JP7789368B2 - Therapeutic antibodies for inflammatory bowel disease - Google Patents

Description

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

Cytokines are generally important for cell differentiation, proliferation, and migration, and because they play a defensive role against tumors and infections, they are deeply involved in chronic inflammatory diseases, wound healing, infectious diseases, cancer, and other conditions. Interleukin-22 (IL-22) is one of the cytokines induced in immune cells upon infection with pathogenic bacteria, and is known to be expressed and secreted by T cells, natural killer cells, dendritic cells, and other cells resident in human lungs, skin, tonsils, uterus, and intestinal tract (small intestine and large intestine) (Non-Patent Document 1). In particular, IL-22 is strongly expressed in the intestine during enteritis, and acts on intestinal epithelial cells expressing the IL-22 receptor (IL-22R), inducing the production of mucus and antibacterial proteins in intestinal epithelial cells via STAT3 (Signal Transducer and Activator of Transcription 3) signaling, thereby suppressing inflammation (Non-Patent Document 1). IL-22 dysfunction has been suggested to be associated with infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, etc., and is well known to be involved in inflammatory bowel diseases, including ulcerative colitis and Crohn's disease (Non-Patent Documents 1 and 2).

IL-22BP is a soluble cytokine receptor, also known as IL-22Ra2, CDR2-10, etc., which 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, intestine, etc., and several animal models and human clinical data suggest that IL-22BP directly inhibits the mucosal protective effect of IL-22 in the intestinal tract (Non-patent Documents 2, 6).

Although there have been studies (Patent Documents 1 and 2) on the use of anti-IL-22BP antibodies to inhibit the binding of IL-22BP to IL-22 for the prevention, treatment, and diagnosis of diseases, the diseases targeted were limited to metabolic disorders such as obesity, diabetes, and dyslipidemia, and only evaluated the effects of rabbit anti-mouse IL-22BP polyclonal antibodies (Patent Document 1), or only suggested that increased expression and function of IL-22 may lead to treatment in the treatment of inflammatory bowel disease caused by microbial infection (Patent Document 2).

International Publication No. WO2007/076422 European Application Publication No. EP2514436A2

Mizoguchi A. Healing of Intestinal inflammation by IL-22, Inflammatory Bowel Disease 2012, Volume 18, Number 9, pp 1777-1784 Mizoguchi A, et al. Clinical importance of IL-22 cascade in IBD, Journal of Gastroenterology, 2018, Volume 53, Issue 4, pp 465-474 Xu W, et al. A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist, Proceedings of the National Academy of Science of the United States of America. 2001, Volume 98, pp 9511-9516 Dumoutier L, et al. Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell derived inducible factor/IL-22, Journal of Immunology. 2001, Volume 166, pp 7090-7095 Kotenko SV, et al. Identification, cloning, and characterization of a novel soluble receptor that binds IL-22 AND neutralizes its activity, Journal of Immunology. 2001, Volume 166, pp 7096-7103 Sugimoto K, et al. IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis, Journal of Clinical Investigation. 2008, Volume 118(2), pp 534-544

The present invention aims to provide an anti-IL-22BP antibody that can treat infectious diseases suggested to be associated with IL-22, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, and inflammatory bowel diseases including ulcerative colitis and Crohn's disease, by enhancing the function of IL-22, as well as pharmaceutical compositions and the like containing the antibody as an active ingredient.

The inventors investigated IL-22BP and found that inhibiting the binding of IL-22BP to IL-22 with an anti-IL-22BP antibody enhances the function of IL-22.

