WO2021241730A1

WO2021241730A1 - Therapeutic antibody for inflammatory bowel disease

Info

Publication number: WO2021241730A1
Application number: PCT/JP2021/020388
Authority: WO
Inventors: 千草吉村; 佑介須知; 充志溝口
Original assignee: 第一三共株式会社; 学校法人久留米大学
Priority date: 2020-05-29
Filing date: 2021-05-28
Publication date: 2021-12-02
Also published as: JPWO2021241730A1; TW202200623A

Abstract

Provided is a novel therapeutic drug for inflammatory bowel disease, etc.　An antibody that has the following characteristics (i) to (iv) or an antigen-binding fragment thereof: (i) being a monoclonal antibody binding to human IL-22BP; (ii) binding to monkey IL-22BP; (iii) having a binding dissociation constant (KD) for human IL-22BP-His of 1x10^-7M or less; and (iv) when STAT3 phosphorylation signals disappear due to the addition of IL-22 and IL-22BP in cells derived from human large intestinal crypt epithelial cells, a simultaneous or preliminary treatment with the antibody resulting in the recovery of the signals.

Description

Therapeutic antibody against inflammatory bowel disease

The present invention is an antibody that binds to a soluble cytokine receptor (IL-22BP), an antibody that exerts a medicinal effect by activating an IL-22 signal attenuated by IL-22BP, and a pharmaceutical composition containing the antibody. Regarding things.

Cytokines are generally important for cell differentiation, proliferation, and migration, and because they play a protective function against tumors and infections, they are deeply involved in chronic inflammatory diseases, wound healing, infectious diseases, and cancer. Interleukin-22 (IL-22) is one of the cytokines induced in immune cells when infected with pathogenic bacteria, and is commonly found in human lungs, skin, tonsils, uterus, and intestinal tract (small intestine, large intestine). It is known that it is expressed and secreted in existing T cells, Natural Killer cells, dendritic cells and the like (Non-Patent Document 1). In particular, IL-22 is strongly expressed in the intestinal tract during enteritis pathology, acts on intestinal epithelial cells expressing the IL-22 receptor (IL-22R), and is a signal of STAT3 (Signal Transducer and Activator of STAT3). It has the effect of inducing the production of mucus and antibacterial proteins in intestinal epithelial cells and suppressing inflammation (Non-Patent Document 1). It has been suggested that IL-22 dysfunction is associated with infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, etc., and ulcerative colitis and Crohn's disease. It is well known that it is involved in inflammatory bowel disease including (Non-Patent Documents 1 and 2).

IL-22BP is a soluble cytokine receptor, also called IL-22Ra2, CDR2-10, etc., and is known to bind to IL-22 with high affinity and inhibit its function (non-patent).

Documents

3, 4, 5).

IL-22BP is expressed in the placenta, skin, lungs, intestinal tract, etc., and from some animal models and human clinical data, IL-22BP may directly inhibit the mucosal protective effect of IL-22 in the intestinal tract. It has been suggested (Non-Patent Documents 2 and 6).

Although there are studies (Patent Documents 1 and 2) for the prevention, treatment, and diagnosis of diseases by inhibiting the binding between IL-22BP and IL-22 with an anti-IL-22BP antibody, the target disease is obesity. Is it limited to metabolic disorders such as diabetes and dyslipidemia and has only evaluated the action of rabbit anti-mouse IL-22BP polyclonal antibody (Patent Document 1), or IL- in the treatment of inflammatory bowel disease due to microbial infection? It merely suggested that the expression and hyperactivity of 22 may lead to treatment (Patent Document 2).

International release number WO 2007/076422 European application publication number EP2514436A2

The present invention relates to inflammation including infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune myocardial disease, bronchitis, uveitis, ulcerative colitis and Crohn's disease, which are suggested to be associated with IL-22. It is an object of the present invention to provide an anti-IL-22BP antibody that makes it possible to treat sexual bowel disease by enhancing the function of IL-22, a pharmaceutical composition containing the antibody as an active ingredient, and the like.

The present inventors have studied IL-22BP and found that the function of IL-22 is enhanced by inhibiting the binding between IL-22BP and IL-22 by an anti-IL-22BP antibody.

That is, the present invention is as follows.
[1] An antibody or a binding fragment thereof having the properties described in (i) to (iv) below:
(I) A monoclonal antibody that binds to human IL-22BP;
(Ii) Binds to monkey IL-22BP;
(Iii) The binding dissociation constant (KD) to human IL-22BP-His is 1 × ^10-7 M or less; and (iv) IL-22 and IL-22BP in cells derived from the human colonic epithelial cell crypt site. Simultaneous or pretreatment with respect to the disappearance of the phosphorylation signal of STAT3 observed at the time of addition restores the signal.
[2] The antibody of [1] or a binding fragment thereof, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.
[3] An antibody of [1] or [2] or a binding fragment thereof that does not cross IL-22BP of rodents.
[4] The antibody or binding fragment thereof according to any one of [1] to [3], wherein the binding dissociation constant (KD) for human IL-22BP-His is 1 × 10 ^{-7 M or less in the SPR method.}
[5] In the BLI method, the binding dissociation constant (KD) for monkey IL-22BP-His is 1 × 10 ^-6 M or less, preferably 5 × 10 ^-7 M or less, [1] to [4]. ] Any antibody or binding fragment thereof.
[6] (a1) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
(B1) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and (c1) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 29.
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d1) SEQ ID NO: 57,
(E1) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 58, and (f1) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 59.
An antibody according to any one of [1] to [5], or a binding fragment thereof, comprising a heavy chain containing.
[7] (a2) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 30.
(B2) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 31, and (c2) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 32.
A light chain comprising, and a heavy chain CDRH1 consisting of (d2) the amino acid sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 105,
(E2) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 61 or SEQ ID NO: 106, and (f2) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 107.
An antibody according to any one of [1] to [5], or a binding fragment thereof, comprising a heavy chain containing.
[8] (a3) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 33,
(B3) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and (c3) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 35,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d3) SEQ ID NO: 63,
(E3) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 64, and (f3) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 65.
An antibody of [1] or [2], or a binding fragment thereof, comprising a heavy chain containing.
[9] (a4) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 36,
(B4) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and (c4) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 38,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d4) SEQ ID NO: 66,
(E4) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 67, and (f4) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 68.
An antibody of [1] or [2], or a binding fragment thereof, comprising a heavy chain containing.
[10] (a5) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 39,
(B5) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 40, and (c5) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 41,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d5) SEQ ID NO: 69,
(E5) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 70, and (f5) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 71.
An antibody according to any one of [1] to [3], or a binding fragment thereof, comprising a heavy chain containing.
[11] (a6) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 42,
(B6) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 43, and (c6) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d6) SEQ ID NO: 72,
(E6) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 73, and (f6) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 74.
An antibody according to any one of [1] to [3], or a binding fragment thereof, comprising a heavy chain containing.
[12] The antibody according to any one of [1] to [6] or a bound fragment thereof, wherein the antibody is a humanized antibody.
[13] An antibody according to any one of [1] to [5] and [7] or a binding fragment thereof, wherein the antibody is a humanized antibody.
[14] The antibody of any one of [1], [2] and [8] or a binding fragment thereof, wherein the antibody is a humanized antibody.
[15] The antibody of any one of [1], [2] and [9] or a binding fragment thereof, wherein the antibody is a humanized antibody.
[16] An antibody according to any one of [1] to [3] and [10] or a binding fragment thereof, wherein the antibody is a humanized antibody.
[17] An antibody according to any one of [1] to [3] and [11] or a binding fragment thereof, wherein the antibody is a humanized antibody.
[18] Containing any one light chain variable region selected from (La1)-(Le1) below and any one heavy chain variable region selected from (Ha1)-(Hc1) [1]. ] To any of [6] and [12] antibodies or binding fragments thereof:
(La1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 121.
(Lb1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122.
(Lc1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 123.
(Ld1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 124.
(Le1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 125,
(Ha1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 138.
(Hb1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 139.
(Hc1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 140.
[19] Containing any one light chain variable region selected from (La2)-(Le2) below and any one heavy chain variable region selected from (Ha2)-(Hc2) [1]. ] Antibodies or binding fragments thereof:
(La2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 121.
(Lb2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122.
(Lc2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 123.
(Ld2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 124.
(Le2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 125.
(Ha2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 138.
(Hb2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 139.
(Hc2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 140.
[20] Containing any one light chain variable region selected from (Lf1) to (Lk1) below and any one heavy chain variable region selected from (Hd1) to (Hf1) [1]. ] To [5], [7] and [13] antibody or binding fragment thereof:
(Lf1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 126,
(Lg1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 127,
(Lh1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 128,
(Li1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 129.
(Lj1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 130.
(Lk1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 131.
(Hd1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 141.
(He1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 142.
(Hf1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 143.
[21] Containing any one light chain variable region selected from (Lf2)-(Lk2) below and any one heavy chain variable region selected from (Hd2)-(Hf2) below [1] ] Antibodies or binding fragments thereof:
(Lf2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 126.
(Lg2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 127.
(Lh2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128.
(Li2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 129.
(Lj2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 130.
(Lk2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 131.
(Hd2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 141.
(He2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 142.
(Hf2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143.
[22] Any of [1] to [6], [12] and [18], which comprises a combination of any heavy chain variable region and light chain variable region selected from (X1) to (X3) below. Antibodies or binding fragments thereof:
(X1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the sequence listing. Variable area,
(X2) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 123 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the sequence listing. Variable area,
(X3) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 140 in the sequence listing. Variable area.
[23] The antibody of [1] or a binding fragment thereof, which comprises a combination of any heavy chain variable region and light chain variable region selected from (X1') to (X3') below:
(X1') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 132.
(X2') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 123 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 132.
(X3') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 140 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 132.
[24] [1] to [5], [7], [13], wherein the antibody comprises a combination of any heavy chain variable region and light chain variable region selected from (X4) to (X6) below. ] And [20] antibody or binding fragment thereof:
(X4) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the sequence listing. Variable area,
(X5) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the sequence listing. Variable area,
(X6) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 130 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 142 in the sequence listing. Variable area.
[25] The antibody of [1] or a binding fragment thereof, wherein the antibody comprises a combination of any heavy chain variable region and light chain variable region selected from (X4') to (X6') below:
(X4') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 137.
(X5') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 137.
(X6') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 130 in the sequence listing, and the amino acid of SEQ ID NO: 142 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 137.
[26] [1] to [6], comprising any one light chain selected from the following (Ll1) to (Lp1) and any one heavy chain selected from (Hg1) to (Hi1). , [12] and [18]:
(Ll1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 121 in the sequence listing.
(Lm1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing.
(Ln1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing.
(Lo1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 124 in the sequence listing.
(Lp1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 125 in the sequence listing.
(Hg1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing.
(Hh1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 139 in the sequence listing.
(Hi1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing.
[27] The antibody of [1], which comprises any one light chain selected from the following (Ll2) to (Lp2) and any one heavy chain selected from (Hg2) to (Hi2):
(Ll2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 121 in the sequence listing.
(Lm2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing.
(Ln2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing.
(Lo2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 124 in the sequence listing.
(Lp2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 125 in the sequence listing.
(Hg2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing.
(Hh2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 139 in the sequence listing.
(Hi2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing.
[28] [1] to [5] comprising any one light chain selected from the following (Lq1) to (Lv1) and any one heavy chain selected from (Hj1) to (Hl1). , [7], [13] and [20]:
(Lq1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing.
(Lr1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 127 in the sequence listing.
(Ls1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing.
(Lt1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 129 in the sequence listing.
(Lu1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing.
(Lv1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 131 in the sequence listing.
(Hj1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 467 of SEQ ID NO: 141 in the sequence listing.
(Hk1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the sequence listing.
(Hl1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing.
[29] The antibody of [1], which comprises any one light chain selected from the following (Lq2) to (Lv2) and any one heavy chain selected from (Hj2) to (Hl2):
(Lq2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing.
(Lr2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 127 in the sequence listing.
(Ls2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing.
(Lt2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 129 in the sequence listing.
(Lu2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing.
(Lv2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 131 in the sequence listing.
(Hj2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 141 in the sequence listing.
(Hk2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the sequence listing.
(Hl2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing.
[30] Any antibody of [1] to [6], [12], [18] and [22], which comprises any of the heavy and light chains selected from the following (Y1) to (Y3). :
(Y1) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing. Heavy chain,
(Y2) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing. Heavy chain,
(Y3) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing. Heavy chain.
[31] The antibody of [1], which comprises any of the heavy and light chains selected from the following (Y1') to (Y3'):
(Y1') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 462.
(Y2') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 462.
(Y3') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 140 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 462.
[32] of [1], which binds to a site on human IL-22BP to which the antibody of [30] binds, or competes with the antibody of [30] or a binding fragment thereof in binding to human IL-22BP. Antibodies or binding fragments thereof.
[33] Of [1] to [5], [7], [13], [20] and [24], which comprises any of the heavy and light chains selected from the following (Y4) to (Y6). Either antibody:
(Y4) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing. Heavy chain,
(Y5) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing. Heavy chain,
(Y6) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the sequence listing. Heavy chain.
[34] The antibody of [1], which comprises any of the heavy and light chains selected from the following (Y4') to (Y6'):
(Y4') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 467.
(Y5') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 467.
(Y6') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing, and the amino acid of SEQ ID NO: 142 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 467.
[35] It binds to a site on human IL-22BP to which the antibody of [33] or a binding fragment thereof binds, or competes with the antibody of [33] or a binding fragment thereof in binding to human IL-22BP. The antibody of [1] or a binding fragment thereof.
[36] The antibody or binding fragment thereof according to any one of [1] to [35], wherein the heavy chain lacks the carboxyl-terminal lysine.
[37] A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in the heavy chain and the light chain of the antibody of the antibody of [1] to [36].
[38] A vector containing the polynucleotide of [37].
[39] A host cell containing the polynucleotide of [37] or the vector of [38].
[40] A method for producing an antibody or a binding fragment thereof according to any one of [1] to [36], which comprises culturing the host cell of [39] and recovering the antibody or the binding fragment thereof from the culture.
[41] An antibody or a binding fragment thereof prepared by the method of [40].
[42] The antibody or binding fragment thereof according to any one of [1] to [36], which forms a complex with a drug.
[43] The antibody or binding fragment thereof according to any one of [1] to [36], which is contained in a multi-characteristic molecule.
[44] The active ingredient is an antibody of any of [1] to [36] and [41] to [43] or a binding fragment thereof, a polynucleotide of [37], a vector of [38], or a cell of [39]. Pharmaceutical composition containing as.
[45] Prevention or prevention of one or more diseases selected from the group consisting of infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, and inflammatory bowel disease. The pharmaceutical composition of [44], which is a therapeutic agent.
[46] The pharmaceutical composition of [45], wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
[47] The pharmaceutical composition according to any one of [44] to [46], which is used in combination with other pharmaceutical products.
[48] A test agent, diagnostic agent or reagent for detecting human IL-22BP, which comprises an antibody of any one of [1] to [36] and [41] to [43] or a binding fragment thereof.
This specification includes the disclosure content of Japanese Patent Application No. 2020-095058, which is the basis of the priority of the present application.

By inhibiting the binding of IL-22BP and IL-22 with an anti-IL-22BP antibody, the function of IL-22 is enhanced.

Inflammatory bowel diseases including infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune myocardial disease, bronchitis, uveitis, ulcerative colitis and Crohn's disease that are suggested to be associated with IL-22. , IL-22 can be treated by enhancing its function.

