / SEQ ID NO: 133, L chain L0 / SEQ ID NO: 56), H0L0-IgG2 (H chain) H0-IgG2 / SEQ ID NO: 134, L chain L0 / SEQ ID NO: 56), H0L0-M14 (H chain H0-M14 / SEQ ID NO: 135, L chain L0 / SEQ ID NO: 56) and H0L0-M58 (H chain) H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO: 56) was prepared. Each antibody was expressed and purified by the method described in Example 4.

  As an evaluation method of heterogeneity, evaluation by cation exchange chromatography was performed. The heterogeneity of the prepared antibody was evaluated by using ProPac WCX-10 (Dionex) as a column, 20 mM Sodium Acetate, pH 5.0 as mobile phase A, 20 mM Sodium Acetate, 1 M NaCl, pH 5 as mobile phase B 0.0 was used with the appropriate flow rate and gradient. The results of evaluation by cation exchange chromatography (IEC) are shown in FIG.

  As shown in FIG. 10, even in the anti-IL-31 receptor antibody, the heterogeneity is increased by converting the constant region from IgG1 to IgG2, and the constant region is converted to M14 or M58. It was also confirmed that heterogeneity can be reduced.

[Example 6] Pharmacokinetic improvement effect of novel constant region M58 in anti-IL-31 receptor antibody As shown in Reference Example 9, in the huPM1 antibody which is an anti-IL-6 receptor antibody, the constant region is converted from IgG1 to M58. Thus, it was found that the binding to human FcRn was improved and the pharmacokinetics was improved in human FcRn transgenic mice. Therefore, it was examined whether the pharmacokinetics of anti-IL-31 receptor antibody could be improved by converting the constant region to M58.

  H0L0-IgG1 prepared in Example 4 and Example 5 (H chain H0-IgG1 / SEQ ID NO: 133, L chain L0 / SEQ ID NO: 56), H0L0-M58 (H chain H0-M58 / SEQ ID NO: 136), The binding to human FcRn was evaluated by the method shown in Reference Example 9 for L chain L0 / SEQ ID NO: 56). The results are shown in Table 3.

  As shown in Table 3, even in H0L0, which is an anti-IL-31 receptor antibody, the ability to bind to human FcRn is improved by converting the constant region from IgG1 to M58, similar to hPM1, which is an anti-IL-6 receptor antibody. Confirmed to do. This indicates that conversion of the constant region from IgG1 to M58 has the potential to improve pharmacokinetics in humans even with anti-IL-31 receptors.

[Example 7] Identification of mutation sites that lower the isoelectric point
Preparation of mutants Each mutant was prepared by a method according to Example 4 or by performing Assemble PCR using PCR. The method using Assemble PCR synthesizes an oligo DNA designed based on the normal and reverse strand sequences including the modification site. 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. Each mutant was generated by joining the two fragments by Assemble PCR. The prepared mutant was inserted into an expression vector that enables expression of the inserted gene in animal cells, 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 Example 4.

Identification of mutation sites
In order to improve the pharmacokinetics of H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56), investigation was made on the modified sites that can lower the isoelectric point of the variable region. . As a result of screening the variable region mutation sites inferred from the three-dimensional structure model, the regions where the isoelectric point of the variable region was reduced without significantly reducing the binding to NR10 were found and summarized in Table 4 (Hp5-VH / SEQ ID NO: 137, Hp7-VH / SEQ ID NO: 138, Hp8-VH / SEQ ID NO: 139, Hp6-VH / SEQ ID NO: 140, Hp9-VH / SEQ ID NO: 141, Hp1-VH / SEQ ID NO: 142 , Hp13-VH / SEQ ID NO: 143, Lp1-VL / SEQ ID NO: 144, Lp2-VL / SEQ ID NO: 145, Lp3-VL / SEQ ID NO: 146, Lp4-VL / SEQ ID NO: 147, Lp7-VL / SEQ ID NO: 148, Lp5-VL / SEQ ID NO: 149, Lp6-VL / SEQ ID NO: 150). Production and purification of each variant was performed by the method described in Example 4.

  The hIL-31 / hNR10 binding inhibitory activity of each variant was evaluated using FMAT. The method was performed according to the method of Example 4. As shown in FIG. 11, the competitive activity of each variant did not show a significant decrease compared to that of H0L0.

  In Table 4 above, * indicates a portion modified to have a human sequence but not related to the isoelectric point.

  An example of a humanized NS22 antibody with a reduced isoelectric point combining these modifications is Hp3Lp15 (H chain Hp3-SKSC / SEQ ID NO: 151, L chain Lp15 / SEQ ID NO: 152). The affinity of Hp3Lp15 for NR10, isoelectric point, and plasma retention in mice were compared to H0L0.

Measurement of affinity The affinity of each antibody for NR10 was determined according to the method of Reference Example 10.
The measured affinity results are shown in Table 5. The affinity of Hp3Lp15 was shown to be almost equivalent to that of H0L0.

Measurement of isoelectric point In order to evaluate the change in isoelectric point of the full-length antibody due to the amino acid modification of the variable region, each antibody was analyzed by isoelectric focusing. The method of isoelectric focusing was as follows.

Phast-Gel Dry IEF (Amersham Biosciences) gel was swollen for about 30 min with the following swelling solution using Phastsystem Cassette (Amersham Biosciences).
Milli-Q water 1.5 mL
Pharmalyte 5-8 for IEF (Amersham Biosciences) 100 μL

Using the swollen gel, electrophoresis was performed by PhastSystem (manufactured by Amersham Biosciences) with the following program. The sample was added to the gel in Step 2. As a pI marker, Calibration Kit for pI (Amersham Biosciences) was used.
Step 1: 2000 V 2.5 mA 3.5 W 15 ° C 75 Vh
Step 2: 200 V 2.5 mA 3.5 W 15 ° C 15 Vh
Step 3: 2000 V 2.5 mA 3.5 W 15 ° C 410 Vh

  The gel after electrophoresis was fixed with 20% TCA, and then silver staining was performed using Silver staining Kit, protein (Amersham Biosciences) according to the protocol attached to the kit. After staining, the isoelectric point of the sample (full-length antibody) was calculated from the known isoelectric point of the pI marker.

  As a result of measuring the isoelectric point by isoelectric focusing, the isoelectric point of H0L0 is approximately 7.8, and the isoelectric point of Hp3Lp15 is approximately 5.5. Therefore, the isoelectric point of Hp3Lp15 is the isoelectric point of H0L0. It was about 2.3 lower than The theoretical isoelectric point of variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION). The theoretical isoelectric point of variable region of H0L0 was 7.76, and the theoretical isoelectric point of variable region of Hp3Lp15 was 4.63. The theoretical isoelectric point of Hp3Lp15 decreased by 3.13 compared with H0L0.