That is, the present invention is as follows.
[1] An antibody or a binding fragment thereof having the following properties (i) to (iv):
(i) a monoclonal antibody that binds to human IL-22BP;
(ii) binds to simian IL-22BP;
(iii) the binding dissociation constant (KD) for human IL-22BP-His is 1 x ^10-7 M or less; and (iv) in cells derived from the crypt region of human colon epithelial cells, the STAT3 phosphorylation signal observed upon addition of IL-22 and IL-22BP is lost, but the signal is restored by simultaneous or pretreatment with IL-22 and IL-22BP.
[2] The antibody or binding fragment thereof according to [1], wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.
[3] The antibody or binding fragment thereof according to [1] or [2], which does not cross-react with rodent IL-22BP.
[4] The antibody or binding fragment thereof according to any one of [1] to [3], which has a binding dissociation constant (KD) for human IL-22BP-His of 1×10 ⁻⁷ M or less in an SPR assay.
[5] The antibody or binding fragment thereof according to any one of [1] to [4], which has a binding dissociation constant (KD) for monkey IL-22BP-His of 1×10 ⁻⁶ M or less, preferably 5×10 ⁻⁷ M or less, in a BLI assay.
[6] (a1) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 27;
(b1) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 28, and (c1) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 29;
(d1) a light chain comprising:
(e1) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 58, and (f1) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 59.
[0022] The antibody or binding fragment thereof according to any one of [1] to [5], wherein the heavy chain comprises:
[7] (a2) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 30;
(b2) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 31, and (c2) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 32;
and (d2) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 60 or SEQ ID NO: 105;
(e2) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 61 or SEQ ID NO: 106, and (f2) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 107.
[0022] The antibody or binding fragment thereof according to any one of [1] to [5], wherein the heavy chain comprises:
[8] (a3) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 33;
(b3) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and (c3) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 35;
and (d3) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 63;
(e3) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 64, and (f3) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 65.
The antibody or binding fragment thereof according to [1] or [2], wherein the heavy chain comprises:
[9] (a4) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 36;
(b4) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and (c4) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 38;
(d4) a light chain comprising a heavy chain CDRH1 having the amino acid sequence shown in SEQ ID NO: 66;
(e4) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 67, and (f4) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 68.
The antibody or binding fragment thereof according to [1] or [2], wherein the heavy chain comprises:
[10] (a5) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 39;
(b5) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 40, and (c5) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 41;
(d5) a light chain comprising a heavy chain CDRH1 having the amino acid sequence shown in SEQ ID NO: 69;
(e5) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 70, and (f5) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 71.
An antibody or a binding fragment thereof according to any one of [1] to [3], wherein the antibody or binding fragment thereof comprises a heavy chain comprising:
[11] (a6) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 42;
(b6) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 43, and (c6) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 44;
and (d6) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 72;
(e6) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 73, and (f6) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 74.
An antibody or a binding fragment thereof according to any one of [1] to [3], wherein the antibody or binding fragment thereof comprises a heavy chain comprising:
[12] The antibody or binding fragment thereof according to any one of [1] to [6], wherein the antibody is a humanized antibody.
[13] The antibody or binding fragment thereof according to any one of [1] to [5] and [7], wherein the antibody is a humanized antibody.
[14] The antibody or binding fragment thereof according to any one of [1], [2] and [8], wherein the antibody is a humanized antibody.
[15] The antibody or binding fragment thereof according to any one of [1], [2] and [9], wherein the antibody is a humanized antibody.
[16] The antibody or binding fragment thereof according to any one of [1] to [3] and [10], wherein the antibody is a humanized antibody.
[17] The antibody or binding fragment thereof according to any one of [1] to [3] and [11], wherein the antibody is a humanized antibody.
[18] An antibody or binding fragment thereof according to any one of [1] to [6] and [12], comprising any one light chain variable region selected from (La1) to (Le1) below, and any one heavy chain variable region selected from (Ha1) to (Hc1):
(La1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 121;
(Lb1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 122;
(Lc1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 123;
(Ld1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 124;
(Le1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 125;
(Ha1) a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 138;
(Hb1) a heavy chain variable region consisting of the amino acid sequence represented by amino acid numbers 20 to 132 of SEQ ID NO: 139;
(Hc1) A heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 140.
[19] The antibody or binding fragment thereof according to [1], comprising any one light chain variable region selected from (La2) to (Le2) below, and any one heavy chain variable region selected from (Ha2) to (Hc2):
(La2) a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by 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 to the amino acid sequence represented by 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 represented by 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 represented by 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 represented by 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 represented by 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 represented by 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 represented by amino acid numbers 20 to 132 of SEQ ID NO: 140.
[20] An antibody or binding fragment thereof according to any one of [1] to [5], [7], and [13], 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):
(Lf1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 126;
(Lg1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 127;
(Lh1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 128;
(Li1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 129;
(Lj1) a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 130;
(Lk1) a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 131;
(Hd1) a heavy chain variable region consisting of the amino acid sequence represented by amino acid numbers 20 to 137 of SEQ ID NO: 141;
(He1) a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 142;
(Hf1) A heavy chain variable region consisting of the amino acid sequence represented by amino acid numbers 20 to 137 of SEQ ID NO: 143.
[21] The antibody or binding fragment thereof according to [1], comprising any one light chain variable region selected from (Lf2) to (Lk2) below, and any one heavy chain variable region selected from (Hd2) to (Hf2):
(Lf2) a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by 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 to the amino acid sequence represented by 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 to the amino acid sequence represented by 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 represented by 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 represented by 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 represented by 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 represented by 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 represented by 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 represented by amino acid numbers 20 to 137 of SEQ ID NO: 143.
[22] Any of the antibodies or binding fragments thereof according to [1] to [6], [12], and [18], comprising a combination of a heavy chain variable region and a light chain variable region selected from the following (X1) to (X3):