It shows the binding activity to human IL-22BP-His, mouse IL-22BP-His, and monkey IL-22BP-His using the ELISA method. The binding activity of rat anti-human IL-22BP antibody (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) to human IL-22BP-His, mouse IL-22BP-His, and monkey IL-22BP-His was evaluated. The vertical axis shows the absorbance at the 490 nm wavelength measured by the ELISA method. The binding activity of the humanized anti-human IL-22BP antibody to human IL-22BP is shown as a dissociation constant. The binding activity of the humanized anti-human IL-22BP antibody to monkey IL-22BP is shown as a dissociation constant. The results of the competitive ELISA method are shown. Humanized anti-human IL-22BP antibody ("hMAb3_H01L03" "hMAb3_H01L04" "hMAb3_H04L03" "hMAb4_H03L01" "hMAb4_H03L03" "hMAb4_H02L05") and human chimeric antibody (by "cMAb3", "cMAb22") Inhibition was performed by a competitive ELISA method. The vertical axis shows the inhibition rate, and the horizontal axis shows the concentration of the added antibody. The inhibition rate was calculated by the following formula. Inhibition rate (%) = (1-maximum absorbance Abs490 when antibody was added / maximum absorbance Abs490 when IL-22 was added alone) × 100 Control IgG (ctrlIgG), HBSor pH 6.0 was used for control. The detection result of phosphorylated STAT3 using the human large intestine epithelial cell crypt is shown. Evaluation was performed using humanized anti-human IL-22BP antibody (“hMAb3_H01L03”, “hMAb3_H01L04”, “hMAb3_H02L05”, “hMAb4_H02L05”, “hMAb4_H03L01”, “hMAb4_H03L03”). It is a figure which shows the sequence of SEQ ID NO: 1 and 2 (primer Dneo-F, Dneo-R), and SEQ ID NO: 3 (FLAGHis tag sequence). It is a figure which shows the sequence of SEQ ID NO: 4 and 5. It is a figure which shows the sequence of SEQ ID NO: 6 and 7. It is a figure which shows the arrangement of SEQ ID NO: 8. It is a figure which shows the arrangement of SEQ ID NO: 9. It is a figure which shows the arrangement of SEQ ID NO: 10. It is a figure which shows the arrangement of SEQ ID NO: 11. It is a figure which shows the arrangement of SEQ ID NO: 12. It is a figure which shows the arrangement of SEQ ID NO: 13. It is a figure which shows the arrangement of SEQ ID NO: 14. It is a figure which shows the sequence of SEQ ID NO: 15-17. It is a figure which shows the arrangement of SEQ ID NO: 18-20. It is a figure which shows the arrangement of SEQ ID NO: 21-26. It is a figure which shows the sequence of SEQ ID NO: 27-38 (CDR of VL of an anti-IL-22BP antibody). It is a figure which shows the sequence of SEQ ID NO: 39-44 (CDR of VL of an anti-IL-22BP antibody). It is a figure which shows the sequence of SEQ ID NO: 45-47. It is a figure which shows the sequence of SEQ ID NO: 48-50. It is a figure which shows the arrangement of SEQ ID NO: 51-56. It is a figure which shows the sequence of SEQ ID NO: 57-68 (CDR of VH of anti-IL-22BP antibody). It is a figure which shows the sequence of SEQ ID NO: 69-74 (CDR of VH of anti-IL-22BP antibody). It is a figure which shows the arrangement of SEQ ID NO: 75. It is a figure which shows the sequence of SEQ ID NO: 76 and 77 (primer 3.3-F1, 3.3-R1). It is a figure which shows the arrangement of SEQ ID NO: 78. It is a figure which shows the arrangement of SEQ ID NO: 79. It is a figure which shows the arrangement of SEQ ID NO: 80. It is a figure which shows the arrangement of SEQ ID NO: 81. It is a figure which shows the arrangement of SEQ ID NO: 82. It is a figure which shows the arrangement of SEQ ID NO: 83. It is a figure which shows the arrangement of SEQ ID NO: 84. It is a figure which shows the arrangement of SEQ ID NO: 85-87. It is a figure which shows the sequence of SEQ ID NO: 88-90. It is a figure which shows the sequence of SEQ ID NO: 91 and 92 (primer CM-LKF, KCL-Inf-R). It is a figure which shows the arrangement of SEQ ID NO: 93. It is a figure which shows the arrangement of SEQ ID NO: 94. It is a figure which shows the arrangement of SEQ ID NO: 95. It is a figure which shows the arrangement of SEQ ID NO: 96. It is a figure which shows the arrangement of SEQ ID NO: 97. It is a figure which shows the arrangement of SEQ ID NO: 98. It is a figure which shows the sequence of SEQ ID NO: 99 and 100. It is a figure which shows the arrangement of SEQ ID NO: 101 and 102. It is a figure which shows the arrangement of SEQ ID NO: 103 and 104. It is a figure which shows the sequence of SEQ ID NO: 105-107 (CDR of VH of an anti-IL-22BP antibody). It is a figure which shows the sequence of SEQ ID NO: 108 and 109 (primer EG-Inf-F, EG1-Inf-R). It is a figure which shows the arrangement of SEQ ID NO: 110. It is a figure which shows the arrangement of arrangement number 111. It is a figure which shows the arrangement of SEQ ID NO: 112. It is a figure which shows the arrangement of SEQ ID NO: 113. It is a figure which shows the arrangement of SEQ ID NO: 114. It is a figure which shows the arrangement of SEQ ID NO: 115. It is a figure which shows the arrangement of SEQ ID NO: 116. It is a figure which shows the arrangement of SEQ ID NO: 117. It is a figure which shows the arrangement of SEQ ID NO: 118. It is a figure which shows the arrangement of SEQ ID NO: 119. It is a figure which shows the arrangement of SEQ ID NO: 120. It is a figure which shows the arrangement of SEQ ID NO: 121-123. It is a figure which shows the sequence of SEQ ID NO: 124-126. It is a figure which shows the sequence of SEQ ID NO: 127-129. It is a figure which shows the arrangement of SEQ ID NO: 130 and 131. It is a figure which shows the arrangement of SEQ ID NO: 132. It is a figure which shows the arrangement of SEQ ID NO: 133. It is a figure which shows the arrangement of SEQ ID NO: 134. It is a figure which shows the arrangement of SEQ ID NO: 135. It is a figure which shows the arrangement of SEQ ID NO: 136. It is a figure which shows the arrangement of SEQ ID NO: 137. It is a figure which shows the sequence of SEQ ID NO: 138 and 139. It is a figure which shows the sequence of SEQ ID NO: 140 and 141. It is a figure which shows the sequence of SEQ ID NO: 142 and 143.

The present invention is an antibody that binds to a soluble cytokine receptor (IL-22BP).
1. 1. Definition In the present invention, "gene" means a nucleic acid molecule containing a nucleotide sequence encoding an amino acid of a protein or a complementary strand thereof, and for example, a nucleotide sequence encoding an amino acid of a protein or a nucleotide complementary to the sequence thereof. Polynucleotides, oligonucleotides, DNAs, mRNAs, cDNAs, cRNAs, etc. having sequences are included in the meaning of "gene". Such genes are single-stranded, double-stranded or triple-stranded or higher nucleotides, which are aggregates of DNA strands and RNA strands, and ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed on one nucleotide strand. Nucleotides and double-stranded or triple-stranded or higher nucleotides containing such nucleotide chains are also included in the meaning of "gene". Examples of the "IL-22BP gene" of the present invention include DNA, mRNA, cDNA, cRNA and the like containing a nucleotide sequence encoding the amino acid sequence of the IL-22BP protein.

In the present invention, "nucleotide", "nucleic acid" and "nucleic acid molecule" are synonymous, and for example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer and the like are also included in their meanings. Such a nucleotide is a nucleotide consisting of a single strand, a double strand, or three or more strands, and an aggregate of a DNA strand and an RNA strand, a ribonucleotide (RNA) and a deoxyribonucleotide (DNA) on one nucleotide strand. Mixtures and aggregates of double or three or more strands containing such nucleotide chains are also included in the meaning of "nucleotide".

In the present invention, "polypeptide", "peptide" and "protein" are synonymous.

In the present invention, "protein" refers to "protein" from any vertebrate source, including mammals such as primates (eg humans and monkeys) and rodents (eg mice and rats), unless otherwise noted. ..

In the present invention, "antigen" may be used to mean "immunogen".

In the present invention, the "cell" also includes various cells derived from individual animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, microorganisms and the like.

2. 2. Antigen protein The IL-22BP protein as an immunogen can be chemically synthesized based on sequence information, or can be obtained as a recombinant protein by a known method based on the DNA sequence information encoding the protein.

3. 3. Antibodies In the present invention, both the antibody that binds to IL-22BP and the antibody that recognizes IL-22BP may be referred to as "anti-IL-22BP antibody" or abbreviated as "IL-22BP antibody". Anti-IL-22BP antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies and the like.

The monoclonal antibody used in the present invention immunizes mammals such as mice, rats, rabbits, hamsters, guinea pigs, horses, monkeys, dogs, pigs, cows, goats, and sheep with the IL-22BP protein or a fragment thereof as an immunogen. A hybridoma can be produced by fusing spleen cells or the like with myeloma, and can be obtained as an antibody produced and secreted by the hybridoma. Hybridomas can be produced by known methods.

The antibody can be screened by any method, but preferably, it may be screened by ELISA in which the IL-22BP protein is immobilized.

The "functional fragment of an antibody" in the present invention means an antibody fragment that plays at least a part of the function of the original antibody. Examples of the "functional fragment of the antibody" include, but are not limited to, Fab, F (ab') 2, scFv, Fab', single-chain immunoglobulin and the like. The functional fragment of such an antibody is a recombinant protein produced in a suitable host cell using a recombinant gene in addition to the one obtained by treating the full-length molecule of the antibody protein with an enzyme such as papain or pepsin. May be. Among the "functional fragments of an antibody", those having an antigen-binding activity to an antigen, that is, human IL-22BP, are referred to as an "antibody-binding fragment".

In the present invention, the "site" to which the antibody binds, that is, the "site" recognized by the antibody means a partial peptide or a partial higher-order structure on the antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or an antibody binding site. Examples of the site on the IL-22BP protein to which the anti-IL-22BP antibody of the present invention binds or recognizes include a partial peptide or a partial higher-order structure on the IL-22BP protein.

It is known that the heavy chain and the light chain of the antibody molecule each have three complementarity determining regions (CDRs). Complementarity determining regions, also called hypervariable domains, are sites within the variable regions of the heavy and light chains of an antibody, where the primary structure variability is particularly high, of the heavy and light chains. On the primary structure of the polypeptide chain, they are usually separated into three locations. In the present invention, the complementarity determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminal side of the heavy chain amino acid sequence, and the complementarity determining regions of the light chain are represented by light chain amino acids. From the amino terminal side of the sequence, they are referred to as CDRL1, CDRL2, and CDRL3. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to which they bind. Heavy chain variable region The part other than CDRH1 to CDRH3 in the amino acid sequence is called the framework region (FR: Framework Region), from the amino terminus to before CDRH1, from after CDRH1 to before CDRH2, from after CDRH2 to CDRH3. The area up to this side and from the position following CDRH3 to the carboxyl terminus are referred to as FRH1 to FRH4, respectively. Similarly, the portion of the light chain variable region amino acid sequence other than CDRL1 to CDRL3 is also FR, from the amino terminus to the front of CDRL1, from the end of CDRL1 to the front of CDRL2, from the next to CDRL2 to the front of CDRL3, and The sequence from the CDRL3 to the carboxyl terminus is referred to as FRL1 to FRL4, respectively. That is, in the variable region (amino acid sequence) of the heavy chain and the light chain, the amino terminus is in the order of FRH1-CDRH1-FRH2-CRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDR3-FRL4. They are lined up continuously from the side toward the carboxyl terminus.

In the present invention, the "antibody variant" has an amino acid sequence in which an amino acid is substituted, deleted, added and / or inserted (hereinafter collectively referred to as "mutation") in the amino acid sequence of the original antibody. And means a polypeptide that binds to the IL-22BP protein of the present invention. The number of mutant amino acids in such antibody variants is 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 From 9, 1 to 10, 1 to 12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50. Such antibody variants are also included in the "antibodies" of the present invention.

In the present invention, "several pieces" in "1 to several pieces" means 3 to 10 pieces.

The antibody used in the present invention also includes a modified antibody. The modified product means an antibody according to the present invention that has been chemically or biologically modified. Chemical modifications include those having a chemical moiety attached to the amino acid skeleton, an N-linked or an O-linked carbohydrate chain having a chemical moiety attached, and the like. Biological modifications include post-translational modifications (eg, N-terminal or O-linked sugar chain addition, N-terminal or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, etc. ), Those with a methionine residue added to the N-terminal by expression using a prokaryotic host cell, and the like. Further, those labeled to enable the detection or isolation of the antibody or antigen according to the present invention, for example, an enzyme-labeled substance, a fluorescent-labeled substance, and an affinity-labeled substance are also included in the meaning of such modified substances. Such a modified antibody according to the present invention is useful for improving the stability and retention of an antibody, reducing the antigenicity, detecting or isolating an antibody or an antigen, and the like.

It is known that the glycine residue at the carboxyl terminus of the heavy chain is deleted in the antibody produced in cultured mammalian cells (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that the two amino acid residues of glycine and lysine at the heavy chain carboxyl terminus are deleted, and the proline residue located at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (Analytical Biochemistry, 360: 75-83). 2007)). However, deletions and modifications of these heavy chain sequences do not affect the antigen-binding ability and effector function status of the antibody (the presence or absence of complement activation and antibody-dependent cellular cytotoxicity, high and low, etc.). Therefore, the antibody according to the present invention also includes the modified antibody and a functional fragment of the antibody, and is a deletion product in which 1 or 2 amino acids are deleted at the heavy chain carboxyl terminus, and the amidated antibody. Deletions (eg, heavy chains in which the proline residue at the carboxyl-terminal site is amidated) and the like are also included. However, as long as the state of antigen-binding ability and effector function (whether present or low or high or low) is maintained, the deletion of the carboxyl end of the heavy chain of the antibody according to the present invention is not limited to the above types. The two heavy chains constituting the antibody according to the present invention may be any one of the heavy chains selected from the group consisting of the full length and the above-mentioned deletion product, or a combination of the two heavy chains. It may be a thing. The amount ratio of each deletion substance can be affected by the type and culture conditions of cultured mammalian cells producing the antibody according to the present invention, but the antibody according to the present invention is preferably carboxyl-terminated in both of the two heavy chains. It can be mentioned that one of the amino acid residues is deleted.

Human Chimera Antibodies and Humanized Antibodies The anti-IL-22BP antibodies of the invention also include human chimeric antibodies and humanized antibodies modified to reduce heterologous antigenicity to humans. Humanized antibodies are also referred to as CDR transplant antibodies.

Human chimeric antibody A human chimeric antibody is an antibody consisting of a light chain variable region and a heavy chain variable region of an antibody of a non-human animal and a light chain constant region and a heavy chain constant region of a human antibody. For the human chimeric antibody, the cDNA encoding the light chain variable region and the cDNA encoding the heavy chain variable region were collected from the hybrid dome that produces anti-IL-22BP, and the cDNA encoding the light chain constant region and the heavy chain constant region of the human antibody were collected. It can be prepared by inserting it into an expression vector having the above to construct a human chimeric antibody expression vector, introducing it into a host cell, and expressing it.
The heavy chain constant region is composed of three

domains

_CH 1, _CH 2 and _CH 3.

Examples of human chimeric antibodies of the anti-human IL-22BP antibody of the present invention include rat anti-human IL-22BP monoclonal antibodies, rMAb3, rMAb4, rMAb8, rMAb14, rMAb20 and human chimeric antibodies having variable regions of rMAb81, cMAb3, cMAb4, cMAb8, Examples thereof include cMAb14, cMAb20 and cMAb81.

A humanized antibody (CDR transplanted antibody) transplants the CDR amino acid sequences of the light chain variable region and heavy chain variable region of a non-human animal antibody into appropriate positions of the light chain variable region and heavy chain variable region of a human antibody. Refers to antibodies that have been developed.

Specifically, a DNA sequence designed to link the CDR of the antibody MAb3, MAb4, MAb8, MAb14, MAb20 or MAb81 with the framework region of the human antibody may be synthesized. The framework region of the human antibody linked via the CDR is selected so that the CDR forms a good antigen binding site. Further, if necessary, amino acids in the framework region in the variable region of the antibody may be substituted so that the CDR of the humanized antibody forms an appropriate antigen binding site. The production of a humanized antibody transplanted with CDR can be performed by a known CDR graphing technique.

The present invention comprises three CDRs (CDRH1) of light chain and heavy chain variable regions comprising the sequences of the three CDRs (CDR1, CDRL2, CDRL3) of the light chain variable regions of the monoclonal antibodies MAb3, MAb4, MAb8, MAb14, MAb20 and MAb81. , CDRH2, CDRH3) contains an antibody consisting of a heavy chain containing the sequence. The amino acid sequences of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 of the monoclonal antibodies MAb3, MAb4, MAb8, MAb14, MAb20 and MAb81 are shown in the following SEQ ID NOs of the sequence listing.

MAb3 MAb4 MAb8 MAb14 MAb20 MAb81
CDRL1
27 30 33 36 39 42
CDRL2
28 31 34 37 40 43
CDRL3
29 32 35 38 41 44
CDRH1
57 60 63 66 69 72
CDRH2
58 61 64 67 70 73
CDRH3
59 62 65 68 71 74

In the heavy chain variable region of MAb4, Ser66 (IMGT numbering; Immunol Today 18 (11), 509 (1997)) was mutated to Ala and Asn106 to Arg for the purpose of avoiding N-type glycosylation, and MAb4'was mutated. Made. The amino acid sequences of CDRH1, CDRH2 and CDRH3 of MAb4'are shown in SEQ ID NO: 105, SEQ ID NO: 106 and SEQ ID NO: 107 of the sequence listing, respectively.