Evaluation of pharmacokinetics of antibodies with reduced isoelectric point in mice To evaluate plasma retention of Hp3Lp15, a modified antibody with reduced isoelectric point, plasma retention of H0L0 and Hp3Lp15 in normal mice A comparison was made. H0L0 and Hp3Lp15 were administered to mice (C57BL / 6J, Nihon Charles River) as a single intravenous dose of 1 mg / kg, and the plasma concentrations were compared. Plasma concentration was measured by ELISA. An immunoplate (Nunc-Immuno Plate, MaxiSorp (manufactured by Nalge nunc International)) on which a calibration curve sample and a plasma measurement sample of an appropriate concentration were immobilized with an anti-human IgG (Fc-specific) antibody (manufactured by Sigma) The solution was dispensed and allowed to stand at room temperature for 1 hour. After reacting Goat Anti-Human IgG-ALP (manufactured by Sigma) for 1 hour at room temperature, a color reaction was performed using BluePhos Microwell Phosphatase Substrates System (manufactured by Kirkegaard & Perry Laboratories) as a substrate, and 650 using a microplate reader. The absorbance at nm was measured. The plasma concentration was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).

  Table 6 shows the pharmacokinetic parameters (AUC, whole body clearance (CL)) calculated from the obtained plasma concentration transition data using the pharmacokinetic analysis software WinNonlin (Pharsight). Compared with H0L0, AUC after intravenous administration of Hp3Lp15 increased by about 14% and clearance decreased by about 12%. Thus, it was found that Hp3Lp15 having a reduced isoelectric point of H0L0 has improved pharmacokinetics.

[Example 8] Influence on biological activity by combination of variable region and constant region In order to evaluate the influence on biological activity by using different constant regions, the following variants were prepared.

  As the H chain, the constant region was SKSC (SEQ ID NO: 62) prepared in Reference Examples 7 and 9, and the M58 (SEQ ID NO: 128) variable region was variable region Hp3 (Hp3-VH / SEQ ID NO: prepared in Example 7). 167) were combined to produce Hp3-M58 (SEQ ID NO: 240) and Hp3-SKSC (SEQ ID NO: 151). Hp3Lp15-SKSC (H chain Hp3-SKSC / SEQ ID NO: 151, L chain Lp15 / SEQ ID NO: 152) and Hp3Lp15 are combined with the prepared H chain and the L chain Lp15 (Lp15 / SEQ ID NO: 152) prepared in Example 7. -M58 (H chain Hp3-M58 / SEQ ID NO: 240, L chain Lp15 / SEQ ID NO: 152) was prepared. Each antibody was expressed and purified by the method described in Example 4.

  H0L0-SKSC (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56) using each antibody prepared above and the constant region SKSC (SEQ ID NO: 62) described in Reference Example 7. H0L0-M58 (H chain H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO: 56) and H0L0-IgG2 (H chain H0-IgG2 / SEQ ID NO: 134, L chain L0 prepared in Example 5) / SEQ ID NO: 56) Then, biological activity using BaF / NR10 was carried out by the method described in Example 2, and the results are summarized in FIG.

  As shown in FIG. 18, no significant difference in biological activity was detected between the constant regions. The combination of the two variable regions H0, Hp3 and each constant region did not affect the biological activity. Therefore, in any variable region to be produced in the future, it is considered that the biological activity does not change with any constant region.

[Example 9] Identification of mutation sites that suppress degradation by thermal acceleration test Although antibodies used for pharmaceuticals are monoclonal antibodies obtained from clones derived from a single antibody-producing cell, heterogeneity exists To do. 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 antibodies as pharmaceuticals, the physical properties of the protein, especially homogeneity and stability, are extremely important, and it is possible to reduce the heterogeneity of the target substance / related substances and to be as single as possible. desired. Therefore, the following experiments were conducted to evaluate the heterogeneity of antibodies under stress conditions and to reduce the heterogeneity.

  In order to evaluate the degradation product, a thermally accelerated product of H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56) was prepared by the following method. The prepared thermal acceleration product and unaccelerated product (initial) were analyzed by cation exchange chromatography by the following method.

・ Method for preparing heat-accelerated product <br/> Buffer solution: PBS
Antibody concentration: 0.2-1.0 mg / mL
Acceleration temperature: 60 ℃
Acceleration period: 1 day

・ Analysis method for cation exchange chromatography Column: ProPac WCX-10, 4 × 250 mm (Dionex)
Mobile phase: (A) 25 mmol / L MES / NaOH, pH 6.1
(B) 25 mmol / L MES / NaOH, 250 mmol / L NaCl, pH 6.1
Flow rate: 0.5 mL / min
Column temperature: 40 ° C
Gradient:% B 0 to 0 (0-5 min) → 0 to 30 (5-80 min)
Detection: 280 nm

  FIG. 19 shows the chromatogram results of the sample before and after thermal acceleration of H0L0. In the sample after thermal acceleration of H0L0, there was a tendency for the basic peak to increase.

  Thus, in order to reduce this peak, as a result of screening, Ha355, Ha356, Ha360, and Ha362 were found, and Ha355L0 (H chain Ha355-SKSC / SEQ ID NO: 242 L chain L0) obtained by combining these H chain variants and L0. / SEQ ID NO: 56), Ha356L0 (H chain Ha356-SKSC / SEQ ID NO: 243 L chain L0 / SEQ ID NO: 56), Ha360L0 (H chain Ha360-SKSC / SEQ ID NO: 244 L chain L0 / SEQ ID NO: 56) Ha362L0 (H chain Ha362-SKSC / SEQ ID NO: 245 L chain L0 / SEQ ID NO: 56) was prepared. The sequence of each variant is summarized in Table 7.

  Expression and purification of each antibody found were performed by the method described in Example 4. As with H0L0, each prepared antibody was prepared as a heat-accelerated product and analyzed by cation exchange chromatography. The results are shown in FIG.

  As a result, the modified antibody containing a modification in which the 101st aspartic acid of the H chain was substituted with glutamic acid produced fewer basic peaks than that of H0L0, which increased after thermal acceleration. These modified antibodies were subjected to biological activity using BaF / NR10 by the method described in Example 2, and the results are shown in FIG. As shown in FIG. 20, the biological activity of these modifications was approximately equal to or greater than that of H0L0. From the above, it was found that the modification of Ha355, Ha356, Ha360, and Ha362 is effective in suppressing degradation products generated by thermal acceleration and enhancing the stability of the antibody.