(X1) a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the Sequence Listing;
(X2) a light chain variable region consisting of the amino acid sequence shown by amino acid numbers 21 to 133 of SEQ ID NO: 123 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence shown by amino acid numbers 20 to 132 of SEQ ID NO: 138 in the Sequence Listing;
(X3) A light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 122 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence represented by amino acid numbers 20 to 132 of SEQ ID NO: 140 in the Sequence Listing.
[23] The antibody or binding fragment thereof according to [1], comprising any combination of heavy chain variable regions and light chain variable regions 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 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 in the Sequence Listing;
(X2') a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by amino acid numbers 21 to 133 of SEQ ID NO: 123 in the Sequence Listing, and a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by amino acid numbers 20 to 132 of SEQ ID NO: 138 in the Sequence Listing;
(X3') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 122 in the Sequence Listing, and a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by amino acid numbers 20 to 132 of SEQ ID NO: 140 in the Sequence Listing.
[24] The antibody or binding fragment thereof of any one of [1] to [5], [7], [13], and [20], wherein the antibody comprises a combination of a heavy chain variable region and a light chain variable region selected from the following (X4) to (X6):
(X4) a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing;
(X5) a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing;
(X6) A light chain variable region consisting of the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 130 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence represented by amino acid numbers 20 to 137 of SEQ ID NO: 142 in the Sequence Listing.
[25] The antibody or binding fragment thereof of [1], wherein the antibody comprises any combination of a heavy chain variable region and a light chain variable region selected from the following (X4') to (X6'):
(X4') a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by amino acid numbers 21 to 133 of SEQ ID NO: 126 in the Sequence Listing, and a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing;
(X5') a light chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by amino acid numbers 21 to 133 of SEQ ID NO: 128 in the Sequence Listing, and a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown by amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing;
(X6') A light chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by amino acid numbers 21 to 133 of SEQ ID NO: 130 in the Sequence Listing, and a heavy chain variable region consisting of an amino acid sequence having 95% or more sequence identity to the amino acid sequence represented by amino acid numbers 20 to 137 of SEQ ID NO: 142 in the Sequence Listing.
[26] Any one of the antibodies of [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 the following (Hg1) to (Hi1):
(Ll1) a light chain comprising the amino acid sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the sequence listing.
[27] The antibody of [1], comprising any one light chain selected from the following (Ll2) to (Lp2) and any one heavy chain selected from the following (Hg2) to (Hi2):
(L12) 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] Any one of the antibodies of [1] to [5], [7], [13] and [20], comprising any one light chain selected from the following (Lq1) to (Lv1) and any one heavy chain selected from the following (Hj1) to (Hl1):
(Lq1) a light chain comprising the amino acid sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence 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 sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing.
[29] The antibody of [1], comprising any one light chain selected from the following (Lq2) to (Lv2) and any one heavy chain selected from the following (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 of the antibodies of [1] to [6], [12], [18], and [22], comprising any heavy chain and light chain selected from the following (Y1) to (Y3):
(Y1) a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the Sequence Listing, and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the Sequence Listing;
(Y2) a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the Sequence Listing, and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the Sequence Listing;
(Y3) A light chain comprising the amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO: 122 in the Sequence Listing, and a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 462 of SEQ ID NO: 140 in the Sequence Listing.
[31] The antibody of [1], comprising any heavy chain and light chain 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 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;
(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 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;
(Y3') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO: 122 in the Sequence Listing, and a heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by amino acid numbers 20 to 462 of SEQ ID NO: 140 in the Sequence Listing.
[32] The antibody or binding fragment thereof of [1], which binds to the site on human IL-22BP to which the antibody of [30] binds, or which competes with the antibody or binding fragment thereof of [30] in binding to human IL-22BP.
[33] Any of the antibodies of [1] to [5], [7], [13], [20] and [24], comprising any heavy chain and light chain selected from the following (Y4) to (Y6):
(Y4) a light chain comprising the amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO: 126 in the Sequence Listing, and a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 467 of SEQ ID NO: 143 in the Sequence Listing;
(Y5) a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the Sequence Listing, and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the Sequence Listing;
(Y6) A light chain comprising the amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO: 130 in the Sequence Listing, and a heavy chain comprising the amino acid sequence represented by amino acid numbers 20 to 467 of SEQ ID NO: 142 in the Sequence Listing.
[34] The antibody of [1], comprising any heavy chain and light chain 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 represented by amino acid numbers 21 to 239 of SEQ ID NO: 126 in the Sequence Listing, and a heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by amino acid numbers 20 to 467 of SEQ ID NO: 143 in the Sequence Listing;
(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 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;
(Y6') A light chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by amino acid numbers 21 to 239 of SEQ ID NO: 130 in the Sequence Listing, and a heavy chain comprising an amino acid sequence having 95% or more sequence identity with the amino acid sequence represented by amino acid numbers 20 to 467 of SEQ ID NO: 142 in the Sequence Listing.
[35] The antibody or binding fragment thereof of [1], which binds to the site on human IL-22BP to which the antibody or binding fragment thereof of [33] binds, or which competes with the antibody or binding fragment thereof of [33] in binding to human IL-22BP.
[36] An antibody or binding fragment thereof according to any one of [1] to [35], wherein the heavy chain lacks a carboxyl-terminal lysine.
[37] A polynucleotide comprising a nucleotide sequence encoding the amino acid sequences contained in the heavy and light chains of the antibody of [1] to [36].
[38] A vector comprising 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], comprising 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 is complexed with a drug.
[43] The antibody or binding fragment thereof according to any one of [1] to [36], which is included in a multi-specific molecule.
[44] A pharmaceutical composition comprising as an active ingredient an antibody or binding fragment thereof of any of [1] to [36] and [41] to [43], a polynucleotide of [37], a vector of [38], or a cell of [39].
[45] The pharmaceutical composition according to [44], which is a preventive or therapeutic agent for 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.
[46] The pharmaceutical composition according to [45], wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
[47] A pharmaceutical composition according to any one of [44] to [46], which is used in combination with other pharmaceuticals.
[48] A test agent, diagnostic agent or reagent for detecting human IL-22BP, comprising the antibody or binding fragment thereof according to any one of [1] to [36] and [41] to [43].
This specification includes the disclosure of Japanese Patent Application No. 2020-095058, from which this application claims priority.