In addition, the light chain amino acid sequence and heavy chain amino acid sequence of human chimeric antibody cMAb3 are assigned to SEQ ID NOs: 85 and 99, the light chain amino acid sequence and heavy chain amino acid sequence of cMAb4 are assigned to SEQ ID NOs: 86 and 100, and the light chain amino acid sequence and heavy chain of cMAb8 are assigned. The chain amino acid sequences are in SEQ ID NOs: 87 and 101, the light chain amino acid sequences and heavy chain amino acid sequences of cMAb14 are in SEQ ID NOs: 88 and 102, the light chain amino acid sequences and heavy chain amino acid sequences of cMAb20 are in SEQ ID NOs: 89 and 103, and cMAb81. The light chain amino acid sequence and the heavy chain amino acid sequence of are shown in SEQ ID NOs: 90 and 104.

The light chain amino acid sequence and the heavy chain amino acid sequence of these human chimeric antibodies include not only the light chain and the heavy chain including the amino acid sequence represented by the above SEQ ID NO:, but also one or an integer number, for example, in the amino acid sequence. The property of the antibody of the present invention comprises an amino acid sequence in which 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid is deleted, substituted, or added. (The above-mentioned [1], preferably [1] and [2], more preferably [1] to [3], even more preferably [1] to [4], and even more preferably [ 1] to [5], etc.), in particular, amino acid sequences contained in light chains and / or heavy chains contained in proteins having an activity of binding to human IL-22BP and the like can also be mentioned. As such an amino acid sequence, at least 85% or more, preferably at least 85% or more, when calculated using the amino acid sequence represented by the above SEQ ID NO: and Clustal W (alignment tool) or the like (for example, default, that is, the default parameter). Examples thereof include those having 90% or more, more preferably 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more sequence identity. It can be said that such an amino acid sequence is substantially the same as the protein having the amino acid sequence represented by the above SEQ ID NO:.

In addition, a humanized antibody hMAb3 was designed based on the monoclonal antibody MAb3 by CDR graphing, and a humanized antibody light chain was designed by connecting the κ chain constant region of human IgG1 to the designed humanized antibody light chain variable region of MAb3. These are referred to as hMAb3_L01, hMAb3_L02, hMAb3_L03, hMAb3_L04, hMAb3_L05, hMAb3_L06, and hMAb3_L07, respectively. Further, those prepared by designing a humanized antibody heavy chain in which the γ chain constant region of human IgG1 is connected to the designed MAb3 humanized antibody heavy chain variable region are referred to as hMAb3_H01, hMAb3_H02, hMAb3_H03, and hMAb3_H04, respectively.

Furthermore, a humanized antibody hMAb4 was designed based on the monoclonal antibody MAb4 by CDR graphing, and a humanized antibody light chain was designed by connecting the κ chain constant region of human IgG1 to the designed humanized antibody light chain variable region of MAb4. These are referred to as hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06, respectively. Designed and produced humanized antibody heavy chains in which the γ chain constant region of human IgG1 is connected to the designed humanized antibody heavy chain variable region are called hMAb4_H01, hMAb4_H02, and hMAb4_H03, respectively.

Antibodies of the present invention include antibodies comprising any of the light chain variable regions of hMAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05 and hMAb3_L07, and any of the heavy chain variable regions of hMAb3_H01, hMAb3_H03 and hMAb3_H04. The amino acid sequences of hMAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05 and hMAb3_L07 are shown in SEQ ID NOs: 121, 122, 123, 124 and 125, respectively, and the amino acid sequences of hMAb3_H01, hMAb3_H03 and hMAb3_H04 are shown in SEQ ID NOs: 138, respectively. show. In hMAb3, the light chain variable region consists of the sequences shown in amino acid numbers 21 to 133 of the amino acid sequence represented by the respective SEQ ID NOs, and the heavy chain variable region consists of the amino acid numbers 20 to 132 of the amino acid sequence represented by the respective SEQ ID NOs. It consists of the sequences shown in.

Further, the antibody of the present invention comprises a light chain variable region of any one of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05 and hMAb4_L06, and an antibody containing any of hMAb4_H01, hMAb4_H02 and hMAb4_H03. The amino acid sequences of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05 and hMAb4_L06 are shown in SEQ ID NOs: 126, 127, 128, 129, 130 and 131, respectively, and the amino acids of hMAb4_H01, hMAb4_H02 and hMAb4_H03, respectively. 142 and 143 are shown. In hMAb4, the light chain variable region consists of the sequences shown in amino acid numbers 21 to 133 of the amino acid sequence represented by the respective SEQ ID NOs, and the heavy chain variable region consists of the amino acid numbers 20 to 137 of the amino acid sequence represented by the respective SEQ ID NOs. It consists of the sequences shown in.

Among these antibodies, the MAb3-based antibody has a light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and amino acid numbers 20 to 132 of SEQ ID NO: 138 in the sequence listing. An antibody containing a heavy chain variable region consisting of the amino acid sequence shown in the above, a light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 123 in the sequence listing, and the amino acid number of SEQ ID NO: 138 in the sequence listing. An antibody containing a heavy chain variable region consisting of the amino acid sequences shown in 20 to 132, a light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and a sequence in the sequence listing. Antibodies containing a heavy chain variable region consisting of the amino acid sequences shown in amino acids Nos. 20 to 132 of No. 140 are preferable.

Further, the MAb4-based antibody is a light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the sequence listing, and the amino acids shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the sequence listing. An antibody containing a heavy chain variable region consisting of a sequence, a light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the sequence listing, and amino acid numbers 20 to 137 of SEQ ID NO: 143 in the sequence listing. An antibody containing a heavy chain variable region consisting of the amino acid sequence shown, a light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 130 in the sequence listing, and an amino acid of SEQ ID NO: 142 in the sequence listing. An antibody containing a heavy chain variable region consisting of the amino acid sequences shown in Nos. 20 to 137 is preferable. Further, the antibody of the present invention includes an antibody comprising the full length of the light chain of any one of MAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05 and hMAb3_L07, and the full length of any heavy chain of hMAb3_H01, hMAb3_H03 and hMAb3_H04. The light chain full-length amino acid sequences of MAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05 and hMAb3_L07 contain the sequences set forth in amino acid numbers 21-239 of the amino acid sequences set forth in SEQ ID NOs: 121, 122, 123, 124 and 125, respectively. The heavy chain full-length amino acid sequences of hMAb3_H01, hMAb3_H03 and hMAb3_H04 include the sequences set forth in amino acid numbers 20-462 of the amino acid sequences set forth in SEQ ID NOs: 138, 139 and 140, respectively. Further, the antibody of the present invention comprises the full length of the light chain of any of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05 and hMAb4_L06, and the full length of any of the heavy chains of hMAb4_H01, hMAb4_H02 and hMAb4_H03. The light chain full-length amino acid sequences of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05 and hMAb4_L06 are the sequences shown in amino acid numbers 21 to 239 of the amino acid sequences set forth in SEQ ID NOs: 126, 127, 128, 129, 130 and 131, respectively. The heavy chain full length amino acid sequences of hMAb4_H01, hMAb4_H02 and hMAb4_H03 are comprising the sequences set forth in amino acid numbers 20-467 of the amino acid sequences set forth in SEQ ID NOs: 141, 142 and 143, respectively.

The MAb3 based antibody specifically, hMAb3_H01L03, hMAb3_H01L04, hMAb3_H03L01, hMAb3_H03L03, hMAb3_H03L05, hMAb3_H04L03, preferably hMAb3_H04L07, MAb4 based antibodies, hMAb4_H01L01, hMAb4_H01L02, hMAb4_H01L04, hMAb4_H01L05, hMAb4_H01L06, hMAb4_H02L01, hMAb4_H02L02, hMAb4_H02L03, hMAb4_H02L04, hMAb4_H02L05, hMAb4_H02L06, hMAb4_H03L01, hMAb4_H03L02, hMAb4_H03L03, hMAb4_H03L04, hMAb4_H03L05, hMAb4_H03L06 are preferable.

Among these antibodies, the MAb3-based antibody is a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing. An antibody comprising a heavy chain comprising the amino acid sequence shown in the above, a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing, and an amino acid number of SEQ ID NO: 138 in the sequence listing. An antibody comprising a heavy chain comprising the amino acid sequences shown in 20 to 462, a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and a sequence in the sequence listing. An antibody comprising a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 462 of No. 140 is more preferable. Specific examples thereof include hMAb3_H01L03, hMAb3_H01L04, and hMAb3_H04L03.

Further, the MAb4-based antibody is a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing, and amino acids shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing. An antibody comprising a heavy chain comprising a sequence, a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing, and amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing. An antibody comprising a heavy chain comprising the amino acid sequence shown, a light chain comprising the amino acid sequences shown in amino acid numbers 21-239 of SEQ ID NO: 130 in the sequence listing, and amino acid number 20 of SEQ ID NO: 142 in the sequence listing. An antibody containing a heavy chain comprising the amino acid sequence shown in ~ 467 is more preferable. Specific examples thereof include hMAb4_H03L01, hMAb4_H03L03, and hMAb4_H02L05.

The light chain amino acid sequence and the heavy chain amino acid sequence, or the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the humanized antibody of the present invention include the light chain and the heavy chain including the amino acid sequence represented by the above SEQ ID NO:. Not only that, in the amino acid sequence, 1 to an integer (the integer is 50 or less, preferably 25 or less, more preferably 20 or less), for example, 1 to 20, 1 to 15, 1 to 10. An amino acid sequence in which 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid is deleted, substituted, or added, and the antibody or a binding fragment thereof (with the above SEQ ID NO:). The activity (including the light chain and heavy chain containing the indicated amino acid sequence) has (the above-mentioned [1], preferably [1] and [2], more preferably [1] to [3], more preferably. [1] to [4], and even more preferably, the properties described in [1] to [5]), particularly included in an antibody or a binding fragment thereof having an activity of binding to human IL-22BP. The range also includes amino acid sequences contained in light and heavy chains. As such an amino acid sequence, at least 85% or more, preferably at least 85% or more, when calculated using the amino acid sequence represented by the above SEQ ID NO: and Clustal W (alignment tool) or the like (for example, default, that is, the default parameter). Examples thereof include those having 90% or more, more preferably 95% or more, particularly preferably 96%, 97% or more, 98% or more, or 99% or more sequence identity. It can be said that the protein having such an amino acid sequence is substantially the same as the protein having the amino acid sequence represented by the above-mentioned SEQ ID NO:.

Examples of the activity / property exhibited by the antibody of the present invention include biological activity, physicochemical property, and the like, and specifically, various biological activities, binding activity to antigens and epitopes, species crossover, and production. And stability at the time of storage, thermal stability and the like can be mentioned.

The antibody of the present invention or a binding fragment thereof has excellent binding activity to IL-22BP. The binding dissociation constant (KD) of the antibody of the present invention or a binding fragment thereof measured by the SPR method for human IL22-BP is 1 × ^10-5 M or less, 5 × ^10-6 M or less, or 2 × ^10-6. M or less, preferably 1 × 10 ^-6 M or less, 5 × 10 ^-7 M or less, 2 × 10 ^-7 M or less, 1 × 10 ^-7 M or less, 5 × 10 ^-8 M or less, 2 × ^10-8 M or less, 1 × ^10-8 M or less.

The antibody of the present invention or a binding fragment thereof preferably binds to monkey IL-22BP, and more preferably to cynomolgus monkey IL-22BP. The binding dissociation constant (KD) of the antibody of the present invention or a binding fragment thereof measured by the BLI method for monkey IL22-BP is 1 × ^10-5 M or less, 5 × ^10-6 M or less, or 2 × ^10-6. M or less, preferably 1 × 10 ^-6 M or less, 5 × 10 ^-7 M or less, 2 × 10 ^-7 M or less, 1 × 10 ^-7 M or less, 5 × 10 ^-8 M or less, 2 × ^10-8 M or less, 1 × ^10-8 M or less. Antibodies that bind to human IL-22BP and monkey IL-22BP are various in terms of efficacy and safety using primates essential for non-clinical development (preclinical development) of pharmaceutical products, especially cynomolgus monkeys (antigen binding, vivo test). It is preferable because it can be used for a test. Further, the antibody of the present invention or a binding fragment thereof that also crosses monkey IL-22BP is useful for the treatment or prevention of various diseases related to IL-22BP such as inflammatory bowel disease in monkeys.

Further, the antibody of the present invention or a binding fragment thereof that binds to human IL-22BP and monkey IL-22BP preferably does not bind to rodent IL-22BP, and more preferably does not bind to mouse IL-22BP. , Cells, tissues, individuals (including transgenic animals, knockout animals, knock-in animals) of rodents such as mice into which human IL-22BP has been introduced, various assays using the antibodies, immunohistochemical studies, etc. It can be carried out without the influence of IL-22BP of rodent animals such as mice as a host, and is preferable for research and development of a drug, veterinary drug or diagnostic agent containing the antibody of the present invention or a binding fragment thereof. In addition, the antibody or binding fragment thereof of the present invention can be obtained by using primates of a pathological model in which intestinal inflammation was induced by administration of sodium dextran sulfate, particularly cynomolgus monkey (Nat Commun. 18; 6: 8020 (2015)). Non-clinical pharmacological activity can be evaluated. Indicators used in clinical practice such as Mayo score can be used to evaluate the pathological condition. Furthermore, changes in signaling mediated by phosphorylation of STAT3 can be detected by pretreatment, concomitant treatment, or posttreatment with respect to the addition of IL-22 by using a biological tissue sample that can be collected at the time of the in vivo pharmacological test or clinically. , Can be evaluated (see ex vivo test of Example 10).

An antibody or binding fragment that binds sites on human IL-22BP to which the antibody of the present invention or a binding fragment thereof binds is also included in the scope of the antibody or binding fragment thereof of the present invention. Such an antibody or a binding fragment thereof preferably has the above-mentioned biological activity, antigen-binding activity, species crossing property, etc. (the properties described in the above-mentioned [1] to [5], etc.). Similarly, an antibody or binding fragment that competes with the antibody of the present invention or a binding fragment thereof in binding to human IL-22BP is also included in the scope of the antibody or binding fragment thereof of the present invention. Such an antibody or a binding fragment thereof preferably has the above-mentioned biological activity, antigen-binding activity, species crossing property, etc. (the properties described in the above-mentioned [1] to [5], etc.).

The antibody of the present invention or a binding fragment thereof can be applied to a multispecific antibody (multispecific molecule) or an antibody drug conjugate. Multispecific antibodies or antibody drug conjugates containing the antibodies of the present invention or binding fragments thereof are also included in the scope of "antibodies" and "binding fragments thereof" of the present invention. That is, the antibody of the present invention or a binding fragment thereof may be in a form formed by forming a complex with a drug or in a form contained in a multispecific antibody (multispecific molecule).

4. Method for producing antibody In the antibody of the present invention or a binding fragment thereof, a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region is inserted into an expression vector, and the vector is used in a host cell for expression. By transforming and culturing the host cell, the cell can be produced as a recombinant antibody.

As the DNA encoding the antibody, a DNA encoding a heavy chain is obtained by linking a DNA encoding a heavy chain variable region and a DNA encoding a heavy chain constant region, and further, a DNA encoding a light chain variable region and a light chain are obtained. By ligating the DNA encoding the chain constant region, the DNA encoding the light chain is obtained.

In the anti-IL-22BP antibody of the present invention, the DNA encoding the heavy chain and the DNA encoding the light chain are inserted into an expression vector, transformed into a host cell for expression using the vector, and the host cell is transformed. Can be cultivated and produced. At this time, the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector, and the host cell may be transformed with the vector, or the DNA encoding the heavy chain may be light. The DNA encoding the strand may be inserted into separate vectors and the two vectors may be used to transform the host cell. At this time, the DNA encoding the heavy chain variable region and the light chain variable region may be introduced into the vector into which the DNA encoding the heavy chain constant region and the DNA encoding the light chain constant region have been introduced in advance. The vector may also contain a DNA encoding a signal peptide that promotes antibody secretion from the host cell, in which case the DNA encoding the signal peptide and the DNA encoding the antibody are linked in-frame. back. By removing the signal peptide after the antibody is produced, the antibody can be obtained as a mature protein.

At this time, the DNA encoding the heavy chain variable region, the DNA encoding the light chain variable region, the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region are linked, and the light chain variable region are encoded. The DNA in which the DNA and the DNA encoding the light chain constant region are linked may be functionally linked to an element such as a promoter, an enhancer, or a polyadenylation signal. Here, functionally connected means that elements are connected so as to perform their functions.