[Example 10] Identification of mutation sites that increase affinity
In order to improve the affinity of H0L0 to NR10, a library in which mutations were introduced into the CDR sequence was prepared and examined. As a result of screening a library in which mutations were introduced into CDRs, mutations that improved the affinity for NR10 were found and are summarized in Table 8. Respective heavy chain variants Ha101-SKSC (SEQ ID NO: 246), Ha103-SKSC (SEQ ID NO: 247), Ha111-SKSC (SEQ ID NO: 248), Ha204-SKSC (SEQ ID NO: 249), Ha219-SKSC ( SEQ ID NO: 250) and L0 (L0 / SEQ ID NO: 56), the respective L chain variants La134 (SEQ ID NO: 251), La130 (SEQ ID NO: 252), La303 (SEQ ID NO: 253), La328 (SEQ ID NO: : 254) and H0 (H0-SKSC / SEQ ID NO: 54) were combined to prepare each antibody. Production and purification of each variant was performed by the method described in Example 4.

  The affinity of each variant for NR10 was evaluated using Biacore and is shown in Table 9. The method was performed by the method described in Reference Example 10. As shown in Table 9, it was found that the KD value of each variant was improved as compared with that of H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56).

  As an example of combining these mutations that improve affinity and mutations that decrease the isoelectric point produced in Example 7, Ha401La402 (H chain Ha401-SKSC / SEQ ID NO: 255, L chain La402 / SEQ ID NO: 256) Or H17L11 (H chain H17-M58 / SEQ ID NO: 222, L chain L11 / SEQ ID NO: 236). Production and purification of each variant was performed by the method described in Example 4.

  The affinity for NR10 of Ha401La402 (H chain Ha401-SKSC / SEQ ID NO: 255, L chain La402 / SEQ ID NO: 256) and the biological activity using BaF / NR10 were carried out by the methods described in Reference Example 10 and Example 2, It was compared with H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56). The measured affinity results are shown in Table 10, and the biological activity using BaF / NR10 is shown in FIG. It was found that both affinity and biological activity were improved from that of H0L0 (H chain H0-SKSC / SEQ ID NO: 54, L chain L0 / SEQ ID NO: 56).

  In addition, the affinity for NR10 of H17L11 (H chain H17-M58 / SEQ ID NO: 222, L chain L11 / SEQ ID NO: 236) and biological activity using BaF / NR10 were determined by the methods described in Example 7 and Example 2. And compared with H0L0 (H chain H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO: 56). The measured affinity results are shown in Table 11, and the biological activity using BaF / NR10 is shown in FIG. It was found that both affinity and biological activity were improved from that of H0L0 (H chain H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO: 56).

[Example 11] Identification of mutation sites for reducing the risk of immunogenicity
Reduced risk of immunogenicity of heavy chain CDR1
The T-cell epitope present in the variable region sequence of H0L0 was analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, it was predicted that there are many T-cell epitopes that bind to HLA in the H chain CDR1 (a sequence with a high risk of immunogenicity exists). Therefore, we examined a modification that reduces the immunogenicity risk of H chain CDR1 in TEPITOPE analysis.By replacing kabat numbering 33rd isoleucine (I) with alanine (A), the risk of immunogenicity is increased. A reduction was found to be altered (Table 12). H19 (H19-M58 / SEQ ID NO: 223) was prepared by adding this modification to H17 prepared in Example 10. The prepared H19 was combined with L12 to prepare H19L12 (H chain H19-M58 / SEQ ID NO: 223, L chain L12 / SEQ ID NO: 237). Each variant was prepared and purified by the method described in Example 4.

  Affinity for NR10 and biological activity using BaF / NR10 were carried out by the methods described in Reference Example 10 and Example 2, and H0L0 (H chain H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO: 56) and Compared. The results of the affinity measured are shown in Table 13, and the biological activity using BaF / NR10 is shown in FIG. Both affinity and biological activity were shown to be almost the same as that of H0L0.

Reduced risk of immunogenicity of light chain CDR1
The kabat numbering 25th threonine (T) present in CDR1 of the L chain is alanine (A) or serine (S) in the germline sequence. Therefore, it is expected that the risk of immunogenicity is lower when the 25th threonine (T) is changed to alanine (A) or serine (S) (Table 14). Therefore, L17 (SEQ ID NO: 238) in which the above modification was added to L12 was prepared. The prepared L17 and H0 were combined to prepare H0L17 (H chain H0-M58 / SEQ ID NO: 136, L chain L17 / SEQ ID NO: 238). Production and purification of the modified product were performed by the method described in Example 4.

  The affinity of each variant for NR10 and the biological activity using BaF / NR10 were carried out by the methods described in Reference Example 10 and Example 2, and H0L0 (H chain H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO. : 56), and H0L12 (H chain H0-M58 / SEQ ID NO: 136, L chain L12 / SEQ ID NO: 237). Since L12 contains a sequence with improved affinity, L12 exhibits an affinity about twice as high as H0L0. The results of the affinity measured are shown in Table 15, and the biological activity using BaF / NR10 is shown in FIG. It was shown that both affinity and biological activity are similar to those of H0L12.

[Example 12] Preparation of fully humanized NS22 antibody Modifications that reduce the pI, modifications that increase affinity, modifications that suppress the degradation of the heavy chain, and modifications that reduce the risk of immunogenicity found in the above examples Various variable regions of NS22 variants were created for H0 (H0-M58 / SEQ ID NO: 136), H1 (H1-M58 / SEQ ID NO: 257), or L0 (L0 / SEQ ID NO: 56). As a result of screening, H28L17 (H chain H28-M58 / SEQ ID NO: 224, L chain L17 / SEQ ID NO: 238), H30L17 (H chain H30-M58 / SEQ ID NO: 225, L chain L17 / SEQ ID NO: 238) ), H34L17 (H chain H34-M58 / SEQ ID NO: 226, L chain L17 / SEQ ID NO: 238), H42L17 (H chain H42-M58 / SEQ ID NO: 227, L chain L17 / SEQ ID NO: 238), H44L17 ( H chain H44-M58 / SEQ ID NO: 228, L chain L17 / SEQ ID NO: 238), H46L17 (H chain H46-M58 / SEQ ID NO: 229, L chain L17 / SEQ ID NO: 238), H57L17 (H chain H57-M58 / SEQ ID NO: 230, L chain L17 / SEQ ID NO: 238), H71L17 (H chain H71-M58 / SEQ ID NO: 231, L chain L17 / SEQ ID NO: 238), H78L17 (H chain H78-M58 / SEQ ID NO: 232, L chain L17 / SEQ ID NO: 238), H92L17 (H chain H92-M58 / SEQ ID NO: 233, L chain L17 / SEQ ID NO: 238), H97L50 (H chain H97 -M58 / SEQ ID NO: 234, L chain L50 / SEQ ID NO: 239), H98L50 (H chain H98-M58 / SEQ ID NO: 235, L chain L50 / SEQ ID NO: 239) were found. Production and purification of the modified product were performed by the method described in Example 4.