Anti-IL-22BP antibodies inhibit the binding of IL-22BP to IL-22, thereby enhancing the function of IL-22.

Infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, and inflammatory bowel diseases including ulcerative colitis and Crohn's disease, which are thought to be related to IL-22, can be treated by enhancing the function of IL-22.

This figure shows the binding activity to human IL-22BP-His, mouse IL-22BP-His, and monkey IL-22BP-His measured by ELISA. The binding activity of rat anti-human IL-22BP antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81) to human IL-22BP-His, mouse IL-22BP-His, and monkey IL-22BP-His was evaluated. The vertical axis shows the absorbance at a wavelength of 490 nm measured by ELISA. 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 competitive ELISA were shown. Inhibition of the binding of IL-22BP to IL-22 by humanized anti-human IL-22BP antibodies (hMAb3_H01L03, hMAb3_H01L04, hMAb3_H04L03, hMAb4_H03L01, hMAb4_H03L03, and hMAb4_H02L05) and human chimeric antibodies (cMAb3 and cMAb4) was measured by competitive ELISA. The vertical axis represents the inhibition rate, and the horizontal axis represents the antibody concentration added. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - absorbance Abs490 upon addition of antibody/maximum absorbance Abs490 upon addition of IL-22 alone) × 100. Control IgG (ctrlIgG) and HBS (pH 6.0) were used as controls. 1 shows the results of detecting phosphorylated STAT3 using human colon epithelial cell crypts. Evaluation was performed using humanized anti-human IL-22BP antibodies (hMAb3_H01L03, hMAb3_H01L04, hMAb3_H02L05, hMAb4_H02L05, hMAb4_H03L01, and hMAb4_H03L03). FIG. 1 shows the sequences of SEQ ID NOs: 1 and 2 (primers Dneo-F and Dneo-R) and SEQ ID NO: 3 (FLAGHis tag sequence). FIG. 1 shows the sequences of SEQ ID NOs: 4 and 5. FIG. 1 shows the sequences of SEQ ID NOs: 6 and 7. FIG. 1 shows the sequence of SEQ ID NO:8. FIG. 1 shows the sequence of SEQ ID NO: 9. FIG. 1 shows the sequence of SEQ ID NO: 10. FIG. 1 shows the sequence of SEQ ID NO: 11. FIG. 1 shows the sequence of SEQ ID NO: 12. FIG. 1 shows the sequence of SEQ ID NO: 13. FIG. 1 shows the sequence of SEQ ID NO: 14. FIG. 1 shows the sequences of SEQ ID NOs: 15-17. FIG. 1 shows the sequences of SEQ ID NOs: 18-20. FIG. 1 shows the sequences of SEQ ID NOs: 21 to 26. FIG. 1 shows the sequences of SEQ ID NOs: 27 to 38 (CDRs of VL of anti-IL-22BP antibodies). FIG. 1 shows the sequences of SEQ ID NOs: 39 to 44 (CDRs of VL of anti-IL-22BP antibodies). FIG. 1 shows the sequences of SEQ ID NOs: 45-47. FIG. 1 shows the sequences of SEQ ID NOs: 48-50. FIG. 1 shows the sequences of SEQ ID NOs: 51 to 56. FIG. 1 shows the sequences of SEQ ID NOs: 57 to 68 (CDRs of VH of anti-IL-22BP antibodies). FIG. 1 shows the sequences of SEQ ID NOs: 69 to 74 (CDRs of VH of anti-IL-22BP antibodies). FIG. 1 shows the sequence of SEQ ID NO: 75. FIG. 1 shows the sequences of SEQ ID NOs: 76 and 77 (primers 3.3-F1, 3.3-R1). FIG. 1 shows the sequence of SEQ ID NO: 78. FIG. 1 shows the sequence of SEQ ID NO: 79. FIG. 1 shows the sequence of SEQ ID NO: 80. FIG. 1 shows the sequence of SEQ ID NO: 81. FIG. 1 shows the sequence of SEQ ID NO: 82. FIG. 1 shows the sequence of SEQ ID NO: 83. FIG. 1 shows the sequence of SEQ ID NO: 84. FIG. 1 shows the sequences of SEQ ID NOs: 85-87. FIG. 1 shows the sequences of SEQ ID NOs: 88-90. FIG. 1 shows the sequences of SEQ ID NOs: 91 and 92 (primers CM-LKF, KCL-Inf-R). FIG. 1 shows the sequence of SEQ ID NO: 93. FIG. 1 shows the sequence of SEQ ID NO: 94. FIG. 1 shows the sequence of SEQ ID NO: 95. FIG. 1 shows the sequence of SEQ ID NO: 96. FIG. 1 shows the sequence of SEQ ID NO: 97. FIG. 1 shows the sequence of SEQ ID NO: 98. FIG. 1 shows the sequences of SEQ ID NOs: 99 and 100. FIG. 1 shows the sequences of SEQ ID NOs: 101 and 102. FIG. 1 shows the sequences of SEQ ID NOs: 103 and 104. FIG. 1 shows the sequences of SEQ ID NOs: 105 to 107 (CDRs of VH of anti-IL-22BP antibodies). FIG. 1 shows the sequences of SEQ ID NOs: 108 and 109 (primers EG-Inf-F, EG1-Inf-R). FIG. 1 shows the sequence of SEQ ID NO: 110. FIG. 1 shows the sequence of SEQ ID NO: 111. FIG. 1 shows the sequence of SEQ ID NO: 112. FIG. 1 shows the sequence of SEQ ID NO: 113. FIG. 1 shows the sequence of SEQ ID NO: 114. FIG. 1 shows the sequence of SEQ ID NO: 115. FIG. 1 shows the sequence of SEQ ID NO: 116. FIG. 1 shows the sequence of SEQ ID NO: 117. FIG. 1 shows the sequence of SEQ ID NO: 118. FIG. 1 shows the sequence of SEQ ID NO: 119. FIG. 1 shows the sequence of SEQ ID NO: 120. FIG. 1 shows the sequences of SEQ ID NOs: 121-123. FIG. 1 shows the sequences of SEQ ID NOs: 124-126. FIG. 1 shows the sequences of SEQ ID NOs: 127-129. FIG. 1 shows the sequences of SEQ ID NOs: 130 and 131. FIG. 1 shows the sequence of SEQ ID NO: 132. FIG. 1 shows the sequence of SEQ ID NO: 133. FIG. 1 shows the sequence of SEQ ID NO: 134. FIG. 1 shows the sequence of SEQ ID NO: 135. FIG. 1 shows the sequence of SEQ ID NO: 136. FIG. 1 shows the sequence of SEQ ID NO: 137. FIG. 1 shows the sequences of SEQ ID NOs: 138 and 139. FIG. 1 shows the sequences of SEQ ID NOs: 140 and 141. FIG. 1 shows the sequences of SEQ ID NOs: 142 and 143.

The present invention is an antibody that binds to a soluble cytokine receptor (IL-22BP).
1. Definition In the present invention, the term "gene" refers to a nucleic acid molecule containing a nucleotide sequence encoding the amino acids of a protein, or a complementary strand thereof. For example, polynucleotides, oligonucleotides, DNA, mRNA, cDNA, cRNA, etc., having a nucleotide sequence encoding the amino acids of a protein or a nucleotide sequence complementary to that sequence are included in the meaning of "gene." Such genes are single-stranded, double-stranded, triple- or more-stranded nucleotides, and the term "gene" also includes an assembly of a DNA strand and an RNA strand, a nucleotide strand in which ribonucleotides (RNA) and deoxyribonucleotides (DNA) are mixed, and double-stranded, triple- or more-stranded nucleotides containing such nucleotide strands. The "IL-22BP gene" of the present invention can include, for example, DNA, mRNA, cDNA, cRNA, etc., 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 include, for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. Such nucleotides are single-stranded, double-stranded, or composed of three or more strands. The term "nucleotide" also includes an aggregate of a DNA strand and an RNA strand, a mixture of ribonucleotides (RNA) and deoxyribonucleotides (DNA) on a single nucleotide strand, and an aggregate of two or more strands containing such a nucleotide strand.