The expression vector is not particularly limited as long as it can be replicated in a host such as an animal cell, a bacterium, or yeast, and examples thereof include known plasmids and phages. Examples of the vector used for constructing the expression vector include pcDNA (trademark) (ThermoFissher SCIENTIFIC), Flexi (registered trademark) vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 ( (Manufactured by Pharmacia), pMAM-neo (manufactured by Clontech) and the like. As the host cell, prokaryotic cells such as Escherichia coli and Bacillus subtilis and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells are preferably used. For example, as animal cells, HEK293 cells, Chinese hamster ovary (CHO) cells, which are human fetal kidney cell lines, and the like may be used. The expression vector may be introduced into a host cell by a known method to transform the host cell. For example, an electroporation method, a calcium phosphate precipitation method, a DEAE-dextran transfection method and the like can be mentioned. The antibody produced can be purified using the separation and purification methods used for conventional proteins. For example, affinity chromatography, other chromatography, filters, ultrafiltration, salting out, dialysis and the like may be appropriately selected and combined.

5. Pharmaceutical Composition The present invention includes a pharmaceutical composition containing the anti-IL-22BP antibody of the present invention or a binding fragment thereof as an active ingredient.

The pharmaceutical composition of the present invention inhibits the binding between IL-22BP and IL-22. When IL-22BP binds to IL-22, the function of IL-22 is inhibited. When IL-22 function is impaired, inflammatory bowel disease including infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune myocardial disease, bronchitis, uveitis, ulcerative colitis and Crohn's disease Etc. are induced. Therefore, the pharmaceutical composition of the present invention includes infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune myocardial disease, bronchitis, uveitis, ulcerative colitis, inflammatory bowel disease including Crohn's disease, etc. It can be used for prevention (administer before onset or exacerbation) or treatment (administer after onset or exacerbation).

The pharmaceutical composition of the present invention can contain a therapeutically effective amount of an anti-IL-22BP antibody, a pharmaceutically acceptable carrier, a diluent, a solubilizer, an emulsifier, a preservative, an auxiliary agent and the like. The "pharmaceutically acceptable carrier" and the like can be appropriately selected from a wide range according to the type of the target disease and the administration form of the drug. The method for administering the pharmaceutical composition of the present invention can be appropriately selected, and for example, it can be administered by injection, such as local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusate injection, etc. Can be adopted. In addition, the solution for injection can be formulated using a carrier consisting of a salt solution, a glucose solution, a mixture of salt water and a glucose solution, various buffer solutions, and the like. Further, it may be formulated in a powder state and mixed with the liquid carrier at the time of use to prepare an injection solution.

Other administration methods can be appropriately selected along with the development of the drug. For example, in the case of oral administration, oral liquids, powders, pills, capsules, tablets and the like can be applied. In the case of oral liquid preparations, as oral liquid preparations such as suspending agents and syrups, water, shoe cloth, sorbitol, sugars such as fructose, glycols such as polyethylene glycol, sesame oil, soybean oil and the like. It can be produced by using oils, preservatives such as alkylparahydroxybenzoate, flavors such as strawberry flavor and peppermint, and the like. Powders, pills, capsules and tablets include excipients such as lactose, glucose, shoe cloth and mannitol, disintegrants such as starch and sodium arginate, lubricants such as magnesium ester and talc, and polyvinyl alcohol. , Hydroxypropyl cellulose, a binder such as gelatin, a surface active agent such as a fatty acid ester, a plastic agent such as glycerin, and the like can be used for the formulation. Tablets and capsules are preferred unit dosage forms in the compositions of the invention in that they are easy to administer. When producing tablets and capsules, a solid production carrier is used.

The amount of antibody or binding fragment effective for treatment varies depending on the nature of the medical condition to be treated, the age and condition of the patient, and may ultimately be decided by the doctor. For example, it is 0.0001 mg to 100 mg per 1 kg of body weight at a time. The predetermined dose may be administered once every 1 to 180 days, or may be administered in divided doses of 2 times, 3 times, 4 times or more per day at appropriate intervals.

The pharmaceutical composition containing the anti-IL-22BP antibody of the present invention or a binding fragment thereof as an active ingredient can be used in combination with other pharmaceutical compositions. Other pharmaceuticals used in combination with the pharmaceutical composition of the present invention include, for example, anti-TNFα antibody, anti-integrin α4β7 antibody, 5-aminosalicylic acid preparation, corticosteroid preparation, thiopurine preparation, calcineurin inhibitor, JAK inhibitor. , Antibacterial agents and the like. Examples of these other drugs include adalimumab, which is an anti-TNFα antibody, vedrizumab, which is an anti-integrin α4β7 antibody, mesalazine, which is a 5-aminosalicylic acid preparation, prednisolone, which is a corticosteroid, azathioprine, which is a thiopurine preparation, and carcinulinin inhibitor. Examples thereof include tacrolimus, tofacitinibu citrate which is a JAK inhibitor, metronidazole which is an antibacterial agent, and the like. In addition, the pharmaceutical composition of the present invention can also be used for the treatment or prevention of inflammatory bowel disease in combination with component nutrition therapy, leukapheresis and the like. These other medicines and therapies may be one type, and a few or more types may be administered or received. Collectively, the pharmaceutical composition of the present invention is referred to as "combination with other pharmaceutical products" or "combination with other pharmaceutical products", and in addition to the antibody of the present invention, a binding fragment thereof or a modified product thereof, other pharmaceutical products are used. The pharmaceutical composition of the present invention, which is contained or used in combination with both, is also included in the present invention as an embodiment of "combination with other pharmaceutical products" or "combination with other pharmaceutical products".

The present invention is the anti-IL-22BP antibody of the present invention or a binding fragment thereof, which is used in "combination with other medicines", and the anti-IL of the present invention, which is used in "combination with other medicines". Includes a pharmaceutical composition comprising a -22BP antibody or a binding fragment thereof as an active ingredient.

Further, a polynucleotide containing a base sequence encoding an amino acid sequence contained in the antibody of the present invention or a binding fragment thereof, a vector containing the polynucleotide, or a polynucleotide or a cell containing the vector (also referred to as a host cell). The pharmaceutical composition containing, is also useful for the treatment or prevention of the above-mentioned diseases such as inflammatory bowel disease, and is included in the present invention.

6. Test Agents, Diagnostic Agents and Reagents The antibodies of the present invention or their bound fragments can also be used as test agents, diagnostic agents, reagents and the like. The amount of IL-22BP in a sample can be measured using the antibody of the present invention or a binding fragment thereof. Furthermore, it is also possible to detect whether or not the function of IL-22 is inhibited in the sample. For example, infection, autoimmune hepatitis, alcoholic liver disease, autoimmune myocardial disease, bronchitis, uveitis, ulcerative colitis, using the amount of IL-22BP or inhibition of IL-22 function as an index. It is possible to test whether or not the patient has inflammatory bowel disease including Crohn's disease or the risk of developing the disease.

Example 1. Preparation of IL-22BP 1) -1 Preparation of expression vector 1) -1-1 Preparation of expression vector p3.3n IL2ss_FLAGHis Vector pcDNA3.3-TOPO / LacZ (manufactured by Thermo Fisher SCIENTIFIC) neomycin expression unit was used as KOD-Plus-Mutagenesis. Deletion was performed using Kit (manufactured by TOYOBO). As primers, 5'-CAAAATAAGCAATAGCATCACAAAATTTC-3'(Dneo-F) (SEQ ID NO: 1) and 5'-CCACAGATAATTTCGCGTTAAATTTTTG-3'(Dneo-R) (SEQ ID NO: 2) were used.

Cleavage of DNA (SEQ ID NO: 4) encoding a polypeptide containing human IL-2 (ACCESSION number: NP_000577) amino acid sequence 1 to 20 and FLAGHis tag DYKDDDDKHHHHHH (SEQ ID NO: 3) with vector restriction enzymes XbaI and PmeI. By inserting between the sites, the expression vector p3.3IL2ss_FLAGHis was prepared.

1) -1-2 Preparation of human IL-22BP-His expression vector A sequence containing a polypeptide in which ENLYFQG is linked to the C-terminal side of positions 1 to 263 of the amino acid sequence of human IL-22BP (ACCESSION number: NP_443194) is encoded. DNA (SEQ ID NO: 5) is inserted between the restriction enzymes XbaI and BamI of the expression vector p3.3IL2ss_FLAGHis using an In-Fusion HD Cloning Kit (manufactured by CLONTECH) to human IL-22BP. (Isoform 1) -His expression vector was prepared. Human IL-22BP (Isoform 2) -His expression by deleting the DNA encoding the 67th to 98th polypeptide of the amino acid sequence from the human IL-22BP (Isoform 1) -His expression vector using the Inverse PCR method. A vector was prepared. Hereinafter, human IL-22BP (Isoform 2) -His will be referred to as human IL-22BP-His.
The nucleotide sequence of human IL-22BP-His is shown in SEQ ID NO: 6, and the amino acid sequence is shown in SEQ ID NO: 7.

1) -1-3 Preparation of monkey IL-22BP-His expression vector 21 of the amino acid sequence of monkey IL-22BP (ACCESSION number: XP_005552009), which is the 1st to 21st amino acid sequences of human IL-22BP (ACCESSION number: NP_443194). Restriction of expression vector p3.3IL2ss_FLAGHis using In-Fusion HD Cloning Kit (manufactured by CLONTECH) for DNA (SEQ ID NO: 8) encoding a sequence containing a polypeptide in which ENLYFQG is ligated to the C-terminal side of the 231st position. A monkey IL-22BP-His expression vector was prepared by insertion between the cleavage sites of the enzymes XbaI and BamI. The nucleotide sequence of monkey IL-22BP-His is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10.

1) -1-4 Preparation of mouse IL-22BP-His expression vector Mouse IL-22BP (ACCESSION number: NP_839989) in which Cys106, which is the 1st to 21st amino acid sequences of human IL-22BP (ACCESSION number: NP_443194), is replaced with Ser. ), A DNA (SEQ ID NO: 11) encoding a sequence containing a polypeptide in which ENLYFQG is linked to the C-terminal side at positions 21 to 230 is expressed using an In-Fusion HD Cloning Kit (manufactured by CLONTECH). A mouse IL-22BP-His expression vector was prepared by inserting the vector p3.3 IL2ss_FLAGHis between the restriction enzymes XbaI and BamI. The nucleotide sequence of mouse IL-22BP-His is shown in SEQ ID NO: 12, and the amino acid sequence is shown in SEQ ID NO: 13.

1) -2 Expression and purification of IL-22BP-His Each IL-22BP-His expression vector was transiently expressed by transfection into FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC). The culture supernatant was completely placed in HisTrap excel (manufactured by GE Healthcare Japan) equilibrated with 3 × PBS (−) (PBS), and then the column was washed with 3 × PBS. It was then eluted with 3 x PBS, 500 mM Imidazole. From the recovered IL-22BP fraction, IL-22BP-His was prepared using HiRoad 26/600 Superdex 200 pg (manufactured by GE Healthcare Japan) equilibrated with 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Purified. The final buffer was prepared in 16 mM Tris-HCl (pH 7.5), 120 mM NaCl, 16% (v / v) Glycerol.

1) -3 Preparation of immune antigen A TEV protease having a His tag added to the N-terminal was added to the purified human IL-22BP-His and reacted at 4 ° C. overnight to cleave FLAGHis. Ni Sepharose 6 FF (GE Healthcare Japan Co., Ltd.) was added to the reaction solution, and 5M imidazole was further added so that the concentration was about 20 mM. The supernatant reaction solution was placed in an empty column, the passing fraction was collected, and the buffer was exchanged with PBS using PD-10 (manufactured by GE Healthcare Japan). The amino acid sequence of human IL-22BP with the tag cleaved is shown in SEQ ID NO: 14.

Example 2. Preparation of rat anti-human IL-22BP antibody 2) -1 Immunization A female WKY / Izm rat (manufactured by Nippon SLC Co., Ltd.) was used for immunization. Lymph nodes and spleens of rats to which a mixture of Recombinant Human IL-22BP antigen protein prepared in Example 1) -3 and Freund's Complete Adjuvant (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was collected. It was used to prepare a hybridoma.

2) -2 Hybridoma production Lymph node cells or spleen cells and mouse myeloma SP2 / 0-ag14 cells (ATCC: CRL-1511) were fused by electric cells using LF301-Cell Fusion Unit (manufactured by BEX), and ClonaCell- The cells were diluted with HY Selection Medium D (manufactured by StemCell Technologies) and cultured. A monoclone hybridoma was produced by collecting the hybridoma colonies that appeared. Each of the recovered hybridoma colonies was cultured, and the obtained hybridoma culture supernatant was used to screen for anti-human IL-22BP antibody-producing hybridomas.

Example 3. Evaluation of rat anti-human IL-22BP antibody 3) -1 antibody screening 3) -1-1 DIRECT-ELISA
The human IL-22BP-His prepared in Example 1) -2 was diluted with 50 mM Tris-HCl (pH 8.5) to 4 μg / ml, and 50 μL was added to Clear Flat-Bottom Immuno 96-Well Plates (manufactured by Thermo Fisher SCIENTIFIC). After each addition, the mixture was solidified overnight at 4 ° C. The next day, the medium of the plate was removed, and after washing twice with PBS containing 5% FBS, 100 μL of PBS containing 5% FBS was added, and the mixture was allowed to stand at room temperature for 1 hour. After washing twice with PBS containing 5% FBS, the hybridoma culture supernatant was added, and the mixture was allowed to stand at room temperature for 2 hours. After washing twice with PBS containing 5% FBS, 50 μL of Anti-rat IgG Peroxidase antibody-produced in rabbit (manufactured by SIGMA-ARDRICH) diluted 500-fold with PBS containing 5% FBS was added, and the mixture was allowed to stand at room temperature for 1 hour. bottom. After washing 5 times with PBS containing 5% FBS, o-phenylenediamine dihydrochloride is added to the OPD color-developing solution (OPD solution (0.05M sodium citrate, 0.1M sodium hydrogen phosphate, 12 water pH 4.5)). (Manufactured by Fujifilm Wako Junyaku Co., Ltd.) and H ₂ O ₂ were dissolved at 0.4 mg / mL and 0.6% (v / v), respectively) at 50 μL / well. The color reaction was carried out with occasional stirring, and 50 μL / well of 1M HCl was added to stop the color reaction, and then the absorbance at 490 nm was measured by Envision (manufactured by PerkinElmer). A hybridoma producing a culture supernatant having an OD value of 0.1 or more and a value more than twice that of a well without human IL-22BP antigen was selected as an anti-human IL-22BP antibody production positive hybridoma.

3) 1-2 Competition ELISA
6 × -His Tag Polyclonal Antibody (Thermo Fisher SCIENTIFIC) was diluted with PBS to 2 μg / mL, and Clear Flat-Bottom Immuno 96-Well Plates (Thermo Fisher) was added to the solution once at 4 ° C. It became a phase. The next day, after washing twice with PBS containing 0.05% Tween-20 (PBS-T), 200 μL of Block BSA in PBS (manufactured by Thermo Fisher SCIENTIFIC) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing twice with PBS-T, 50 μL of human IL-22BP-His diluted to 2 μg / mL with PBS-T (diluting buffer) containing 1% BSA was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing 5 times with PBS-T, the selected anti-human IL-22BP antibody production positive hybridoma culture supernatant was added, and subsequently diluted to 1 μg / mL with a diluting buffer, Recombinant Human IL-22 Protein (R & D Systems). ] Or only the diluted buffer solution was added in an amount of 5 μL each, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing 5 times with PBS-T, 50 μL of Human IL-22 Antibody (manufactured by R & D Systems) diluted to 0.5 μg / mL with a diluting buffer was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing 5 times with PBS-T, 50 μL of Goat Anti-Mouse IgG Antibody, HRP conjugate, and Species Advanced (Merck) diluted to 0.8 μg / mL with a diluting buffer was added, and the temperature was 37 ° C. for 1 hour. It was left still. After washing 6 times with PBS-T, 100 μL of OPD color-developing solution was added, and after standing for 6 minutes, 100 μL of 1M HCl was added, and the absorbance at 490 nm was measured by Envision (manufactured by Perkin Elmer). To select hybridomas that specifically inhibit the binding of Recombinant Human IL-22 Protein to human IL-22BP, hybridomas that produce culture supernatants that show lower absorbance compared to control's dilution buffer, IL- It was selected as a positive anti-human IL-22BP antibody-producing hybridoma having a binding inhibitory activity between 22 and IL-22BP. As a result, 6 types of hybridomas producing rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81 were selected from 2000 clones of hybridomas.