  The affinity of each variant for NR10 and the biological activity using BaF / NR10 were carried out by the methods described in Reference Example 10 and Example 2, and H0L0 (H chain H0-M58 / SEQ ID NO: 136, L chain L0 / SEQ ID NO. : 56). The measured affinity results are shown in Table 16, and the biological activity using BaF / NR10 is shown in FIGS. 25-1 and 25-2. It was shown that the affinity and biological activity of any antibody showed almost the same or higher than that of H0L0.

[Example 13] Analysis of binding domain of anti-NR10 neutralizing antibody (1) Preparation of human / mouse wild type and chimeric antigens Human and mouse wild type and chimeric NR10 extracellular region (hhh (SEQ ID NO: 258), mmm (SEQ ID NO: 259), hhm (SEQ ID NO: 260), mmh (SEQ ID NO: 261), hmm (SEQ ID NO: 262), mhm (SEQ ID NO: 263), mhh (SEQ ID NO: 264)) The gene was inserted into an animal cell expression vector with a His tag and Myc tag (HHHHHHEQKLISEEDL / SEQ ID NO: 287) at the C-terminus, and transiently expressed with the FreeStyle 293 Expression System (invitrogen ^™ ). A schematic diagram of these human / mouse wild type and chimeric NR10-ECD is shown in FIG.

  Purification of human / mouse wild type and chimeric antigens (hhh, mmm, hhm, mmh, hmm, mhm, mhh) from the culture supernatant was performed by Ni-NTA Superflow column chromatography. That is, 1 mL of Ni-NTA Superflow (QIAGEN) is packed into a polyprep empty column (BioRad), 30 mL of each culture supernatant is added, and D-PBS (Dulbecco's Phosphate containing 150 mM sodium chloride and 20 mM imidazole) is added. -Buffered Salines) and then eluted with D-PBS containing 150 mM sodium chloride and 250 mM imidazole. The eluted fraction was substituted with D-PBS with Amicon-Ultra (Millipore) having a molecular weight cut off of 10 K, and then concentrated.

(2) Detection of bound antigen by Western Blot The prepared human / mouse wild type and chimeric antigens were electrophoresed on 3 sheets of 4-20% polyacrylamide gel (Daiichi Kagaku) corresponding to 0.5 μg / lane each. The sample was electrically transferred to a PVDF membrane (Millipore) with a semi-dry blotting apparatus and blocked with TBS containing 5% SkimMilk. One (humanized anti-human NR10 antibody detection system) is 5μg / mL H44M58L17, one (mouse anti-human NR10 antibody detection system) is 5μg / mL ND41, and one (Myc tag detection system) is The mixture was reacted with an anti-Myc antibody (SantaCruz, Cat. # Sc-789) diluted 500 times with TBS containing 5% SkimMilk at room temperature for 1 hour.

After washing 3 times for 3 minutes with TBS containing 0.05% Tween ^™ 20, the secondary antibody was reacted. Alkaline phosphatase-labeled goat anti-human IgGγ (BIOSOURCE, Cat. # AHI0305) is used for the humanized anti-human NR10 antibody detection system, and alkaline phosphatase-labeled goat anti-mouse IgG (SantaCruz, Cat. # Sc) for the mouse anti-human NR10 antibody detection system. -2008), alkaline phosphatase-labeled goat anti-rabbit IgG (SantaCruz, Cat. # Sc-2057) was used for the Myc tag detection system and reacted at room temperature for 1 hour. After washing 4 times for 3 minutes with TBS containing 0.05% Tween ^™ 20, color was developed with BCIP / NBT Phosphatase substrate, 1-Component System (KPL). The TBS (Tris-Buffered Saline) used here was prepared by dissolving 1 TBS (Tris-Buffered-Saline) powder (TaKaRa) in 1 L of distilled water. The results are shown in FIG.

  When humanized antibodies and mouse antibodies were used, binding was detected only in the extracellular regions of NR10, hhh, hhm, and hmm.

[Reference Example 1] Isolation of cynomolgus monkey NR10, OSMR, IL-31 genes For the safety evaluation in the preclinical stage, cross-reactivity and neutralization activity to cynomolgus monkeys are considered important, and cynomolgus monkey NR10 genes, OSMR genes An attempt was made to isolate. Primers were designed from the published rhesus monkey genome information, and NR10 and OSMR genes were successfully amplified from cynomolgus monkey pancreatic cDNA by PCR. The gene sequences of isolated cynomolgus monkeys NR10, OSMR, and IL-31 are shown in SEQ ID NOs: 65, 69, and 67. The amino acid sequences of cynomolgus monkey NR10, OSMR, and IL-31 are shown in SEQ ID NOs: 66, 70, and 68.

[Reference Example 2] Establishment of NR10 and OSMR-expressing Ba / F3 cell lines Human full-length NR10 cDNA (SEQ ID NO: 75) was incorporated into an expression vector pCOS1 (Biochem Biophys Res Commun. 228, p838-45, 1996) and pCosNR10.3 It was. Oncostatin M receptor cDNA (OSMR, GenBank accession No. NM003999) was isolated from a human placenta library by PCR, and similarly, an expression vector pCos1-hOSMR was constructed. 10 μg of each vector was simultaneously introduced into the mouse IL-3-dependent pro-B cell-derived cell line Ba / F3 by the electroporation method (BioRad Gene Pulser, 960 μF, 0.33 kV). After the introduction, human IL-31 (R & D Systems) was added and cultured to obtain a cell line (hNR10 / hOSMR / BaF3 cells) showing proliferation in an IL-31-dependent manner. Moreover, the cynomolgus monkey IL-31 gene (SEQ ID NO: 67) was incorporated into an expression vector for mammalian cells and introduced into the CHO cell line DG44 to obtain cynomolgus monkey IL-31 as its culture supernatant. Using this culture supernatant, similarly to hNR10 / hOSMR / BaF3, cynomolgus monkey full-length NR10 and cynomolgus OSMR genes were inserted into the expression vector pCOS1, respectively, and expressed in Ba / F3 cells to express cynomolgus IL-31-dependent cell lines ( cynNR10 / cynOSMR / BaF3 cells) were established.

[Reference Example 3] Establishment of NR10-expressing CHO cell line Expression of mammalian region-deficient human NR10 gene (SEQ ID NO: 73) and intracellular region-deficient cynomolgus monkey NR10 gene (SEQ ID NO: 71), respectively, for mammalian cells The vector was inserted into a vector and linearized with a restriction enzyme, and then introduced into the CHO cell line DG44 by electroporation (BioRad Gene Pulser, 25 μF, 1.5 kV). NR10-expressing cells were selected and established by FCM analysis using anti-human NR10 antibody. The amino acid sequence encoded by the nucleotide sequence of the intracellular region-deficient human NR10 gene (SEQ ID NO: 73) is represented by SEQ ID NO: 74, and the nucleotide sequence of the intracellular region-deleted cynomolgus monkey NR10 gene (SEQ ID NO: 71). The encoded amino acid sequence is shown in SEQ ID NO: 72.