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

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

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

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

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

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

The monoclonal antibodies used in the present invention can be obtained by immunizing 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, fusing spleen cells and myelomas to produce hybridomas, and then producing and secreting antibodies from the hybridomas. Hybridomas can be produced by known methods.

Antibodies can be screened by any method, but preferably, screening is performed by ELISA using immobilized IL-22BP protein.

In the present invention, a "functional fragment of an antibody" refers to an antibody fragment that exhibits at least a portion of the function exhibited by the original antibody. Examples of "functional fragments of an antibody" include, but are not limited to, Fab, F(ab')2, scFv, Fab', and single-chain immunoglobulins. Such functional fragments of antibodies may be obtained by treating full-length antibody protein molecules with enzymes such as papain and pepsin, as well as recombinant proteins produced in appropriate host cells using recombinant genes. Among "functional fragments of antibodies," those that have binding activity to an antigen, i.e., human IL-22BP, are referred to as "binding fragments of an antibody."

In the present invention, the "site" to which an antibody binds, i.e., the "site" recognized by an antibody, refers to a partial peptide or partial higher-order structure on an antigen to which the antibody binds or recognizes. In the present invention, such a site is also referred to as an epitope or 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 partial higher-order structure on the IL-22BP protein.

It is known that the heavy and light chains of an antibody molecule each contain three complementarity-determining regions (CDRs). Complementarity-determining regions, also known as hypervariable domains, are located within the variable regions of the heavy and light chains of an antibody and are regions with particularly high variability in their primary structure. They are typically separated into three regions in the primary structure of the heavy and light polypeptide chains. In the present invention, the complementarity-determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 from the amino-terminus of the heavy chain amino acid sequence, and CDRL1, CDRL2, and CDRL3 from the amino-terminus of the light chain amino acid sequence. These regions are adjacent to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind. The portion of the heavy chain variable region amino acid sequence other than CDRH1 to CDRH3 is called the framework region (FR), and the portions from the amino terminus to just before CDRH1, from after CDRH1 to just before CDRH2, from after CDRH2 to just before CDRH3, and from after CDRH3 to the carboxyl terminus are called FRH1 to FRH4, respectively. Similarly, the portion of the light chain variable region amino acid sequence other than CDRL1 to CDRL3 is also FR, and the portions from the amino terminus to just before CDRL1, from after CDRL1 to just before CDRL2, from after CDRL2 to just before CDRL3, and from after CDRL3 to the carboxyl terminus are called FRL1 to FRL4, respectively. That is, in the heavy chain and light chain variable regions (amino acid sequences), the amino acids are arranged consecutively from the amino terminus to the carboxyl terminus in the order FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4.

In the present invention, the term "antibody mutant" refers to a polypeptide that has an amino acid sequence in which amino acids have been substituted, deleted, added, and/or inserted (hereinafter collectively referred to as "mutations") in the amino acid sequence of the original antibody, and that binds to the IL-22BP protein of the present invention. The number of mutated amino acids in such an antibody mutant is 1, 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 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 mutants are also encompassed by the "antibody" of the present invention.

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

The antibodies used in the present invention also include modified antibodies. The term "modified antibody" refers to an antibody of the present invention that has been chemically or biologically modified. Chemical modifications include those in which a chemical moiety is attached to the amino acid backbone or an N- or O-linked carbohydrate chain. Biological modifications include those that have undergone post-translational modification (e.g., addition of an N- or O-linked glycan, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, etc.), and those in which a methionine residue has been added to the N-terminus by expression in a prokaryotic host cell. Also included within the meaning of such modifications are those labeled to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme-labeled, fluorescent-labeled, or affinity-labeled antibodies. Such modified antibody forms of the present invention are useful for improving antibody stability and blood retention, reducing antigenicity, and detecting or isolating antibodies or antigens.

It is known that antibodies produced in cultured mammalian cells lose the lysine residue at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions and modifications of the heavy chain sequence do not affect the antigen-binding ability or effector function of the antibody (the presence or absence or level of complement activation or antibody-dependent cellular cytotoxicity, etc.). Therefore, the antibodies of the present invention include antibodies and functional fragments of such antibodies that have undergone such modifications, including deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and amidated deletions (e.g., heavy chains in which the proline residue at the carboxyl terminus is amidated). However, as long as the antigen-binding ability and effector function (whether present or absent or high or low) are maintained, the carboxyl-terminal deletions of the heavy chains of the antibody of the present invention are not limited to the above types. The two heavy chains constituting the antibody of the present invention may be any one type of heavy chain selected from the group consisting of full-length and the above-mentioned deletions, or a combination of any two types. The quantitative ratio of each deletion may be affected by the type and culture conditions of the cultured mammalian cells producing the antibody of the present invention, but preferred antibodies of the present invention include those in which one amino acid residue is deleted at the carboxyl terminus of each of the two heavy chains.

Human Chimeric Antibodies and Humanized Antibodies The anti-IL-22BP antibodies of the present invention also include human chimeric antibodies and humanized antibodies that have been modified to reduce xenoantigenicity to humans. Humanized antibodies are also called CDR-grafted antibodies.

Human Chimeric Antibody A human chimeric antibody is an antibody consisting of the light chain variable region and heavy chain variable region of an antibody of an animal other than human and the light chain constant region and heavy chain constant region of a human antibody. A human chimeric antibody can be produced by obtaining cDNA encoding the light chain variable region and cDNA encoding the heavy chain variable region from a hybridoma producing anti-IL-22BP, inserting them into an expression vector containing cDNA encoding the light chain constant region and heavy chain constant region of a human antibody to construct a human chimeric antibody expression vector, and introducing the vector into a host cell for expression.
The heavy chain constant region is comprised of three domains, _CH1 , _CH2 and _CH3 .