3) -2 Antibody isotype determination Isotypes of the antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) produced by the binding inhibitory activity-positive anti-human IL-22BP antibody-producing hybridoma selected in Example 3) -1-2. Was determined by the Rapid monoclonal Isotyping kit (manufactured by Antibody Pharmaceuticals). As a result, it was confirmed that only rMAb8 had IgG2a and κ chains as isotypes, and all others had IgG1 and κ chains.

3) -3 Preparation of purified antibody 3) 3-1 Preparation of culture supernatant The anti-human IL-22BP monoclonal antibody was purified from the culture supernatant of rat hybridoma. A rat anti-human IL-22BP monoclonal antibody-producing hybridoma was grown to a sufficient amount in ClonaCell-HY Selection Medium E (manufactured by StemCell Technologies), and Ultra Low IgG FBS (manufactured by Thermo Fisher SCIENTIFIC) was added. after medium change Company Ltd.) were seeded and cultured for 7 days hybridomas 8 ~ 9 × 10 ⁷ cells 1272Cm ² flasks (CORNING Co.). The culture supernatant was collected by centrifugation, passed through a 0.8 μm filter, and then sterilized through a 0.45 μm filter (manufactured by CORNING).

3) -3-2 Purification of antibody From the hybridoma culture supernatant obtained in Example 3) 3-1, rat anti-human IL-22BP antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) were added from 4 to 4. Purified by one step of Protein G affinity chromatography at 6 ° C. The buffer replacement step after protein G affinity chromatography purification was performed at 4-6 ° C. First, the hybridoma culture supernatant was applied to a column packed with Protein G HP (manufactured by GE Healthcare Japan) equilibrated with PBS. After the culture supernatant was completely contained in the column, the column was washed with PBS having a column volume of 2 times or more. Next, it was eluted with 0.1 M glycine / hydrochloric acid aqueous solution and pH 2.3, and the fraction containing the antibody was collected. 1M Tris-HCl, pH 9.0 was added to adjust the pH to 7.0 to 7.5, and then concentrated at 4 to 6 ° C. with Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut off UF10K, manufactured by Sartorius). Buffer replacement with PBS was performed. After replacement with PBS, the IgG concentration was adjusted to 1 mg / mL to prepare a purified sample.

3) -4 Binding evaluation using purified antibody 3) -4-1 Measurement of binding activity with human IL-22BP (BLI method)
The binding activity of the rat anti-human IL-22BP monoclonal antibody prepared in Example 3) -3-2 and the human IL-22BP-His prepared in Example 1) -2 was measured by Octet RED 384 (manufactured by MOLECULAR DEVICES). ) Is used to capture the antigen as a ligand in the biosensor Anti-Penta-HIS (HIS1K) (manufactured by MOLECULAR DEVICES), and the rat antibody is used as an analyst and PBS-T (Takara Bio) as a buffer solution for measurement. It was measured using (manufactured by the company). The biosensor was reacted with 5 μg / mL human IL-22BP-His for 600 seconds, and then reacted with a diluted series solution of rat anti-human IL-22BP monoclonal antibody (7 concentration series from 500 nM to a common ratio of 2) for 600 seconds. As a regeneration solution, 10 mM Glycine pH 1.5 (manufactured by GE Healthcare Japan) was reacted with the biosensor for 25 seconds. The data was analyzed by equilibrium value analysis, and the binding dissociation constant KD was calculated.

3) 4-2 Species cross-reactivity measurement (DIRECT-ELISA) for mouse IL-22BP
Example 3) Species cross-reactivity measurement between the rat anti-human IL-22BP monoclonal antibody prepared in -3-2 and mouse and monkey IL-22BP was carried out in mouse IL-22BP-His and monkey prepared in Example 1) -2. Using IL-22BP-His, the measurement was performed by DIRECT-ELISA in the same manner as in Example 3) -1-1. The results of the binding activity of each clone to mouse, monkey, and human IL-22BP are shown in FIG. All the clones evaluated bind to monkey and human IL-22BP, while the clones that also bind to mouse IL-22BP were rMAb8 and rMAb14.

Example 4. Determination of the nucleotide sequence of the cDNA encoding the rat anti-human IL-22BP antibody variable region 4) -1 Preparation of total RNA from hybridomas producing rat anti-human IL-22BP antibody (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81). In order to amplify the cDNA containing the variable region, total RNA was prepared from a rat anti-human IL-22BP antibody-producing hybridoma using Direct-zol RNA Miniprep kit (manufactured by ZYMO RESEARCH).

4)-Amplification and sequence determination of cDNA containing light chain variable region of rat anti-human IL-22BP antibody (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) by 25'-RACE PCR The following reaction is SMARTerTM RACE. It was carried out using a 5'/3'Kit (manufactured by CLONTECH).

The 1st strand cDNA was synthesized from Total RNA. The cDNA containing the variable region of the light chain of the rat anti-human IL-22BP antibody was amplified by 5'-RACE PCR using the 1st strange cDNA synthesized with the combination of the following primers as a template. As primers, oligonucleotides having the sequences UPM (attached to Kit) and 5'-TCAGTAACACTGTCCAGGACACCACTTC-3'(RKR5) were used. RKR5 was designed from the sequence of the constant region of the rat light chain in the database.

The PCR fragment amplified by 5'-RACE PCR was cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Thermo Fisher SCIENTIFIC), and the nucleotide sequence of the cDNA containing the variable region of the cloned light chain was sequenced. bottom. RKR5 and NUP (attached to Kit) were used as sequence primers.

Sequence of the nucleotide sequence of the variable region (MAb3_VL, MAb4_VL, MAb8_VL, MAb14_VL, MAb20_VL, MAb81_VL) of the light chain of the rat anti-human IL-22BP antibody (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81). In 20, the amino acid sequence is shown in SEQ ID NOs: 21 to 26 of the sequence listing. Further, the amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb3_VL are arranged in SEQ ID NOs: 27 to 29, the amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb4_VL are arranged in SEQ ID NOs: 30 to 32, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb8_VL are arranged. The amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb14_VL are assigned to SEQ ID NOs: 36 to 38, the amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb20_VL are assigned to SEQ ID NOs: 39 to 41, and the amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb81_VL are assigned to numbers 33 to 35. The amino acid sequences are shown in SEQ ID NOs: 42 to 44, respectively.

4)-3.5'-RACE PCR to amplify and sequence cDNA containing heavy chain variable regions of rat anti-human IL-22BP antibody (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) The following reaction is SMARTerTM RACE It was carried out using a 5'/3'Kit (manufactured by CLONTECH).

The 1st strand cDNA was synthesized from Total RNA. The cDNA containing the variable region of the heavy chain of the rat anti-human IL-22BP antibody was amplified by 5'-RACE PCR using the 1st strange cDNA synthesized with the combination of the following primers as a template. PCR was performed using KOD-Plus- (manufactured by TOYOBO) as Polymerase. As primers, oligonucleotides having the sequences of UPM (attached to Kit) and 5'-CTCCAGAGTTCCAGGTCACGGGTGACTGGC-3'(RG2AR3) were used. RG2AR3 was designed from the sequence of the constant region of the rat heavy chain in the database.

The PCR fragment amplified by 5'-RACE PCR was cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Thermo Fisher SCIENTIFIC), and the nucleotide sequence of the cDNA containing the cloned heavy chain variable region was sequenced. bottom. RG2AR3 and NUP (attached to Kit) were used as sequence primers.

Sequence of the nucleotide sequence of the heavy chain variable region (MAb3_VH, MAb4_VH, MAb8_VH, MAb14_VH, MAb20_VH, MAb81_VH) of the rat anti-human IL-22BP antibody (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81). At 50, the amino acid sequences are shown in SEQ ID NOs: 51-56 of the sequence listing. Further, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb3_VH are arranged in SEQ ID NOs: 57 to 59, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb4_VH are arranged in SEQ ID NOs: 60 to 62, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb8_VH are arranged. The amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb14_VH are assigned to Nos. 63 to 65, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb20_VH are assigned to SEQ ID NOs: 69 to 71, and the amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb81_VH are assigned to SEQ ID NOs: 66 to 68. The amino acid sequences are shown in SEQ ID NOs: 72 to 74, respectively.

Example 5. Preparation of human chimeric anti-human IL-22BP antibody 5) -1 Construction of light chain expression vector pCMA-LK Approximately 5 obtained by digesting plasmid pcDNA3.3-TOPO / LacZ (manufactured by Thermo Fisher SCIENTIFIC) with restriction enzymes XbaI and PmeI. . Binding of a DNA fragment containing a 4 kbp fragment and a DNA sequence encoding the human κ chain secreting signal shown in SEQ ID NO: 75 of the sequence listing and the human κ chain constant region using an In-Fusion Antibody PCR cloning kit (manufactured by CLONTECH). Then, cDNA 3.3 / LK was prepared.

PCR was performed with the following primer set using pcDNA3.3 / LK as a template, and the obtained fragment of about 3.8 kbp was phosphorylated and self-ligated to determine the signal sequence, cloning site, and human kappa chain downstream of the CMV promoter. An expression vector pCMA-LK having a normal region was constructed.
Primer set 5'-TATACCGTCGACCCTAGCTAGACTTGGC-3'(Primer 3.3-F1: SEQ ID NO: 76)
5'-GCTATGGCAGGGCCTCGCCGCCCCGACGTTG-3'(Primer 3.3-R1: SEQ ID NO: 77)

5) -2 Construction of the heavy chain expression vector pCMA-G1 A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the κ chain secretion signal and the human κ chain constant region, and the human heavy chain secretion shown in SEQ ID NO: 78. A DNA fragment containing a signal and a DNA sequence encoding an amino acid in the human IgG1 constant region was bound using an In-Fusion Advantage PCR cloning kit (manufactured by CLONTECH), and the signal sequence, cloning site, and human IgG1 were bound downstream of the CMV promoter. An expression vector pCMA-G1 having a heavy chain constant region was constructed.

5) -3 Construction of light chain expression vector for human chimeric anti-human IL-22BP antibody A DNA fragment containing a DNA sequence encoding the light chain variable region (MAb3_VL, MAb4_VL, MAb8_VL, MAb14_VL, MAb20_VL, MAb81_VL) of each clone was synthesized. (Artificial gene synthesis service manufactured by GENEART). Using the synthesized DNA fragment as a template, amplify the DNA fragment containing the DNA sequence encoding MAb3_VL, MAb4_VL, MAb8_VL, MAb14_VL, MAb20_VL, MAb81_VL with KOD-Plus- (manufactured by TOYOBO) and the following primer set, and express the light chain. The light chains of cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81 are expressed by inserting the vector pCMA-hLK into a site cleaved with the restriction enzymes BsiWI and SacII using an In-Fusion HD Cloning Kit (manufactured by CLOSETECH). Constructed a vector. The nucleotide sequences of the cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81 light chains are shown in SEQ ID NOs: 79 to 84 in the sequence listing, and the amino acid sequences are shown in SEQ ID NOs: 85 to 90.
Primer set 5'-CTGTGGATCTCCGGCGCGTACCGGC-3'(Primer CM-LKF: SEQ ID NO: 91)
5'-GGAGGGGGCGCGCCACCGTACG-3'(Primer KCL-Inf-R: SEQ ID NO: 92)

5) -4 Construction of heavy chain expression vector for human chimeric anti-human IL-22BP antibody MAb4_VH transferred Ser66 (IMGT numbering; Immunol Today 18 (11), 509 (1997)) to Ala for the purpose of avoiding N-glycosylation. Asn106 was mutated to Arg and used as MAb4'_VH.

A DNA fragment containing a DNA sequence encoding a heavy chain variable region (MAb3_VH, MAb4'_VH, MAb8_VH, MAb14_VH, MAb20_VH, MAb81_VH) of each clone was synthesized (artificial gene synthesis service manufactured by GENEART). Using the synthesized DNA fragment as a template, the DNA fragment containing the DNA sequences encoding MAb3_VH, MAb4'_VH, MAb8_VH, MAb14_VH, MAb20_VH, and MAb81_VH is amplified with KOD-Plus- (manufactured by TOYOBO) and the following primer set. The heavy chains of cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81 are expressed by inserting the chain expression vector pCMA-G1 into a site cleaved with the restriction enzyme BlpI using an In-Fusion HD Cloning Kit (manufactured by CLONTECH). An expression vector was constructed. The nucleotide sequences of the cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81 heavy chains are sequenced in SEQ ID NOs: 93 to 98, the amino acid sequences are sequenced in SEQ ID NOs: 99 to 104, and the MAb4'_VH CDRH1, CDRH2, and CDRH3 are sequenced. It is shown in 105-107.
Primer set 5'-AGCTCCCAGATGGGTGCTGAGC-3' (Primer EG-Inf-F: SEQ ID NO: 108)
5'-GGGCCTTGGTGGGAGGCTGAGC-3'(Primer EG1-Inf-R: SEQ ID NO: 109)

5) -5 Production of human chimeric anti-human IL-22BP antibody FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) were subcultured and cultured according to the manual.

1.2 × 10 ⁹ FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) in the logarithmic growth phase were inoculated into a 3L Fernbach Erlenmeyer Flask (manufactured by CORNING) and diluted with FreeStyle 293Express (Made by CORNING). After preparation, the cells were shake-cultured at 37 ° C. in an 8% CO ₂ incubator at 95 rpm for 1 hour. 1.8 mg of Polyethylenimine (manufactured by Polyscience) was dissolved in Opti-Pro SFM (Thermo Fisher SCIENTIFIC) to make 20 mL, and then a PureLink HiPure plasmid kit (Thermo Fisher SCIENT) was used to prepare 0.2 mg of the PureLink HiPure Plusmid kit (ThermoFisher SCIENT). And 0.36 mg of heavy chain expression vector was suspended in Opti-Pro SFM (manufactured by Thermo Fisher SCIENTIFIC) to make 20 mL. To 20 mL of the Polyethylenimine / Opti-Pro SFM mixture, 20 mL of the expression vector / Opti-Pro SFM mixture was added, the mixture was gently stirred, and the mixture was allowed to stand for another 5 minutes before being added to FreeStyle 293F cells. After culturing in an 8% CO ₂ incubator at 37 ° C. for 4 hours at 95 rpm, 600 mL of EX-CELL VPRO medium (manufactured by SAFC Biosciences) and 30 mL of BD Recharge CD (manufactured by BD Biosciences) were added, and 37 ° C. The culture supernatant obtained by shaking in an 8% CO ₂ incubator at 95 rpm for 6 days was filtered through a Disposable Capsule Filter (manufactured by ADVANTEC).

5) -6 Purification of human chimeric anti-human IL-22BP antibody The antibody was purified from the culture supernatant obtained in Example 5) -5 by a one-step process of rProtein A affinity chromatography at 4 to 6 ° C. The buffer replacement step after rProteinA affinity chromatography purification was performed at 4-6 ° C. First, the culture supernatant was applied to a column packed with MabSelectSuRe (manufactured by GE Healthcare Japan) equilibrated with PBS. After all the culture was in the column, the column was washed with PBS at least twice the column volume. Next, it was eluted with 2M arginine hydrochloride solution (pH 4.0), and the fraction containing the antibody was collected. The fraction was dialyzed against a Slide-A-Lyzer Diarysis Cassette (manufactured by Thermo Fisher SCIENTIFIC) and replaced with HBSor (25 mM histidine, 5% sorbitol, pH 6.0). Finally, the sample was concentrated at Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut-off UF10K, manufactured by Sartorius) at 4 ° C. to adjust the IgG concentration to 10 mg / mL and used as a purified sample.

Example 6. Human chimeric anti-human IL-22BP antibody in vitro evaluation 6) -1 Binding activity evaluation (human): SPR
The binding dissociation constant between the human chimeric anti-human IL-22BP antibody prepared in Example 5) -6 and the human IL-22BP-His prepared in Example 1) -2 was measured by Biacore T200 (manufactured by GE Healthcare Japan). The human chimeric antibody was captured as a ligand in an anti-human IgG (Fc) antibody immobilized using Human Antibody Capital Kit (manufactured by GE Healthcare Japan), and the antigen was measured as an analyte. .. HBS-EP + (manufactured by GE Healthcare Japan) was used as the running buffer. A human chimeric anti-human IL-22BP antibody diluted to 2 μg / mL was added onto the sensor chip at 10 μL / min for 30 seconds, and then a diluted series solution of human IL-22BP-His as an antigen (from 243 nM to a 5 concentration series with a common ratio of 3). ) Was added at a flow rate of 30 μL / min for 300 seconds, and the dissociated phase was subsequently measured for 600 seconds. As a regeneration solution, 3M magnesium chloride (manufactured by GE Healthcare Japan Co., Ltd.) was added at a flow rate of 20 μL / min for 30 seconds. For the analysis of the data, a 1: 1 binding model was used to calculate the binding rate constant ka, the dissociation rate constant cd, and the binding dissociation constant KD.