[Reference Example 4] Preparation of NR10 protein (extracellular region) Using human NR10 cDNA as a template, only the extracellular region is amplified by the PCR method, and a FLAG tag sequence is added to the C-terminus, which is used as an expression vector for mammalian cells. Incorporated. 10 μg of this linearized vector was introduced into Chinese hamster ovary cell line DG44 by electroporation (BioRad Gene PulserII, 25 μF, 1.5 kV) to obtain a cell line exhibiting high expression. A soluble NR10 was obtained by purifying the cell line from a culture supernatant obtained by mass culture using an anti-FLAG antibody column (manufactured by SIGMA) and gel filtration. The nucleotide sequence of soluble NR10 is shown in SEQ ID NO: 77, and the amino acid sequence is shown in SEQ ID NO: 78.

[Reference Example 5] Preparation of anti-human NR10 antibody Human NR10 protein (extracellular region) [described in Reference Example 4] was immunized to a mouse, and a hybridoma was prepared by a conventional method. The culture supernatant of these hybridomas was evaluated by neutralizing activity using the human IL-31 dependent cell line (hNR10 / hOSMR / BaF3 cells) shown in Reference Example 2 to obtain NA633 having NR10 neutralizing activity. It was.

  In addition, using a mammalian expression vector into which the full-length human NR10 gene (SEQ ID NO: 75) was inserted, DNA immunization was performed using a He gas injection gene gun, and hybridomas were prepared by a conventional method. The culture supernatant of these hybridomas was evaluated by neutralizing activity using the human IL-31-dependent cell line (hNR10 / hOSMR / BaF3 cells) shown in Reference Example 2 to obtain ND41 having NR10 neutralizing activity. It was.

[Reference Example 6] Preparation of human chimeric antibody
The amino acid sequence of the heavy chain variable region of NA633 is shown in SEQ ID NO: 104, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 108. The amino acid sequence of CDR1 of the heavy chain variable region of NA633 is SEQ ID NO: 105, the amino acid sequence of CDR2 is SEQ ID NO: 106, the amino acid sequence of CDR3 is SEQ ID NO: 107, and the amino acid sequence of CDR1 of the light chain variable region The sequence is shown in SEQ ID NO: 109, the amino acid sequence of CDR2 is shown in SEQ ID NO: 110, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 111. Further, according to a conventional method, a chimeric antibody comprising these mouse variable regions and human constant regions (γ1 for H chain and κ for L chain) was prepared.

[Reference Example 7] Production of huPM1-SKSC with reduced heterogeneity of wild-type IgG2 without reducing stability
Since NS22 antibody is an NR10 neutralizing antibody, binding to Fcγ receptor may be unfavorable in consideration of immunogenicity and side effects. In order to reduce the binding to the Fcγ receptor, a method of selecting IgG2 or IgG4 instead of IgG1 as an isotype of the constant region is considered (Ann Hematol. 1998 Jun; 76 (6): 231-48.). From the viewpoint of plasma retention, IgG2 was considered preferable to IgG4 (Nat Biotechnol. 2007 Dec; 25 (12): 1369-72). On the other hand, when developing antibodies as pharmaceuticals, the physical properties of the protein, especially homogeneity and stability, are extremely important, and it has been reported that the IgG2 isotype has a large amount of heterogeneity derived from disulfide bonds in the hinge region. (J Biol Chem. 2008 Jun 6; 283 (23): 16206-15.). It is not easy to manufacture a large amount as a pharmaceutical product while maintaining the heterogeneity of the target substance / related substance derived from this, and it is not easy and leads to an increase in cost. Therefore, in developing an IgG2 isotype antibody as a pharmaceutical, it is desirable that the heterogeneity derived from disulfide bonds is reduced without reducing stability.

  In order to reduce the heterogeneity of wild type IgG2, the cysteine in the hinge part of IgG2 and the cysteine present in the CH1 domain were modified. As a result of examination of various variants, among the wild type IgG2 constant region sequences, EU numbering (Sequences of proteins of immunological interest, NIH Publication No.91-3242) existing in the CH1 domain of the H chain 131st cysteine and 133rd SKSC (SEQ ID NO: 62), which is a constant region in which the arginine was modified to serine and lysine and the EU numbering 219th cysteine in the upper hinge of the H chain was changed to serine, without reducing stability. It is considered possible to reduce heterogeneity. On the other hand, as a method for reducing heterogeneity, a method in which only the EU numbering 219th cysteine present in the upper hinge of the H chain is changed to serine, and a method in which only the 220th cysteine is changed to serine can be considered. Therefore, SC (SEQ ID NO: 153), which is a constant region in which the 219th cysteine of IgG2 is changed to serine, and CS (SEQ ID NO: 153), which is a constant region in which the 220th cysteine of IgG2 is changed to serine. 154).

  As the H chain, each constant region prepared above, IgG1 (SEQ ID NO: 60), IgG2 (SEQ ID NO: 132), and variable region of humanized anti-IL-6 receptor antibody (H chain variable region huPM1-VH / sequence) No. 155, L chain variable region huPM1-VL / SEQ ID NO: 156) (Cancer Res. 1993 Feb 15; 53 (4): 851-6.) In combination, huPM1-SC (SEQ ID NO: 157), huPM1- CS (SEQ ID NO: 158), huPM1-IgG1 (SEQ ID NO: 159), huPM1-IgG2 (SEQ ID NO: 160) and huPM1-SKSC (SEQ ID NO: 161) are used, and huPM1-L (SEQ ID NO: : 162) to prepare each antibody. Each antibody was expressed and purified by the method described in Example 4.

  The heterogeneity of each antibody was compared. As a method for evaluating the heterogeneity of huPM1-IgG1, huPM1-IgG2, huPM1-SC, huPM1-CS, and huPM1-SKSC, evaluation by cation exchange chromatography was performed. Use ProPac WCX-10 (Dionex) as the column, 20 mM Sodium Acetate, pH 5.0 as mobile phase A, 20 mM Sodium Acetate, 1 M NaCl, pH 5.0 as mobile phase B, This was carried out using a gradient. The results of evaluation by cation exchange chromatography are shown in FIG.

  As a result, as shown in FIG. 12, the heterogeneity increased by converting the constant region from IgG1 to IgG2, but the heterogeneity was greatly reduced by converting the constant region to SKSC. On the other hand, when the constant region was SC, the heterogeneity was greatly reduced as in the case where the constant region was SKSC. However, when the constant region was CS, the heterogeneity was not sufficiently improved.