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

A humanized antibody (CDR-grafted antibody) is an antibody in which the amino acid sequences of the CDRs of the light chain variable region and heavy chain variable region of an antibody from a non-human animal have been transplanted into appropriate positions in the light chain variable region and heavy chain variable region of a human antibody.

Specifically, a DNA sequence designed to link the CDRs of antibody MAb3, MAb4, MAb8, MAb14, MAb20, or MAb81 to the framework regions of a human antibody can be synthesized. The framework regions of the human antibody linked via the CDRs are selected so that the CDRs form a good antigen-binding site. Furthermore, if necessary, amino acids in the framework regions of the antibody variable regions can be substituted so that the CDRs of the humanized antibody form a suitable antigen-binding site. CDR-grafted humanized antibodies can be produced using known CDR grafting techniques.

The present invention includes antibodies comprising a light chain containing the sequences of the three CDRs (CDRL1, CDRL2, CDRL3) of the light chain variable region of monoclonal antibodies MAb3, MAb4, MAb8, MAb14, MAb20, and MAb81, and a heavy chain containing the sequences of the three CDRs (CDRH1, CDRH2, CDRH3) of the heavy chain variable region. The amino acid sequences of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 of monoclonal antibodies MAb3, MAb4, MAb8, MAb14, MAb20, and MAb81 are set forth in the following SEQ ID NOs in 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 order to avoid N-glycosylation, the heavy chain variable region of MAb4 was mutated from Ser66 (IMGT numbering; Immunol Today 18(11), 509(1997)) to Ala and Asn106 to Arg to produce MAb4'. 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, respectively, in the Sequence Listing.

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

These human chimeric antibody light chain amino acid sequences and heavy chain amino acid sequences include not only light chains and heavy chains comprising the amino acid sequences set forth in the above SEQ ID NOs., but also include amino acid sequences in which one or an integer number of amino acids, for example, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid, have been deleted, substituted, or added to the amino acid sequences, and which are contained in the light chains and/or heavy chains of proteins that have the properties possessed by the antibodies of the present invention (such as the properties described in [1] above, preferably [1] and [2], more preferably [1] to [3], even more preferably [1] to [4], and even more preferably [1] to [5]), in particular the activity of binding to human IL-22BP. Such amino acid sequences include those that have at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in the above SEQ ID NO: when calculated using CLUSTAL W (alignment tool) or the like (for example, default i.e. initial setting parameters). Such amino acid sequences can be said to be substantially identical to the protein having the amino acid sequence shown in the above SEQ ID NO:.

Furthermore, a humanized antibody, hMAb3, was designed by CDR grafting based on the monoclonal antibody MAb3, and the humanized antibody light chains were designed and prepared by connecting the human IgG1 kappa chain constant region to the humanized antibody light chain variable region of the designed MAb3. These humanized antibody light chains are designated hMAb3_L01, hMAb3_L02, hMAb3_L03, hMAb3_L04, hMAb3_L05, hMAb3_L06, and hMAb3_L07, respectively. Furthermore, humanized antibody heavy chains were designed and prepared by connecting the human IgG1 gamma chain constant region to the humanized antibody heavy chain variable region of the designed MAb3. These humanized antibody heavy chains are designated hMAb3_H01, hMAb3_H02, hMAb3_H03, and hMAb3_H04, respectively.

Furthermore, a humanized antibody, hMAb4, was designed by CDR grafting based on the monoclonal antibody MAb4. The humanized antibody light chains, in which the humanized antibody light chain variable region of the designed MAb4 was ligated with the human IgG1 kappa chain constant region, were designed and produced, and are designated hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06, respectively. The humanized antibody heavy chains, in which the humanized antibody heavy chain variable region of the designed MAb4 was ligated with the human IgG1 gamma chain constant region, were designed and produced, and are designated hMAb4_H01, hMAb4_H02, and hMAb4_H03, respectively.

The antibodies of the present invention include antibodies comprising the light chain variable region of any of hMAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05, and hMAb3_L07, and the heavy chain variable region of any 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, 139, and 140, respectively. In hMAb3, the light chain variable region consists of the sequence shown by amino acid numbers 21 to 133 of the amino acid sequence shown by each SEQ ID NO, and the heavy chain variable region consists of the sequence shown by amino acid numbers 20 to 132 of the amino acid sequence shown by each SEQ ID NO.

The antibodies of the present invention also include antibodies comprising the light chain variable region of any one of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06, and the heavy chain variable region of any one 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 acid sequences of hMAb4_H01, hMAb4_H02, and hMAb4_H03 are shown in SEQ ID NOs: 141, 142, and 143, respectively. In hMAb4, the light chain variable region consists of the sequence represented by amino acid numbers 21 to 133 of the amino acid sequence represented by each SEQ ID NO, and the heavy chain variable region consists of the sequence represented by amino acid numbers 20 to 137 of the amino acid sequence represented by each SEQ ID NO.

Among these antibodies, preferred MAb3-based antibodies are antibodies comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the Sequence Listing and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the Sequence Listing; antibodies comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 123 in the Sequence Listing and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 138 in the Sequence Listing; and antibodies comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 122 in the Sequence Listing and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 132 of SEQ ID NO: 140 in the Sequence Listing.