6) -2 Species crossover evaluation (monkey, mouse):
6) -2-1 Binding activity evaluation (monkey): BLI
For the measurement of the binding dissociation constant between the human chimeric anti-human IL-22BP antibody prepared in Example 5) -6 and the monkey IL-22BP-His prepared in Example 1) -2, Antigen RED 384 (manufactured by MOLECULAR DEVICES) was used. The human chimeric antibody was captured as a ligand in the biosensor Protein A (ProA) (manufactured by MOLECULAR DEVICES), and PBS-T (manufactured by Takara Bio Co., Ltd.) was used as a buffer for measurement using the antigen as an analyte. Was measured. After reacting the biosensor with a human chimeric anti-human IL-22BP antibody diluted to 3 μg / mL for 120 seconds, a diluted series solution of monkey IL-22BP-His (7.81, 15.6, 31.3, 62.5). , 125, 250, 500 nM) for 900 seconds. As a regeneration solution, 10 mM Glycine pH 1.5 (manufactured by GE Healthcare Japan) was reacted with the biosensor for 25 seconds. The data was analyzed by equilibrium value analysis, and the binding dissociation constant KD was calculated.

6) -2-2 Binding activity evaluation (mouse): SPR
The binding dissociation constant between the human chimeric anti-human IL-22BP antibody prepared in Example 5) -6 and the mouse IL-22BP-His prepared in Example 1) -2 was measured by Biacore T200 (manufactured by GE Healthcare Japan). The antigen was captured as a ligand in Anti-6 × His tag antibody [AD1.1.10] (manufactured by Abcam) immobilized using Amine Coupling Kit, type2 (manufactured by GE Healthcare Japan). (Captured), and the human chimeric antibody was measured as an analyte. HBS-EP + (manufactured by GE Healthcare Japan) was used as the running buffer. After adding mouse IL-22BP-His diluted to 1 μg / mL on the sensor chip at 10 μL / min for 60 seconds, a diluted series solution of human chimeric anti-human IL-22BP antibody (5 concentration series from 1 μM to a common ratio of 2) was added. The mixture was added at a flow rate of 30 μL / min for 300 seconds. As a regeneration solution, 3M magnesium chloride (manufactured by GE Healthcare Japan Co., Ltd.) was added at a flow rate of 20 μL / min for 45 seconds. The data was analyzed by equilibrium value analysis, and the binding dissociation constant KD was calculated.

Example 7. Design of humanized anti-human IL-22BP antibody 7) -1 Design of humanization: For each clone, each H, L chain 7) -1-1 Molecular modeling of variable region Known method as homology modeling (Methods in Enzymemogy, 203,121-153 (1991)) was used. Registered in Protein data Bank (Nuc. Acid Res. 35, D301-D303 (2007)), which has high sequence homology to variable regions, using a commercially available protein three-dimensional structure analysis program Discovery Studio (manufactured by Dassault Systèmes). I searched for the structure that is being used. A three-dimensional model structure was created using the hit heavy chain, light chain, and the interface structure between the heavy chain and the light chain as a template.

7) -1-2 Design method of humanized antibody The construction of humanized anti-human IL-22BP antibody is generally referred to as CDR graphing (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). It was carried out by a known method. The framework regions of MAb3 are the human kappa chains IGKV2D-30 * 01, IGKJ4 * 01 and KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) to have a high consensus sequence from the human γ chain subgroup 1 of the human γ chain subgroup. They were selected as MAb3 light and heavy chain acceptors, respectively. The framework area of MAb4 is KABAT et al. Since they have high homology to the consensus sequences of human κ chain subgroup 1, human κ chain subgroup 2 and human κ chain subgroup 4 and human γ chain subgroup 3 defined by, they are the light chains of MAb4. And selected as heavy chain acceptors, respectively. Donor residues to be transferred onto the acceptor are described in Queen et al. The three-dimensional model was analyzed with reference to the criteria given by (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)) and independently designed according to each sequence.

7) -1-3 Humanization of MAb3 light chain A humanized antibody light chain was designed by connecting the κ chain constant region of human IgG1 to the designed MAb3 humanized antibody light chain variable region, and hMAb3_L01, hMAb3_L02, and hMAb3_L03, respectively. , HMAb3_L04, hMAb3_L05, hMAb3_L06, hMAb3_L07. The full-length amino acid sequence of hMAb3_L01 is set forth in SEQ ID NO: 121. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 121 is set forth in SEQ ID NO: 110. The full-length amino acid sequence of hMAb3_L03 is set forth in SEQ ID NO: 122. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 122 is set forth in SEQ ID NO: 111. The full-length amino acid sequence of hMAb3_L04 is set forth in SEQ ID NO: 123. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 123 is set forth in SEQ ID NO: 112. The full-length amino acid sequence of hMAb3_L05 is set forth in SEQ ID NO: 124. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 124 is set forth in SEQ ID NO: 113. The full-length amino acid sequence of hMAb3_L07 is set forth in SEQ ID NO: 125. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 125 is set forth in SEQ ID NO: 114.

7) -1-4 Humanization of MAb3 heavy chain A humanized antibody heavy chain in which the γ chain constant region of human IgG1 is connected to the designed MAb3 humanized antibody heavy chain variable region is designed, and hMAb3_H01, hMAb3_H02, hMAb3_H03, respectively. It was named hMAb3_H04. The full-length amino acid sequence of hMAb3_H01 is set forth in SEQ ID NO: 138. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138 is set forth in SEQ ID NO: 132. The full-length amino acid sequence of hMAb3_H03 is set forth in SEQ ID NO: 139. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 139 is set forth in SEQ ID NO: 133. The full-length amino acid sequence of hMAb3_H04 is set forth in SEQ ID NO: 140. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 140 is set forth in SEQ ID NO: 134.

7) -1-5 Humanization of MAb4 light chain A humanized antibody light chain was designed by connecting the κ chain constant region of human IgG1 to the designed MAb4 humanized antibody light chain variable region, and hMAb4_L01, hMAb4_L02, respectively. They were named hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06. The full-length amino acid sequence of hMAb4_L01 is described in 126. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 126 is set forth in SEQ ID NO: 115. The full-length amino acid sequence of hMAb4_L02 is described in 127. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 127 is set forth in SEQ ID NO: 116. The full-length amino acid sequence of hMAb4_L03 is described in 128. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 128 is set forth in SEQ ID NO: 117. The full-length amino acid sequence of hMAb4_L04 is described in 129. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129 is set forth in SEQ ID NO: 118. The full-length amino acid sequence of hMAb4_L05 is described in 130. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 130 is set forth in SEQ ID NO: 119. The full-length amino acid sequence of hMAb4_L06 is described in 131. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 131 is set forth in SEQ ID NO: 120.

7) -1-6 Humanization of MAb4 heavy chain A humanized antibody heavy chain in which the γ chain constant region of human IgG1 is connected to the designed humanized antibody heavy chain variable region of MAb4 was designed, and hMAb4_H01, hMAb4_H02, and hMAb4_H03, respectively. I named it. The full-length amino acid sequence of hMAb4_H01 is described in 141. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 141 is set forth in SEQ ID NO: 135. The full-length amino acid sequence of hMAb4_H02 is described in 142. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 142 is set forth in SEQ ID NO: 136. The full-length amino acid sequence of hMAb4_H03 is described in 143. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 143 is set forth in SEQ ID NO: 137.

Example 8. Construction of expression vector for humanized anti-human IL-22BP antibody and preparation of antibody 8) -1 Construction of light chain expression vector for humanized anti-human IL-22BP antibody Humanized anti-human shown in SEQ ID NOs: 110-120 of the sequence listing. A DNA fragment containing a DNA sequence encoding a variable region in the nucleotide sequence of the IL-22BP antibody light chain was synthesized (artificial gene synthesis service manufactured by GENEART). Using the synthesized DNA fragment as a template, amplify the DNA fragment containing the DNA sequence encoding the variable region with KOD-Plus- (manufactured by TOYOBO) and the following primer set, and use the light chain expression vector pCMA-LK with the restriction enzyme BsiWI. A vector expressing the light chain of the humanized anti-human IL-22BP antibody was constructed by inserting it into the cut site using an In-Fusion HD Cloning Kit (manufactured by CLONTECH). The amino acid sequences of the humanized anti-human IL-22BP antibody light chain are shown in SEQ ID NOs: 121-131 in the sequence listing.
Primer set 5'-CTGTGGATCTCCGGCGCGTACCGGC-3'(Primer CM-LKF: SEQ ID NO: 91)
5'-GGAGGGGGCGCGCCACCGTACG-3'(Primer KCL-Inf-R: SEQ ID NO: 92)

8) -2 Construction of heavy chain expression vector for humanized anti-human IL-22BP antibody A DNA fragment containing a DNA sequence encoding a variable region in the heavy chain nucleotide sequence shown in SEQ ID NOs: 132 to 137 of the sequence table was synthesized. (Artificial gene synthesis service manufactured by GENEART). Using the synthesized DNA fragment as a template, amplify the DNA fragment containing the DNA sequence encoding the variable region with KOD-Plus- (manufactured by TOYOBO) and the following primer set, and use the heavy chain expression vector pCMA-G1 with the restriction enzyme BlpI. An expression vector expressing the heavy chain of the humanized anti-human IL-22BP antibody was constructed by inserting it into the cut site using an In-Fusion HD Cloning Kit (manufactured by CLONTECH). The amino acid sequence of the humanized anti-human IL-22BP antibody heavy chain is shown in SEQ ID NOs: 138 to 143 of the sequence listing.
Primer set 5'-AGCTCCCAGATGGGTGCTGAGC-3' (Primer EG-Inf-F: SEQ ID NO: 108)
5'-GGGCCTTGGTGGGAGGCTGAGC-3'(Primer EG1-Inf-R: SEQ ID NO: 109)

8) -3 Small-scale production of humanized anti-human IL-22BP antibody FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) were subcultured and cultured according to the manual.

1 × 10 ⁷ FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) in the logarithmic growth phase were diluted with 9.6 mL of FreeStyle 293 expression medium (manufactured by Thermo Fisher SCIENTIFIC) and then seeded with 9.6 mL of Nalgene Kitchen (manufactured by Thermo Fisher SCIENTIFIC). , 37 ° C., in an 8% CO ₂ incubator, 90 rpm, 1 hour shaking culture. 30 μg of Polyethyleneimine (manufactured by Polyscience) was dissolved in 200 μL of Opti-Pro SFM (manufactured by Thermo Fisher SCIENTIFIC), and then a PureLink HiPure Plusmid kit (manufactured by Thermo Fisher SCIENTIFIC) was used to prepare a purelink HiPure plasmid kit (Thermo Fisher SCI) expression chain expression vector SCIEN. 4 μg was added to 200 μL of Opti-Pro SFM (manufactured by Thermo Fisher SCIENTIFIC). To 200 μL of the Polyethylenimine / Opti-Pro SFM mixture, 200 μL of the expression vector / Opti-Pro SFM mixture was added, the mixture was gently stirred, and the mixture was allowed to stand for another 5 minutes before being added to FreeStyle 293F cells. The culture supernatant obtained by shaking in an 8% CO ₂ incubator at 37 ° C. for 7 days at 90 rpm was filtered through a Minisart-Plus filter (manufactured by Sartorius) to prepare a sample for evaluation.

The humanized antibody obtained by the combination of hMAb3_H01 and hMAb3_L03 was obtained by "hMAb3_H01L03", the humanized antibody obtained by the combination of hMAb3_H01 and hMAb3_L04 was obtained by "hMAb3_H01L04", and the humanized antibody obtained by the combination of hMAb3_H03 and hMAb3_L01 was obtained by the combination of hMAb3_H03 and hMAb3_L01. "HMAb3_H03L01", the humanized antibody obtained by the combination of hMAb3_H03 and hMAb3_L03 was "hMAb3_H03L03", and the humanized antibody obtained by the combination of hMAb3_H03 and hMAb3_L05 was "hMAb3_H03L05" and the combination of hMAb3_H03 and hMAb3_H04. The humanized antibody is "hMAb3_H04L03", the humanized antibody obtained by the combination of hMAb3_H04 and hMAb3_L07 is "hMAb3_H04L07", and the humanized antibody obtained by the combination of hMAb4_H01 and hMAb4_L01 is "hMAb4_H01L01", hMAb4_H01L01. The humanized antibody obtained by the combination of "hMAb4_H01L02", hMAb4_H01 and hMAb4_L04 was "hMAb4_H01L04", and the humanized antibody obtained by the combination of hMAb4_H01 and hMAb4_L05 was "hMAb4_H01L05", hMAb4_H01L05. The humanized antibody obtained by the combination with hMAb4_L06 is "hMAb4_H01L06", the humanized antibody obtained by the combination of hMAb4_H02 and hMAb4_L01 is "hMAb4_H02L01", and the humanized antibody obtained by the combination of hMAb4_H02 and hMAb4_L02 is the humanized antibody "hMAb4_L02". , The humanized antibody obtained by the combination of hMAb4_H02 and hMAb4_L03 is "hMAb4_H02L03", the humanized antibody obtained by the combination of hMAb4_H02 and hMAb4_L04 is "hMAb4_H02L04", and the combination of hMAb4_H02 and hMAb4_L05. The humanized antibody obtained by combining the antibody with "hMAb4_H02L05" and hMAb4_H02 and hMAb4_L06 by "hMAb4_H02L06", by the combination of hMAb4_H03 and hMAb4_L01. The humanized antibody obtained by the combination of "hMAb4_H03L01", hMAb4_H03 and hMAb4_L02 was "hMAb4_H03L02", and the humanized antibody obtained by the combination of hMAb4_H03 and hMAb4_L03 was "hMAb4_H03L03", hMAb4_H03L03. The humanized antibody obtained by the combination with hMAb4_L04 is "hMAb4_H03L04", the humanized antibody obtained by the combination of hMAb4_H03 and hMAb4_L05 is "hMAb4_H03L05", and the humanized antibody obtained by the combination of hMAb4_H03 and hMAb4_L06 is "hMAb4_L06". I named it.

8) -4 Production of humanized anti-human IL-22BP antibody "hMAb3_H01L03", "hMAb3_H01L04", "hMAb3_H04L03", "hMAb4_H02L05", "hMAb4_H03L01", "hMAb4_H03L03" in the same manner as in Example 5) -5. bottom.

FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) were subcultured and cultured according to the manual.

1.2 × 10 ⁹ FreeStyle 293F cells (manufactured by Thermo Fisher SCIENTIFIC) in the logarithmic growth phase were inoculated into a 3L Fernbach Erlenmeyer Flask (manufactured by CORNING), diluted with FreeStyle 293Express (manufactured by CORNING), and diluted with FreeStyle 293 Express After preparation, the cells were shake-cultured at 37 ° C. in an 8% CO ₂ incubator at 95 rpm for 1 hour. 1.8 mg of Polyethylenimine (manufactured by Polyscience) was dissolved in Opti-Pro SFM (manufactured by Thermo Fisher SCIENTIFIC) to make 20 mL, and then a PureLink HiPure plasmid kit (manufactured by Thermo Fisher SCIENTIFIC) was used to prepare a Vector 0 vector prepared by Thermo Fisher (Thermo Fisher SCI). 24 mg and 0.36 mg of heavy chain expression vector were suspended in Opti-Pro SFM (manufactured by Thermo Fisher SCIENTIFIC) to make 20 mL. To 20 mL of the Polyethylenimine / Opti-Pro SFM mixture, 20 mL of the expression vector / Opti-Pro SFM mixture was added, the mixture was gently stirred, and the mixture was allowed to stand for another 5 minutes before being added to FreeStyle 293F cells. After culturing in an 8% CO ₂ incubator at 37 ° C. for 4 hours at 95 rpm, 600 mL of EX-CELL VPRO medium (manufactured by SAFC Biosciences) and 30 mL of BD Recharge CD (manufactured by BD Biosciences) were added, and 37 ° C. The culture supernatant obtained by shaking in an 8% CO ₂ incubator at 95 rpm for 6 days was filtered through a Disposable Capsule Filter (manufactured by ADVANTEC).