  In general, in order to develop an antibody as a pharmaceutical product, it is desirable to have high stability in order to prepare a stable preparation in addition to low heterogeneity. Therefore, as an evaluation method of stability, the thermal denaturation intermediate temperature (Tm value) was evaluated by differential scanning calorimetry (DSC) (VP-DSC, manufactured by Microcal). Thermal denaturation intermediate temperature (Tm value) is an indicator of stability, and it is desirable that the thermal denaturation intermediate temperature (Tm value) is high in order to produce a stable pharmaceutical product (J Pharm Sci. 2008 Apr; 97 (4): 1414-26.). Therefore, huPM1-IgG1, huPM1-IgG2, huPM1-SC, huPM1-CS, and huPM1-SKSC were dialyzed (EasySEP, TOMY) against a solution of 20 mM sodium acetate, 150 mM NaCl, pH 6.0, and about 0.1 DSC measurement was performed at a temperature increase rate of 1 ° C./min from 40 ° C. to 100 ° C. at a protein concentration of mg / mL. The resulting DSC denaturation curve is shown in FIG. 13 and the Tm value of the Fab portion is shown in Table 17 below.

  The Tm values of huPM1-IgG1 and huPM1-IgG2 were about the same, about 94 ° C (IgG2 was about 1 ° C lower), whereas the Tm values of huPM1-SC and huPM1-CS were about 86 ° C. Yes, the Tm value was significantly reduced compared to huPM1-IgG1 and huPM1-IgG2. On the other hand, the Tm value of huPM1-SKSC was about 94 ° C., which was almost equivalent to huPM1-IgG1 and huPM1-IgG2. Since huPM1-SC and huPM1-CS are significantly less stable than IgG2, it was considered that huPM1-SKSC in which the cysteine of the CH1 domain was also changed to serine was preferable for development as a pharmaceutical product. The reason why the Tm values of huPM1-SC and huPM1-CS were significantly reduced compared to IgG2 was considered to be because huPM1-SC and huPM1-CS took a different form from the disulfide bond pattern of IgG2.

  In addition, when DSC denaturation curves were compared, the denaturation peaks of the Fab portion of huPM1-IgG1 and huPM1-SKSC were sharp, whereas huPM1-SC and huPM1-CS were compared with these, The denaturation peak was broad, and huPM1-IgG2 had a shoulder peak on the low temperature side of the denaturation peak of the Fab portion. The DSC denaturation peak usually shows a sharp denaturation peak in the case of a single component, but it is considered that the denaturation peak becomes broad when there are multiple components having different Tm (that is, heterogeneity). That is, huPM1-IgG2, huPM1-SC, and huPM1-CS have a plurality of components, suggesting that huPM1-SC and huPM1-CS may not have sufficiently reduced the heterogeneity of natural IgG2. This suggests that the natural IgG2 heterogeneity involves not only the cysteine in the hinge part but also the cysteine present in the CH1 domain. To reduce heterogeneity on the DSC, the hinge It was considered necessary to modify not only the partial cysteine but also the cysteine of the CH1 domain. In addition, as described above, it is possible to have stability equivalent to that of natural IgG2 only by modifying not only the cysteine in the hinge part but also the cysteine in the CH1 domain.

  Based on the above, as constant regions with reduced heterogeneity derived from the IgG2 hinge region, SC and CS, which are constant regions in which only the cysteine in the hinge portion is replaced with serine, are insufficient in terms of heterogeneity and stability. It is considered that the heterogeneity can be significantly reduced while maintaining the same stability as IgG2 for the first time by replacing the cysteine at the 131st EU numbering in the CH1 domain with serine. It was issued. An example of such a constant region was SKSC.

[Reference Example 8] Preparation and evaluation of Fcγ receptor non-binding optimized constant region M14
As for the constant region of IgG2, EU numbering: 233, 234, 235, 236 of Fcγ receptor binding sites is non-binding type, whereas EU numbering of Fcγ receptor binding sites: 327, 330, and 331st are non-binding type IgG4. EU numbering: the amino acids 327, 330 and 331 need to be modified to the IgG4 sequence (G2Δa in Eur J Immunol. 1999 Aug; 29 (8): 2613-24). However, IgG4 is EU numbering: 339th amino acid is alanine, whereas IgG2 is threonine. EU numbering: 327, 330, 331 amino acids are naturally changed to IgG4 sequence. A new peptide sequence of 9 amino acids that can be a non-existing T-cell epitope peptide appears, resulting in the risk of immunogenicity. Thus, in addition to the above-mentioned modification, it was found that IgG2 EU numbering: it is possible to prevent the appearance of a new peptide sequence by modifying the 339th threonine to alanine. In addition to these mutations, a mutation was introduced from the 397th methionine to valine in the EU numbering of IgG2, which improves the stability of IgG2 under acidic conditions. Furthermore, SKSC (SEQ ID NO: 62), which improves the heterogeneity derived from the disulfide bond in the hinge region prepared in Reference Example 7, is a T-cell epitope peptide that does not exist in nature with the 131st and 133rd mutations. Since a new peptide sequence of 9 amino acids that could become an immunological risk arises, by introducing the EU numbering mutation 137th glutamic acid to glycine and the 138th serine to glycine mutation, The peptide sequence from the 131st to the 139th region was the same as IgG1. A constant region sequence M14 (SEQ ID NO: 129) into which all of these mutations were introduced was prepared.

Expression and purification of huPM1-M14 using huPM1-M14 as the H chain and huPM1-L (SEQ ID NO: 162) as the L chain were carried out by the method described in Reference Example 7. Evaluation of heterogeneity of the prepared huPM1-M14 (SEQ ID NO: 163) and huPM1-IgG1 and huPM1-IgG2 was carried out by the method described in Reference Example 7 using cation exchange chromatography.
As shown in FIG. 14, huPM1-M14 also showed reduced heterogeneity, similar to huPM1-SKSC.

[Reference Example 9] Production of huPM1-M58 with improved pharmacokinetics by reducing heterogeneity on the C-terminal side of the H chain
Preparation of huPM1-M58 molecule
huPM1 is an IgG1 antibody. As a heterogeneity of the H chain C-terminal sequence of IgG antibody, deletion of lysine residue of C-terminal amino acid and amidation of C-terminal amino group due to deletion of both glycine and lysine of C-terminal 2 amino acids have been reported. (Anal Biochem. 2007 Jan 1; 360 (1): 75-83.). Even in huPM1, the main component is a sequence in which the lysine of the C-terminal amino acid present on the base sequence is deleted by post-translational modification, but the C-terminal amino group due to the loss of both glycine and lysine and the secondary component in which lysine remains The amidated subcomponent of is also present as a heterogeneity. It is not easy to manufacture a large amount as a pharmaceutical product while maintaining the heterogeneity of the target substance / related substance, leading to increased costs, and it is desirable that it be a single substance as much as possible. For development, it is desirable that these heterogeneities are reduced. Therefore, it is desirable that there is no H chain C-terminal heterogeneity when developing as a pharmaceutical. In order to reduce the dose of the antibody, it is desirable to increase the plasma half-life of the antibody.