Furthermore, preferred MAb4-based antibodies include antibodies comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 126 in the Sequence Listing and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing; antibodies comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the Sequence Listing and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing; and antibodies comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 130 in the Sequence Listing and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 142 in the Sequence Listing. Furthermore, the antibodies of the present invention include antibodies comprising the full-length light chain of any one of MAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05, and hMAb3_L07, and the full-length heavy chain of any one of hMAb3_H01, hMAb3_H03, and hMAb3_H04. The full-length light chain amino acid sequences of MAb3_L01, hMAb3_L03, hMAb3_L04, hMAb3_L05, and hMAb3_L07 comprise the sequences shown in amino acid numbers 21 to 239 of the amino acid sequences shown in SEQ ID NOs: 121, 122, 123, 124, and 125, respectively, and the full-length heavy chain amino acid sequences of hMAb3_H01, hMAb3_H03, and hMAb3_H04 comprise the sequences shown in amino acid numbers 20 to 462 of the amino acid sequences shown in SEQ ID NOs: 138, 139, and 140, respectively. Furthermore, the antibodies of the present invention include antibodies comprising the full-length light chain of any of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06, and the full-length heavy chain of any of hMAb4_H01, hMAb4_H02, and hMAb4_H03. The full-length light chain amino acid sequences of hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06 comprise the sequences shown in amino acid numbers 21 to 239 of the amino acid sequences shown in SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively, and the full-length heavy chain amino acid sequences of hMAb4_H01, hMAb4_H02, and hMAb4_H03 comprise the sequences shown in amino acid numbers 20 to 467 of the amino acid sequences shown in SEQ ID NOs: 141, 142, and 143, respectively.

Specifically, preferred MAb3-based antibodies are hMAb3_H01L03, hMAb3_H01L04, hMAb3_H03L01, hMAb3_H03L03, hMAb3_H03L05, hMAb3_H04L03, and hMAb3_H04L07, and preferred MAb4-based antibodies are hMAb4_H01L01, hMAb4_H01L02, hMAb4_H01L04, and hMAb4_H01L0. 5, hMAb4_H01L06, hMAb4_H02L01, hMAb4_H02L02, hMAb4_H02L03, hMAb4_H02L04, hMAb4_H02L05, hMAb4_H02L06, hMAb4_H03L01, hMAb4_H03L02, hMAb4_H03L03, hMAb4_H03L04, hMAb4_H03L05, and hMAb4_H03L06 are preferred.

Among these antibodies, more preferred MAb3-based antibodies include antibodies comprising a light chain comprising the amino acid sequence set forth in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the Sequence Listing and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the Sequence Listing; antibodies comprising a light chain comprising the amino acid sequence set forth in amino acid numbers 21 to 239 of SEQ ID NO: 123 in the Sequence Listing and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 20 to 462 of SEQ ID NO: 138 in the Sequence Listing; and antibodies comprising a light chain comprising the amino acid sequence set forth in amino acid numbers 21 to 239 of SEQ ID NO: 122 in the Sequence Listing and a heavy chain comprising the amino acid sequence set forth in amino acid numbers 20 to 462 of SEQ ID NO: 140 in the Sequence Listing. Specific examples include hMAb3_H01L03, hMAb3_H01L04, and hMAb3_H04L03.

More preferred MAb4-based antibodies include antibodies comprising a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 126 in the Sequence Listing and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the Sequence Listing; antibodies comprising a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the Sequence Listing and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the Sequence Listing; and antibodies comprising a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 130 in the Sequence Listing and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 142 in the Sequence Listing. Specific examples include hMAb4_H03L01, hMAb4_H03L03, and hMAb4_H02L05.

The light chain amino acid sequences and heavy chain amino acid sequences, or light chain variable region amino acid sequences and heavy chain variable region amino acid sequences, of the humanized antibodies of the present invention not only refer to light chains and heavy chains containing the amino acid sequences set forth in the above SEQ ID NOs. The scope also includes amino acid sequences in which one to an integer (the integer is 50 or less, preferably 25 or less, and more preferably 20 or less), for example, 1 to 20, 1 to 15, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid has been deleted, substituted, or added, and which have the activity of the above antibody or binding fragment thereof (including the light chain and heavy chain containing the amino acid sequence set forth in the above SEQ ID NOs.) (e.g., the properties described in [1], preferably [1] and [2], more preferably [1] to [3], even more preferably [1] to [4], and even more preferably [1] to [5] above), in particular the activity of binding to human IL-22BP. Such amino acid sequences include those that have at least 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 96%, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence set forth in the above SEQ ID NO: when calculated using CLUSTAL W (alignment tool) or the like (for example, default i.e., initial setting parameters). Proteins having such amino acid sequences can be said to be substantially identical to proteins having the amino acid sequences set forth in the above SEQ ID NO:.

The activities and properties exhibited by the antibodies of the present invention include, for example, biological activity and physicochemical properties, specifically various biological activities, binding activity to antigens or epitopes, species cross-reactivity, stability during production and storage, and thermal stability.

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

The antibody or binding fragment thereof of the present invention preferably also binds to monkey IL-22BP, more preferably cynomolgus monkey IL-22BP. The binding dissociation constant (KD) of the antibody or binding fragment thereof of the present invention to monkey IL-22BP, measured by BLI, is 1×10 ⁻⁵ M or less, 5×10 ⁻⁶ M or less, or 2×10 ⁻⁶ M or less, and 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, or 1×10 ⁻⁸ M or less. Antibodies that bind to human IL-22BP and monkey IL-22BP are preferred because they can be subjected to various efficacy and safety tests using primates, particularly cynomolgus monkeys (antigen binding, in vivo tests), which are essential for non-clinical (pre-clinical) development of pharmaceuticals. Furthermore, the antibodies of the present invention or binding fragments thereof that also cross-react with monkey IL-22BP are useful for treating or preventing various diseases associated with IL-22BP, such as inflammatory bowel disease in monkeys.

Furthermore, the antibodies or binding fragments thereof of the present invention that bind to human IL-22BP and monkey IL-22BP preferably do not bind to rodent IL-22BP, more preferably mouse IL-22BP. Therefore, various assays, immunohistochemical studies, etc. can be performed using cells, tissues, or individuals (including transgenic, knockout, and knock-in animals) of rodents such as mice into which human IL-22BP has been introduced, as well as the antibodies, without the influence of IL-22BP in the host rodent, such as mice. This is preferable for research and development of pharmaceuticals, veterinary drugs, or diagnostic agents comprising the antibodies or binding fragments thereof of the present invention. Furthermore, the non-clinical pharmacological activity of the antibodies or binding fragments thereof can be evaluated using a pathological model of primates, particularly cynomolgus monkeys, in which intestinal inflammation is induced by the administration of dextran sulfate sodium (Nat Commun. 18;6:8020 (2015)). Clinically used indicators, such as the Mayo score, can be used to evaluate the pathological condition. Furthermore, by using biological tissue samples that can be collected during the in vivo pharmacological test or in clinical settings, changes in signaling mediated by STAT3 phosphorylation can be evaluated by pre-treatment, simultaneous treatment, or post-treatment with IL-22 (see ex vivo test in Example 10).