8) -5 Purification of humanized anti-human IL-22BP antibody The culture supernatant obtained in Example 8) -4 was subjected to rProteinA affinity chromatography at 4 to 6 ° C. and ceramic hydroxylapatite at room temperature. Purified in a step step. The buffer replacement step after the purification of rProteinA affinity chromatography and the purification of ceramic hydroxylapatite was carried out at 4 to 6 ° C. First, the culture supernatant was applied to MabSelectSuRe (manufactured by GE Healthcare Japan) equilibrated with PBS. After the culture medium was completely contained in the column, the column was washed with PBS having a column volume of 2 times or more. Next, it was eluted with 2M arginine hydrochloride solution at pH 4.0, and the fraction containing the antibody was collected. The fraction was dialyzed against PBS using Slide-A-Lyzer Diarysis Cassette (manufactured by ThermoFiser SCIENTIFIC), and then the antibody solution diluted 5-fold with a buffer of 5 mM sodium phosphate, 50 mM MES and pH 7.0 was prepared. It was applied to a ceramic hydroxyl apatite column Bio-Scale CHT Type-I Hydroxyapatite Solution (manufactured by Nippon Biorad Co., Ltd.) equilibrated with a buffer of 5 mM NaPi, 50 mM MES, 30 mM NaCl, and pH 7.0. A linear concentration gradient elution with sodium chloride was performed and fractions containing the antibody were collected. The fraction was replaced with HBSor (25 mM histidine, 5% sorbitol, pH 6.0) by dialysis using Slide-A-Lyzer Dialysis Cassette (manufactured by Thermo Fisher SCIENTIFIC). Finally, the sample was concentrated at Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut-off UF10K, manufactured by Sartorius) at 4 ° C., and the IgG concentration was adjusted to 30 or 50 mg / mL or more to prepare a purified sample.

Example 9. In vitro evaluation of humanized anti-human IL-22BP antibody 9) -1 Binding activity evaluation (human): SPR
The binding dissociation constant between the humanized anti-human IL-22BP antibody prepared in Example 8) -5 and the human IL-22BP-His prepared in Example 1) -2 is the SPR method as in Example 6) -1. It was measured and calculated in. As shown in FIG. 2, the binding dissociation constant of the humanized antibody of the hMAb3 line is wide from 13.8 nM to 513 nM, and the binding dissociation constant of the humanized antibody of the hMAb4 line is 8.14 nM to 12.5 nM, which is generally stronger than that of the hMAb3 line. Shown binding.

9) -2 Species crossover evaluation (monkey): BLI
The binding dissociation constant between the humanized anti-human IL-22BP antibody prepared in Example 8) -5 and the monkey IL-22BP-His prepared in Example 1) -2 was measured by Octet RED 384 (manufactured by MOLECULAR DEVICES). The humanized antibody was captured as a ligand in the biosensor Protein A (ProA) (manufactured by MOLECULAR DEVICES), and the antigen was used as an analyze and PBS-T (manufactured by Takara Bio) as a buffer solution for measurement. Was measured using. After reacting the biosensor with a humanized anti-human IL-22BP antibody diluted to 3 μg / mL for 120 seconds, a diluted series solution of monkey IL-22BP-His (7.0, 20.0, 40.0, 75. It reacted with 0, 150, 270, 500 nM) for 900 seconds. As a regeneration solution, 10 mM Glycine pH 1.5 (manufactured by GE Healthcare Japan) was reacted with the biosensor for 25 seconds. The data was analyzed by equilibrium value analysis, and the binding dissociation constant KD was calculated. FIG. 3 shows a list of bond dissociation constants.

9) -3 Competitive inhibition experiment using Competition ELISA 6 × -His Tag Polyclonal Antibody (Thermo Fisher SCIENTIFIC) diluted with PBS to 2 μg / mL and added to 96-well Maxi-Sorp plates (Nunc) by 50 After that, it was solidified overnight at 4 ° C. The next day, after washing twice with PBS containing 0.05% Tween-20 (PBS-T), 200 μL of Block BSA in PBS (manufactured by Thermo Fisher SCIENTIFIC) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing twice with PBS-T, add 50 μL of human IL-22BP-His diluted to 2 μg / mL with PBS-T (buffer for dilution) containing 1% BSA (manufactured by JACKSON IMMUNO RESEARCH) at 37 ° C. It was allowed to stand for 1 hour. After washing 5 times with PBS-T, add 50 μL each of a diluted series solution of humanized anti-human IL-22BP antibody (75 μg / mL to 5 concentration series with a common ratio of 5) prepared with a buffer for dilution, and then dilute. Recombinant diluted to 1 μg / mL with buffer solution

Only 5 μL of Human IL-22 Protein (manufactured by R & D Systems) or a buffer for dilution was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing 5 times with PBS-T, 50 μL of Human IL-22 Antibody (manufactured by R & D Systems) diluted to 0.5 μg / mL with a diluting buffer was added, and the mixture was allowed to stand at 37 ° C. for 1 hour. After washing 5 times with PBS-T, 50 μL of Goat Anti-Mouse IgG Antibody, HRP conjugate, and Species Advanced (Merck) diluted to 0.8 μg / mL with a diluting buffer was added, and the temperature was 37 ° C. for 1 hour. It was left still. After washing 6 times with PBS-T, 100 μL of OPD color-developing solution was added, and after standing for 6 minutes, 100 μL of 1M HCl was added, and the absorbance at 490 nm was measured with Enspire (manufactured by PerkinElmer). The inhibition rate was calculated by the following formula.
Inhibition rate (%) = (1-Maximum absorbance Abs490 when antibody is added / Maximum absorbance Abs490 when IL-22 is added alone) × 100

Control IgG (ctrlIgG), HBSor pH 6.0 was used for control. As shown in FIG. 4, all the antibodies showed antibody concentration-dependent inhibitory activity, and the humanized antibodies hMAb3H01L03, hMAb3H01L04, and hMAb3H04L03 designed from the human chimeric antibody cMAb3 and the antibody were humanized from the human chimeric antibody cMAb4 and the antibody. It showed a stronger inhibitory tendency than the antibodies hMAb4H03L01, hMAb4H03L03, and hMAb4H02L05.

Example 10. Ex vivo evaluation of humanized anti-human IL-22BP antibody 10) -1 Extraction of human colonic epithelial cell crypt site
Extraction of the crypt site of human colonic epithelial cells was performed by Mizoguchi E, et al. (Gastroenterology 125: 148-161 (2003)) was used as a reference. A tissue having a size of 2 cm × 2 cm was collected from a human colon surgery excised specimen, and then washed with PBS. In order to increase the penetration efficiency of EDTA, the lamina propria was exfoliated from the muscular layer, and then the exfoliated lamina propria was washed again with PBS. The isolated lamina propria was soaked in 30 mL of 30 mM EDTA / HANKS solution and left at room temperature for 15 minutes, and then shaken vigorously for about 30 seconds by hand to confirm that the epithelium was exfoliated in the crypt structure.

The solution containing the crypts was filtered through a 20 μm pore filter, and the crypts remaining on the filter were collected and collected by immersing them in a 4% FBS / RPMI solution.

10) -2 Detection of phosphorylated STAT3 10) After counting the cells of the human colonic epithelial cell crypts recovered in -1, 6 × 10 ⁵ crypts were placed in 200 μL of 4% FBS / RPMI solution and Recombinant Human IL. -22 Protein (manufactured by R & D Systems) was stimulated at 37 ° C. for 15 minutes at 10 ng / mL. At that time, the human IL-22BP (10 ng / mL) prepared in Example 1) -2 and the humanized anti-human IL22-BP antibody (1, 10 to 100 μg / mL) prepared in Examples 8) -5. Was added simultaneously or 20 minutes before the addition of IL-22 to evaluate the action of the humanized anti-human IL22-BP antibody. By adding human IL22-BP and humanized anti-human IL-22BP antibody after the addition of IL-22, it is also possible to evaluate the action of the antibody in post-treatment.

After collecting the cells, the cells were washed twice with PBS, Lysis buffer [50 mM Tris (pH 8.0), 0.5% NP-40, 1 mM EDTA, 150 mM NaCl, 1 mM sodium vanate, 50 mM sodium pyrophosphate, 1 mM PMSF, 1 mM It was dissolved in [table of protease inhibitor chloride], homogenized, and then rapidly frozen on dry ice. The Western blotting method for detecting phosphorylated STAT3 is described by Mizoguchi A, et al. (Immunity.16 (2): 219-230 (2002)) was used as a reference. The frozen specimen was rapidly thawed at 37 ° C., the cell membrane was completely destroyed by sonication, and then the supernatant was recovered by centrifugation at 12,000 rpm and 4 ° C. for 30 minutes. After developing each supernatant with SDS-PAGE, it was transferred to a nitrocellulose membrane, and a phosphorylated STAT3-specific band was detected with an ECL detection reagent (manufactured by GE Healthcare Japan). For detection, an anti-phosphorylated STAT3 antibody manufactured by Cell Signaling Technology was used.

FIG. 5 shows the detection result of phosphorylated STAT3 when the evaluation antibody was pretreated. Compared with the blank group, the phosphorylation signal of STAT3 in cells derived from the crypt site of human colonic epithelial cells was strongly observed in the IL-22-added group, whereas phosphorylation was observed when an equal amount of IL-22BP was added. The signal band has disappeared. On the other hand, it was confirmed that the phosphorylation signal was restored by adding each humanized anti-human IL-22BP antibody. The total STAT3 band indicates the total amount of STAT3 protein signal.

The antibody of the present invention is useful for the treatment of inflammatory bowel disease and the like.

SEQ ID NO: 1: Primer Dneo-F
SEQ ID NO: 2: Primer Dneo-R
SEQ ID NO: 3: FLAGHis tag sequence SEQ ID NO: 4: DNA encoding a polypeptide containing DYKDDDDKHHHHH, positions 1 to 20 of the amino acid sequence of human IL2.
SEQ ID NO: 5: DNA encoding a sequence containing a polypeptide in which ENLYFQG is linked to the C-terminal side of positions 1 to 263 of the amino acid sequence of human IL-22BP (Isoform 1).
SEQ ID NO: 6: Human IL-22BP-His nucleotide sequence SEQ ID NO: 7: Amino acid sequence of human IL-22BP-His SEQ ID NO: 8: Amino acid sequence of monkey IL-22BP at positions 1 to 21 of the amino acid sequence of human IL-22BP. DNA encoding a sequence containing a polypeptide in which ENLYFQG is linked to the C-terminal side of the 21st to 231st positions of
SEQ ID NO: 9: Binary sequence of monkey IL-22BP-His SEQ ID NO: 10: Amino acid sequence of monkey IL-22BP-His SEQ ID NO: 11: Mice in which Cys106, 1st to 21st of the amino acid sequence of human IL-22BP, is replaced with Ser. DNA encoding a sequence containing a polypeptide in which ENLYFQG is linked to the C-terminal side of positions 21 to 230 of the amino acid sequence of IL-22BP.
SEQ ID NO: 12: Binary sequence of mouse IL-22BP-His SEQ ID NO: 13: Amino acid sequence of mouse IL-22BP-His SEQ ID NO: 14: Amino acid sequence of human IL-22BP with cleaved tags SEQ ID NO: 15: Rat anti-human IL- 22BP antibody rMAb3 light chain variable region nucleotide sequence SEQ ID NO: 16: rat anti-human IL-22BP antibody rMAb4 light chain variable region nucleotide sequence SEQ ID NO: 17: rat anti-human IL-22BP antibody rMAb8 light chain variable Amino Acid SEQ ID NO: 18: Vessel SEQ ID NO: 19 of the variable region of the light chain of the rat anti-human IL-22BP antibody rMAb14: Amino acid SEQ ID NO: 20 of the variable region of the light chain of the rat anti-human IL-22BP antibody rMAb20: Amino acid sequence of variable region of light chain of rat anti-human IL-22BP antibody rMAb81 SEQ ID NO: 21: Amino acid sequence of variable region of light chain of rat anti-human IL-22BP antibody rMAb3 SEQ ID NO: 22: Rat anti-human IL-22BP antibody rMAb4 Amino acid sequence of variable region of light chain SEQ ID NO: 23: Amino acid of variable region of light chain of rat anti-human IL-22BP antibody rMAb8 SEQ ID NO: 24: Amino acid of variable region of light chain of rat anti-human IL-22BP antibody rMAb14 Distribution SEQ ID NO: 25: Amino acid of variable region of light chain of rat anti-human IL-22BP antibody rMAb20 SEQ ID NO: 26: Amino acid of variable region of light chain of rat anti-human IL-22BP antibody rMAb81 SEQ ID NO: 27: CDRL1 of MAb3_VL Amino acid version of SEQ ID NO: 28: Amino acid version of CDRL2 of MAb3_VL SEQ ID NO: 29: Amino acid version of CDRL3 of MAb3_VL SEQ ID NO: 30: Amino acid version of CDRL1 of MAb4_VL SEQ ID NO: 31: Amino acid version of CDRL2 of MAb4_VL. 32: Amino acid version of CDRL3 of MAb4_VL SEQ ID NO: 33: Amino acid version of CDRL1 of MAb8_VL SEQ ID NO: 34: Amino acid version of CDRL2 of MAb8_VL SEQ ID NO: 35: Amino acid version of CDRL3 of MAb8_VL SEQ ID NO: 36: CDRL1 of MAb14_VL Amino acid version SEQ ID NO: 37: Amino acid version of CDRL2 of MAb14_VL SEQ ID NO: 38: Amino acid version of CDRL3 of MAb14_VL SEQ ID NO: 39: Amino acid version of CDRL1 of MAb20_VL Noic acid version SEQ ID NO: 40: Amino acid version of CDRL2 of MAb20_VL SEQ ID NO: 41: Amino acid version of CDRL3 of MAb20_VL SEQ ID NO: 42: Amino acid version of CDRL1 of MAb81_VL SEQ ID NO: 43: Amino acid version of CDRL2 of MAb81_VL. 44: Amino acid version of CDRL3 of MAb81_VL SEQ ID NO: 45: Binary of variable region of heavy chain of rat anti-human IL-22BP antibody rMAb3 SEQ ID NO: 46: Substance of variable region of heavy chain of rat anti-human IL-22BP antibody rMAb4 SEQ ID NO: 47: Amino acid of the variable region of the heavy chain of the rat anti-human IL-22BP antibody rMAb8 SEQ ID NO: 48: Amino acid of the variable region of the heavy chain of the rat anti-human IL-22BP antibody rMAb14 SEQ ID NO: 49: Rat anti-human IL -22BP antibody rMAb20 heavy chain variable region nucleotide sequence SEQ ID NO: 50: rat anti-human IL-22BP antibody rMAb81 heavy chain variable region nucleotide sequence SEQ ID NO: 51: rat anti-human IL-22BP antibody rMAb3 heavy chain Amino acid sequence of variable region 52: Amino acid sequence of variable region of heavy chain of rat anti-human IL-22BP antibody rMAb4 SEQ ID NO: 53: Amino acid sequence of variable region of heavy chain of rat anti-human IL-22BP antibody rMAb8 : Amino acid sequence of variable region of heavy chain of rat anti-human IL-22BP antibody rMAb14 SEQ ID NO: 55: Amino acid sequence of variable region of heavy chain of rat anti-human IL-22BP antibody rMAb20 SEQ ID NO: 56: Amino acid sequence of rat anti-human IL-22BP antibody Amino acid sequence of variable region of heavy chain of rMAb81 SEQ ID NO: 57: Amino acid version of CDRH1 of MAb3_VH SEQ ID NO: 58: Amino acid version of CDRH2 of MAb3_VH SEQ ID NO: 59: Amino acid version of CDRH3 of MAb3_VH SEQ ID NO: 60: CDRH1 of MAb4_VH Amino acid version of SEQ ID NO: 61: Amino acid version of MAb4_VH Amino acid version of SEQ ID NO: 62: Amino acid version of MAb4_VH for Amino acid version of SEQ ID NO: 63: Amino acid version of MAb8_VH for CDRH1 SEQ ID NO: 64: Amino acid version of MAb8_VH for amino acid version of SEQ ID NO. 65: Amino acid version of CDRH3 of MAb8_VH SEQ ID NO: 66: Amino acid version of CDRH1 of MAb14_VH SEQ ID NO: 67: CDRH of MAb14_VH Amino acid version of 2 SEQ ID NO: 68: Amino acid version of CDRH3 of MAb14_VH SEQ ID NO: 69: Amino acid version of CDRH1 of MAb20_VH SEQ ID NO: 70: Amino acid version of CDRH2 of MAb20_VH SEQ ID NO: 71: Amino acid version of CDRH3 of MAb20_VH No. 72: Amino acid version of CDRH1 of MAb81_VH SEQ ID NO: 73: Amino acid version of MAb81_VH of CDRH2 SEQ ID NO: 74: Amino acid version of MAb81_VH of CDRH3 SEQ ID NO: 75: Encodes human κ chain secretory signal and human κ chain constant region Nucleotide sequence containing DNA SEQ ID NO: 76: Primer 3.3-F1
SEQ ID NO: 77: Primer 3.3-R1
SEQ ID NO: 78: Substance sequence containing a human heavy chain signal sequence and a DNA sequence encoding an amino acid in the human IgG1 constant region SEQ ID NO: 79: Human chimeric anti-human IL-22BP antibody cMAb3 light chain nucleotide sequence No. 80: Human chimeric anti-human IL- 22BP antibody cMAb4 light chain nucleotide sequence No. 81: human chimeric anti-human IL-22BP antibody cMAb8 light chain nucleotide sequence No. 82: human chimera anti-human IL-22BP antibody cMAb14 light chain nucleotide sequence No. 83: human chimera anti-human IL-22BP antibody cMAb20 light chain nucleotide sequence No. 84: human chimeric anti-human IL-22BP antibody cMAb81 light chain nucleotide sequence No. 85: human chimera anti-human IL-22BP antibody cMAb3 light chain amino acid sequence No. 86: human chimera anti-human IL-22BP antibody cMAb4 light Chain amino acid sequence SEQ ID NO: 87: Human chimeric anti-human IL-22BP antibody cMAb8 light chain amino acid SEQ ID NO: 88: Human chimera anti-human IL-22BP antibody cMAb14 light chain amino acid sequence number 89: Human chimera anti-human IL-22BP antibody cMAb20 light chain amino acid SEQ ID NO: 90: Human chimera anti-human IL-22BP antibody cMAb81 Light chain amino acid SEQ ID NO: 91: Primer CM-LKF
SEQ ID NO: 92: Primer KCL-Inf-R
SEQ ID NO: 93: Human chimera anti-human IL-22BP antibody cMAb3 heavy chain nucleotide sequence No. 94: Human chimera anti-human IL-22BP antibody cMAb4 heavy chain nucleotide sequence number 95: Human chimera anti-human IL-22BP antibody cMAb8 heavy chain nucleotide sequence number 96: Human chimeric anti-human IL-22BP antibody cMAb14 heavy chain nucleotide sequence number 97: Human chimeric anti-human IL-22BP antibody cMAb20 heavy chain nucleotide sequence number 98: Human chimeric anti-human IL-22BP antibody cMAb81 heavy chain nucleotide sequence number 99: Human chimera anti-human IL-22BP antibody cMAb3 heavy chain amino acid sequence number 100: Human chimera anti-human IL-22BP antibody cMAb4 heavy chain amino acid sequence number 101: Human chimera anti-human IL-22BP antibody cMAb8 heavy chain amino acid sequence number 102: human chimera anti Human IL-22BP antibody cMAb14 heavy chain amino acid sequence number 103: human chimeric anti-human IL-22BP antibody cMAb20 heavy chain amino acid sequence number 104: human chimeric anti-human IL-22BP antibody cMAb81 heavy chain amino acid sequence number 105: MAb4'_VH Amino acid version of CDRH1 SEQ ID NO: 106: Amino acid version of CDRH2 of MAb4'_VH SEQ ID NO: 107: Amino acid version of CDRH3 of MAb4'_VH SEQ ID NO: 108: Primer EG-Inf-F
SEQ ID NO: 109: Primer EG1-Inf-R
SEQ ID NO: 110: nucleotide sequence number 111 of hMAb3_L01: nucleotide sequence number 112 of hMAb3_L03: nucleotide sequence number 113 of hMAb3_L04: nucleotide sequence number 114 of hMAb3_L05: nucleotide sequence number 115 of hMAb3_L07: nucleotide sequence number 115 of hMAb4_L01 116: nucleotide sequence number 117 of hMAb4_L02: nucleotide sequence number 118 of hMAb4_L03: nucleotide sequence number 119 of hMAb4_L04: nucleotide sequence number 120 of hMAb4_L05: nucleotide sequence number 121 of hMAb4_L06: amino acid sequence number 122 of hMAb3_L01: hMAb3_L03 amino acid sequence SEQ ID NO: 123: hMAb3_L04 amino acid sequence SEQ ID NO: 124: hMAb3_L05 amino acid sequence SEQ ID NO: 125: hMAb3_L07 amino acid sequence SEQ ID NO: 126: hMAb4_L01 amino acid sequence SEQ ID NO: 127: hMAb4_L02 amino acid sequence number 128: 03hMAb Amino acid SEQ ID NO: 129: Amino acid sequence number 130 of hMAb4_L04: Amino acid sequence number 131 of hMAb4_L05: Amino acid sequence number 132 of hMAb4_L06: Binary sequence number 133 of hMAb3_H01: Baselet sequence number 134 of hMAb3_H04 SEQ ID NO: 135: nucleotide sequence number 136 of hMAb4_H01: nucleotide sequence number 137 of hMAb4_H02: nucleotide sequence number 138 of hMAb4_H03: amino acid sequence number 139 of hMAb3_H01: amino acid sequence number 140 of hMAb3_H04: amino acid sequence number 140: hMAb3_H04 141: Amino acid sequence of hMAb4_H01 SEQ ID NO: 142: Amino acid sequence of hMAb4_H02 SEQ ID NO: 143: Amino acid sequence of hMAb4_H03 All publications, patents and patent applications cited herein are incorporated herein by reference. ..