  Therefore, a new constant region with reduced heterogeneity on the C-terminal side of the H chain, improved pharmacokinetics compared with huPM1-IgG1, and reduced heterogeneity derived from wild-type IgG2 without reducing stability The following modifications were introduced for the purpose of producing:

  Specifically, EU numbering 137th glutamic acid and 138th serine glycine against huPM1-SKSC with high stability and reduced heterogeneity related to IgG2 isotype constant region antibody. In addition, 268th histidine is replaced with glutamine, 355th arginine is replaced with glutamine, 419th glutamine is replaced with glutamic acid, and in addition, 446th glycine and HuPM1-M58 (SEQ ID NO: 164) lacking the 447th lysine was found. Expression and purification of huPM1-M58 using huPM1-M58 as the H chain and huPM1-L (SEQ ID NO: 162) as the L chain were carried out by the method described in Example 4.

  Evaluation of heterogeneity of the prepared huPM1-M58, huPM1-IgG1, and huPM1-IgG2 was performed by cation exchange chromatography, and stability was evaluated by DSC, respectively, by the method described in Example 5. .

  The results of DSC are shown in Table 18. Further, as shown in FIGS. 13 and 16, also in huPM1-M58, similar to huPM1-SKSC, heterogeneity is reduced without impairing stability.

Evaluation of plasma retention of huPM1-M58
The retention of IgG molecules in plasma is long (disappears slowly) because FcRn, known as an IgG molecule salvage receptor, is functioning (Nat Rev Immunol. 2007 Sep; 7 (9): 715 -twenty five). IgG molecules taken into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions (around pH 6.0) in endosomes. IgG molecules that could not bind to FcRn proceed to lysosomes and are degraded by lysosomes, but IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma (around pH 7.4). Return to the plasma again.

  IgG1, IgG2, IgG3, and IgG4 isotypes are known as IgG type antibodies, but the plasma half-life in these humans is about 36 days for IgG1, IgG2, about 29 days for IgG3, and 16 for IgG4. (Nat Biotechnol. 2007 Dec; 25 (12): 1369-72.), And IgG1 and IgG2 are considered to have the longest retention in plasma. In general, antibody drug isotypes are IgG1, IgG2, and IgG4. As a method to further improve the pharmacokinetics of these IgG antibodies, the binding to the above-mentioned human FcRn is improved by modifying the sequence of the constant region of IgG. (J Biol Chem. 2007 Jan 19; 282 (3): 1709-17, J Immunol. 2006 Jan 1; 176 (1): 346-56).

  Because there are species differences between mouse FcRn and human FcRn (Proc Natl Acad Sci US A. 2006 Dec 5; 103 (49): 18709-14), the retention of IgG antibodies with modified constant region sequences in human plasma To predict human FcRn binding and plasma retention in human FcRn transgenic mice (Int Immunol. 2006 Dec; 18 (12): 1759-69) .

Evaluation of binding to human FcRn
FcRn is a complex of FcRn and β2-microglobulin. Oligo DNA primers were prepared based on the published human FcRn gene sequence (J. Exp. Med. 180 (6), 2377-2381 (1994)). A human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) was used as a template, and a DNA fragment encoding the entire gene length was prepared by PCR using the prepared primer. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular region (Met1-Leu290) including a signal region was amplified by PCR and inserted into an animal cell expression vector (human FcRn amino acid sequence / SEQ ID NO: 165). ). Similarly, oligo DNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. USA 99 (26), 16899-16903 (2002)). Using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as a template, a DNA fragment encoding the entire gene length was prepared by PCR using the prepared primers. Using the obtained DNA fragment as a template, a DNA fragment encoding β2-microglobulin full length (Met1-Met119) including the signal region was amplified by PCR and inserted into an animal cell expression vector (human β2-microglobulin amino acid sequence / sequence) Number: 166).

  Soluble human FcRn is expressed by lipofection method using human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) and 10% Fetal Bovine Serum (Invitrogen). did. The obtained culture supernatant was collected and purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) according to the method of (J Immunol. 2002 Nov 1; 169 (9): 5171-80.). Thereafter, purification was performed using HiTrap Q HP (GE Healthcare).

  Biacore 3000 was used to evaluate the binding to human FcRn, and human FcRn was interacted with human FcRn as an analyte to protein L immobilized on the sensor chip or to the antibody bound to rabbit anti-human IgG Kappa chain antibody. The affinity (KD) was calculated from the amount of binding. Specifically, Protein L was immobilized on sensor chip CM5 (BIACORE) by an amine coupling method using 50 mM Na-phosphate buffer containing 150 mM NaCl, pH 6.0 as a running buffer. Thereafter, the antibody was diluted with a running buffer containing 0.02% Tween20 and injected to bind the antibody to the chip. Then, human FcRn was injected, and the binding of the antibody to human FcRn was evaluated.

  Affinity was calculated using software, BIAevaluation. From the obtained sensorgram, the amount of hFcRn binding to the antibody immediately before the end of human FcRn injection was determined, and this was fitted by the steady state affinity method to calculate the affinity of human FcRn to the antibody.

Predictive evaluation of plasma retention in humans using human FcRn of huPM1-IgG1 and huPM1-M58
The binding of huPM1-IgG1 and huPM1-M58 to human FcRn was evaluated by BIAcore. As shown in Table 19, the binding of huPM1-M58 was about 1.4 times better than huPM1-IgG1.

Evaluation of plasma retention in human FcRn transgenic mice Evaluation of pharmacokinetics in human FcRn transgenic mice (B6.mFcRn-/-. HFcRn Tg line 276 + / + mice, Jackson Laboratories) was performed as follows. The antibody was administered intravenously at a dose of 1 mg / kg to mice and blood was collected in a timely manner. The collected blood was immediately centrifuged at 4 ° C. and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set to −20 ° C. or lower until measurement was performed. Plasma concentrations were measured using the ELISA method.

Predictive evaluation of plasma retention in humans using huPM1-IgG1 and huPM1-M58 human FcRn transgenics
The plasma retention of huPM1-IgG1 and huPM1-M58 in human FcRn transgenic mice was evaluated. As a result, as shown in FIG. 17, huPM1-M58 was confirmed to have improved pharmacokinetics as compared to huPM1-IgG1. It was suggested that the binding to human FcRn and plasma retention in human FcRn transgenic mice were correlated.