Claims

An antibody or a binding fragment thereof having the properties described in (i) to (iv) below:
(I) A monoclonal antibody that binds to human IL-22BP;
(Ii) Binds to monkey IL-22BP;
(Iii) The binding dissociation constant (KD) to human IL-22BP-His is 1 × 10-7 M or less; and (iv) IL-22 and IL-22BP in cells derived from the human colonic epithelial cell crypt site. Simultaneous or pretreatment with respect to the disappearance of the phosphorylation signal of STAT3 observed at the time of addition restores the signal.
The antibody according to claim 1 or a bound fragment thereof, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.
The antibody or binding fragment thereof according to claim 1 or 2, which does not intersect with IL-22BP of rodents.
The antibody or binding fragment thereof according to any one of claims 1 to 3, wherein the binding dissociation constant (KD) for human IL-22BP-His is 1 × 10 -7 M or less in the SPR method.
Any one of claims 1 to 4, wherein in the BLI method, the binding dissociation constant (KD) for monkey IL-22BP-His is 1 × 10 -6 M or less, preferably 5 × 10 -7 M or less. The antibody or binding fragment thereof according to the section.
(A1) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 27,
(B1) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and (c1) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 29.
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d1) SEQ ID NO: 57,
(E1) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 58, and (f1) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 59.
The antibody according to any one of claims 1 to 5, or a binding fragment thereof, comprising a heavy chain containing the above.
(A2) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 30.
(B2) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 31, and (c2) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 32.
A light chain comprising, and a heavy chain CDRH1 consisting of (d2) the amino acid sequence set forth in SEQ ID NO: 60 or SEQ ID NO: 105,
(E2) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 61 or SEQ ID NO: 106, and (f2) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 107.
The antibody according to any one of claims 1 to 5, or a binding fragment thereof, comprising a heavy chain containing the above.
(A3) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 33,
(B3) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and (c3) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 35,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d3) SEQ ID NO: 63,
(E3) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 64, and (f3) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 65.
The antibody according to claim 1 or 2, or a binding fragment thereof, comprising a heavy chain containing the above.
(A4) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 36,
(B4) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and (c4) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 38,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d4) SEQ ID NO: 66,
(E4) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 67, and (f4) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 68.
The antibody according to claim 1 or 2, or a binding fragment thereof, comprising a heavy chain containing the above.
(A5) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 39,
(B5) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 40, and (c5) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 41,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d5) SEQ ID NO: 69,
(E5) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 70, and (f5) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 71.
The antibody according to any one of claims 1 to 3, or a binding fragment thereof, comprising a heavy chain containing the above.
(A6) Light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 42,
(B6) Light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 43, and (c6) Light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44,
A light chain comprising, and a heavy chain CDRH1 consisting of the amino acid sequence set forth in (d6) SEQ ID NO: 72,
(E6) Heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 73, and (f6) Heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 74.
The antibody according to any one of claims 1 to 3, or a binding fragment thereof, comprising a heavy chain containing the above.
The antibody or a binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a humanized antibody.
The antibody or a binding fragment thereof according to any one of claims 1 to 5 and 7, wherein the antibody is a humanized antibody.
The antibody or a binding fragment thereof according to any one of claims 1, 2 and 8, wherein the antibody is a humanized antibody.
The antibody or a binding fragment thereof according to any one of claims 1, 2 and 9, wherein the antibody is a humanized antibody.
The antibody or a binding fragment thereof according to any one of claims 1 to 3 and 10, wherein the antibody is a humanized antibody.
The antibody or a binding fragment thereof according to any one of claims 1 to 3 and 11, wherein the antibody is a humanized antibody.
Claims 1 to 6 include any one light chain variable region selected from (La1) to (Le1) below and any one heavy chain variable region selected from (Ha1) to (Hc1). And the antibody according to any one of 12 or a binding fragment thereof:
(La1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 121.
(Lb1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122.
(Lc1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 123.
(Ld1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 124.
(Le1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 125,
(Ha1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 138.
(Hb1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 139.
(Hc1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 140.
1. Antibodies or binding fragments thereof:
(La2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 121.
(Lb2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122.
(Lc2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 123.
(Ld2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 124.
(Le2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 125.
(Ha2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 138.
(Hb2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 139.
(Hc2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 140.
Claims 1 to 5 comprising any one light chain variable region selected from (Lf1) to (Lk1) below and any one heavy chain variable region selected from (Hd1) to (Hf1) below. , 7 and 13, the antibody or binding fragment thereof according to any one of:
(Lf1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 126,
(Lg1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 127,
(Lh1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 128,
(Li1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 129.
(Lj1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 130.
(Lk1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 131.
(Hd1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 141.
(He1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 142.
(Hf1) A heavy chain variable region consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 143.
1. Antibodies or binding fragments thereof:
(Lf2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 126.
(Lg2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 127.
(Lh2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128.
(Li2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 129.
(Lj2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 130.
(Lk2) A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 131.
(Hd2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 141.
(He2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 142.
(Hf2) A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143.
The antibody according to any one of claims 1 to 6, 12 and 18, which comprises a combination of any heavy chain variable region and light chain variable region selected from (X1) to (X3) below. Bonded fragment:
(X1) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the sequence listing. Variable area,
(X2) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 123 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the sequence listing. Variable area,
(X3) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 132 of SEQ ID NO: 140 in the sequence listing. Variable area.
The antibody or binding fragment thereof according to claim 1, which comprises a combination of any heavy chain variable region and light chain variable region selected from (X1') to (X3') below:
(X1') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 132.
(X2') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 123 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 132.
(X3') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 140 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 132.
13. The antibody described or a binding fragment thereof:
(X4) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the sequence listing. Variable area,
(X5) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the sequence listing. Variable area,
(X6) A light chain variable region consisting of the amino acid sequences shown in amino acid numbers 21 to 133 of SEQ ID NO: 130 in the sequence listing, and a heavy chain consisting of the amino acid sequences shown in amino acid numbers 20 to 137 of SEQ ID NO: 142 in the sequence listing. Variable area.
The antibody or binding fragment thereof according to claim 1, wherein the antibody comprises a combination of any heavy chain variable region and light chain variable region selected from (X4') to (X6') below:
(X4') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 137.
(X5') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 137.
(X6') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 130 in the sequence listing, and the amino acid of SEQ ID NO: 142 in the sequence listing. A heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 137.
Claims 1 to 6, 12 and 18, comprising any one light chain selected from the following (Ll1) to (Lp1) and any one heavy chain selected from (Hg1) to (Hi1). The antibody according to any one of the following items:
(Ll1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 121 in the sequence listing.
(Lm1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing.
(Ln1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing.
(Lo1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 124 in the sequence listing.
(Lp1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 125 in the sequence listing.
(Hg1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing.
(Hh1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 139 in the sequence listing.
(Hi1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing.
The antibody according to claim 1, which comprises any one light chain selected from the following (Ll2) to (Lp2) and any one heavy chain selected from (Hg2) to (Hi2):
(Ll2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 121 in the sequence listing.
(Lm2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing.
(Ln2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing.
(Lo2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 124 in the sequence listing.
(Lp2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 125 in the sequence listing.
(Hg2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing.
(Hh2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 139 in the sequence listing.
(Hi2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing.
Claims 1 to 5, 7, 13 and comprising any one light chain selected from the following (Lq1) to (Lv1) and any one heavy chain selected from (Hj1) to (Hl1). The antibody according to any one of 20:
(Lq1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing.
(Lr1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 127 in the sequence listing.
(Ls1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing.
(Lt1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 129 in the sequence listing.
(Lu1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing.
(Lv1) A light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 131 in the sequence listing.
(Hj1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 467 of SEQ ID NO: 141 in the sequence listing.
(Hk1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the sequence listing.
(Hl1) A heavy chain comprising the amino acid sequences shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing.
The antibody according to claim 1, which comprises any one light chain selected from the following (Lq2) to (Lv2) and any one heavy chain selected from (Hj2) to (Hl2):
(Lq2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing.
(Lr2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 127 in the sequence listing.
(Ls2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing.
(Lt2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 129 in the sequence listing.
(Lu2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing.
(Lv2) A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 131 in the sequence listing.
(Hj2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 141 in the sequence listing.
(Hk2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the sequence listing.
(Hl2) A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing.
The antibody according to any one of claims 1 to 6, 12, 18 and 22, which comprises any heavy chain and light chain selected from the following (Y1) to (Y3):
(Y1) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid sequences shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing. Heavy chain,
(Y2) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the sequence listing. Heavy chain,
(Y3) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing. Heavy chain.
The antibody according to claim 1, which comprises any of the heavy and light chains selected from the following (Y1') to (Y3'):
(Y1') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 462.
(Y2') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the sequence listing, and the amino acid of SEQ ID NO: 138 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 462.
(Y3') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the sequence listing, and the amino acid of SEQ ID NO: 140 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 462.
10. The first aspect of claim 1, wherein the antibody of claim 30 binds to a site on human IL-22BP to which it binds, or competes with the antibody of claim 30 or a binding fragment thereof in binding to human IL-22BP. Antibodies or binding fragments thereof.
The antibody according to any one of claims 1 to 5, 7, 13, 20 and 24, which comprises any heavy chain and light chain selected from the following (Y4) to (Y6):
(Y4) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing. Heavy chain,
(Y5) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing. Heavy chain,
(Y6) Containing a light chain comprising the amino acid sequences shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing, and the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the sequence listing. Heavy chain.
The antibody according to claim 1, which comprises any of the heavy and light chains selected from the following (Y4') to (Y6'):
(Y4') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 467.
(Y5') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing, and the amino acid of SEQ ID NO: 143 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 467.
(Y6') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the sequence listing, and the amino acid of SEQ ID NO: 142 in the sequence listing. A heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in Nos. 20 to 467.
Claim 33, which binds to a site on human IL-22BP to which the antibody or binding fragment thereof binds, or competes with the antibody or binding fragment thereof according to claim 33 in binding to human IL-22BP. Item 1. The antibody or binding fragment thereof.
The antibody or binding fragment thereof according to any one of claims 1 to 35, wherein the heavy chain lacks the carboxyl-terminal lysine.
A polynucleotide containing a nucleotide sequence encoding an amino acid sequence contained in the heavy chain and light chain of the antibody according to any one of claims 1 to 36.
A vector containing the polynucleotide according to claim 37.
A host cell comprising the polynucleotide according to claim 37 or the vector according to claim 38.
The method for producing an antibody or a bound fragment thereof according to any one of claims 1 to 36, which comprises culturing the host cell according to claim 39 and recovering the antibody or the bound fragment thereof from the culture.
An antibody or a binding fragment thereof prepared by the method according to claim 40.
The antibody or binding fragment thereof according to any one of claims 1 to 36, which forms a complex with a drug.
The antibody or binding fragment thereof according to any one of claims 1 to 36, which is contained in a multi-characteristic molecule.
A pharmaceutical composition comprising the antibody according to any one of claims 1 to 36 and 41 to 43 or a binding fragment thereof, the polynucleotide according to claim 37, the vector according to claim 38, and the cell according to claim 39 as an active ingredient. thing.
A prophylactic or therapeutic agent for one or more diseases selected from the group consisting of infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune myocardial disease, bronchitis, uveitis, and inflammatory bowel disease. The pharmaceutical composition according to claim 44.
The pharmaceutical composition according to claim 45, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
The pharmaceutical composition according to any one of claims 44 to 46, which is used in combination with other pharmaceutical products.
A test agent, diagnostic agent or reagent for detecting human IL-22BP, which comprises the antibody according to any one of claims 1 to 36 and 41 to 43 or a binding fragment thereof.