[Reference Example 10] Affinity measurement of antigen-antibody reaction using Biacore
Kinetic analysis of antigen-antibody reaction was performed using Biacore T100 (GE Healthcare Bioscience). Rec-Protein A (ZYMED) (hereinafter referred to as Protein A) was immobilized on the sensor chip, the antibody was captured by the immobilized Protein A, and the antigen was reacted as an analyte, and the interaction between the antibody and the antigen was measured. . As the antigen, rhNR10 prepared at various concentrations was used. The association rate constant k _a (1 / Ms) and dissociation rate constant k _d (1 / s), which are kinetic parameters, are calculated from the sensorgram obtained by the measurement, and K _D (M ) Was calculated. Each parameter was calculated using Biacore T100 Evaluation Software version 1.1 (GE Healthcare Bioscience).

Protein A immobilized on the sensor chip Protein A was immobilized on all flow cells of the sensor chip CM5 (GE Healthcare Bioscience) by the amine coupling method. The running buffer was HBS-EP + (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20), and the experiment was performed at a flow rate of 10 μL / min. The carboxyl group of carboxymethyldextran on the sensor chip is 100 μL of a 1: 1 mixture of 75 mg / mL EDC (N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 11.5 mg / mL NHS (N-hydroxysuccinimide). Then, Protein A prepared at 50 μg / mL with 10 mM acetate buffer (pH 4.5) was allowed to flow therethrough for reaction. Thereafter, 100 μL of 1 M ethanolamine hydrochloride (pH 8.5) was flowed to inactivate unreacted active groups. Finally, about 4000-5000 RU was immobilized. All experiments were performed at 25 ° C.

HBS-EP + was used as an affinity measurement running buffer for antigen-antibody reaction between antibody captured by Protein A and rhNR10 . Each antibody was prepared to bind to 0.25 μg / mL or about 100 RU of Protein A. RhNR10 used as the analyte was prepared to 0, 38.5, 77.0, 154 nM, or 0, 19.25, 77.01 nM using HBS-EP +. In the measurement, the antibody solution was first captured by Protein A, and the analyte solution was reacted for 3 min at a flow rate of 20 μL / min, then switched to HBS-EP + and the 5 min dissociation phase was measured. After measurement of the dissociation phase, the sensor chip was regenerated by washing with 10 mM glycine-HCl (pH 1.5). From the obtained sensorgram, kinetic analysis was performed using Biacore T100 Evaluation Software Version 1.1, which is Biacore's dedicated data analysis software.

  The anti-NR10 antibody obtained by the present inventor exhibits effective neutralizing activity against NR10, and is useful, for example, as a therapeutic agent for inflammatory diseases.

Claims (5)

The anti-NR10 antibody according to any one of the following (1) to (12);
(1) an antibody (H28L17) comprising a heavy chain variable region (H28) having the amino acid sequence set forth in SEQ ID NO: 206 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(2) an antibody (H30L17) comprising a heavy chain variable region (H30) having the amino acid sequence set forth in SEQ ID NO: 207 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(3) an antibody (H34L17) comprising a heavy chain variable region (H34) having the amino acid sequence set forth in SEQ ID NO: 208 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(4) an antibody (H42L17) comprising a heavy chain variable region (H42) having the amino acid sequence set forth in SEQ ID NO: 209 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(5) an antibody (H44L17) comprising a heavy chain variable region (H44) having the amino acid sequence set forth in SEQ ID NO: 210 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(6) an antibody (H46L17) comprising a heavy chain variable region (H46) having the amino acid sequence set forth in SEQ ID NO: 211 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(7) an antibody (H57L17) comprising a heavy chain variable region (H57) having the amino acid sequence set forth in SEQ ID NO: 212 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(8) an antibody (H71L17) comprising a heavy chain variable region (H71) having the amino acid sequence set forth in SEQ ID NO: 213 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(9) an antibody (H78L17) comprising a heavy chain variable region (H78) having the amino acid sequence set forth in SEQ ID NO: 214 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(10) an antibody (H92L17) comprising a heavy chain variable region (H92) having the amino acid sequence set forth in SEQ ID NO: 215 and a light chain variable region (L17) having the amino acid sequence set forth in SEQ ID NO: 220;
(11) an antibody (H97L50) comprising a heavy chain variable region (H97) having the amino acid sequence set forth in SEQ ID NO: 216 and a light chain variable region (L50) having the amino acid sequence set forth in SEQ ID NO: 221;
(12) An antibody (H98L50) comprising a heavy chain variable region (H98) having the amino acid sequence set forth in SEQ ID NO: 217 and a light chain variable region (L50) having the amino acid sequence set forth in SEQ ID NO: 221. A humanized antibody, anti-NR10 antibody of claim 1. Any of the following anti-NR10 antibodies (1) to (12);
(1) an antibody (H28L17) comprising a heavy chain (H28) having the amino acid sequence set forth in SEQ ID NO: 224 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238;
(2) an antibody (H30L17) comprising a heavy chain (H30) having the amino acid sequence set forth in SEQ ID NO: 225 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238;
(3) an antibody (H34L17) comprising a heavy chain (H34) having the amino acid sequence set forth in SEQ ID NO: 226 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238,
(4) an antibody (H42L17) comprising a heavy chain (H42) having the amino acid sequence set forth in SEQ ID NO: 227 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238,
(5) an antibody (H44L17) comprising a heavy chain (H44) having the amino acid sequence set forth in SEQ ID NO: 228 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238,
(6) an antibody (H46L17) comprising a heavy chain (H46) having the amino acid sequence set forth in SEQ ID NO: 229 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238,
(7) an antibody (H57L17) comprising a heavy chain (H57) having the amino acid sequence set forth in SEQ ID NO: 230 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238,
(8) an antibody (H71L17) comprising a heavy chain (H71) having the amino acid sequence set forth in SEQ ID NO: 231 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238,
(9) an antibody (H78L17) comprising a heavy chain (H78) having the amino acid sequence set forth in SEQ ID NO: 232 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238;
(10) an antibody (H92L17) comprising a heavy chain (H92) having the amino acid sequence set forth in SEQ ID NO: 233 and a light chain (L17) having the amino acid sequence set forth in SEQ ID NO: 238;
(11) an antibody (H97L50) comprising a heavy chain (H97) having the amino acid sequence set forth in SEQ ID NO: 234 and a light chain (L50) having the amino acid sequence set forth in SEQ ID NO: 239,
(12) An antibody (H98L50) comprising a heavy chain (H98) having the amino acid sequence set forth in SEQ ID NO: 235 and a light chain (L50) having the amino acid sequence set forth in SEQ ID NO: 239. The pharmaceutical composition containing the antibody in any one of Claims 1-3 . The pharmaceutical composition according to claim 4 , which is a therapeutic agent for inflammatory diseases.

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