Antibodies or binding fragments thereof that bind to the site on human IL-22BP to which the antibodies or binding fragments thereof of the present invention bind are also encompassed within the scope of the antibodies or binding fragments thereof. Such antibodies or binding fragments thereof preferably have the above-mentioned biological activity, antigen-binding activity, species cross-reactivity, etc. (e.g., the properties described in [1] to [5] above). Similarly, antibodies or binding fragments thereof that compete with the antibodies or binding fragments thereof of the present invention in binding to human IL-22BP are also encompassed within the scope of the antibodies or binding fragments thereof. Such antibodies or binding fragments thereof preferably have the above-mentioned biological activity, antigen-binding activity, species cross-reactivity, etc. (e.g., the properties described in [1] to [5] above).

Antibodies or binding fragments thereof of the present invention can be used in multispecific antibodies (multispecific molecules) or antibody-drug conjugates. Multispecific antibodies or antibody-drug conjugates containing antibodies or binding fragments thereof of the present invention are also included within the scope of the "antibodies" and "binding fragments thereof" of the present invention. In other words, antibodies or binding fragments thereof of the present invention may be in a form in which they are conjugated with a drug, or in a form in which they are included in a multispecific antibody (multispecific molecule).

4. Method for Producing Antibodies The antibodies of the present invention or binding fragments thereof can be produced in cells as recombinant antibodies by inserting DNA encoding the heavy chain variable region or DNA encoding the light chain variable region into an expression vector, transforming host cells for expression with the vector, and culturing the host cells.

DNA encoding an antibody can be obtained by linking DNA encoding a heavy chain variable region with DNA encoding a heavy chain constant region, and DNA encoding a light chain can be obtained by linking DNA encoding a light chain variable region with DNA encoding a light chain constant region.

The anti-IL-22BP antibody of the present invention can be produced by inserting the DNA encoding the heavy chain and the DNA encoding the light chain into an expression vector, transforming host cells for expression with the vector, and culturing the host cells. In this case, the DNA encoding the heavy chain and the DNA encoding the light chain may be introduced into the same expression vector and the host cells may be transformed with the vector, or the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into separate vectors and the host cells may be transformed with the two vectors. In this case, the DNA encoding the heavy chain variable region and the DNA encoding the light chain variable region may be introduced into a vector into which DNA encoding the heavy chain constant region and DNA encoding the light chain constant region have already been introduced. The vector may also contain DNA encoding a signal peptide that promotes antibody secretion from host cells. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are linked in-frame. After antibody production, the signal peptide is removed to obtain the antibody as a mature protein.

In this case, the DNA encoding the heavy chain variable region, the DNA encoding the light chain variable region, the DNA combining the DNA encoding the heavy chain variable region and the DNA encoding the heavy chain constant region, and the DNA combining the DNA encoding the light chain variable region and the DNA encoding the light chain constant region may be operatively linked to elements such as a promoter, enhancer, polyadenylation signal, etc. Here, "operatively linked" means that the elements are linked in such a way that they can perform their functions.

The expression vector is not particularly limited as long as it can replicate in a host such as an animal cell, bacteria, or yeast, and examples include known plasmids and phages. Examples of vectors used to construct expression vectors include pcDNA™ (ThermoFisher SCIENTIFIC), Flexi™ Vector (Promega), pUC19, pUEX2 (Amersham), pGEX-4T, pKK233-2 (Pharmacia), and pMAM-neo (Clontech). While prokaryotic cells such as Escherichia coli and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, are both acceptable host cells, eukaryotic cells are preferred. For example, animal cells such as HEK293 cells, a human embryonic kidney cell line, or Chinese hamster ovary (CHO) cells may be used. The expression vector can be introduced into the host cell using known methods to transform the host cell. Examples of such methods include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The produced antibodies can be purified using separation and purification methods commonly used for proteins. For example, affinity chromatography, other chromatography, filtering, ultrafiltration, salting out, dialysis, and the like may be appropriately selected and combined.

5. Pharmaceutical Compositions The present invention encompasses pharmaceutical compositions comprising the anti-IL-22BP antibody or binding fragment thereof of the present invention as an active ingredient.

The pharmaceutical composition of the present invention inhibits the binding of IL-22BP to IL-22. When IL-22BP binds to IL-22, the function of IL-22 is inhibited. Inhibition of IL-22 function induces infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, ulcerative colitis, inflammatory bowel diseases including Crohn's disease, and the like. Therefore, the pharmaceutical composition of the present invention can be used for the prevention (administered before the onset, exacerbation, etc.) or treatment (administered after the onset, exacerbation, etc.) of infectious diseases, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, ulcerative colitis, inflammatory bowel diseases including Crohn's disease, and the like.

The pharmaceutical composition of the present invention may contain a therapeutically effective amount of an anti-IL-22BP antibody and pharmaceutically acceptable carriers, diluents, solubilizers, emulsifiers, preservatives, adjuvants, etc. Pharmaceutically acceptable carriers may be selected from a wide range of options depending on the type of target disease and the dosage form of the drug. The method of administration of the pharmaceutical composition of the present invention may be selected as appropriate, including injection, local injection, intraperitoneal administration, selective intravenous injection, intravenous injection, subcutaneous injection, and organ perfusion injection. Injectable solutions may also be formulated using carriers such as salt solutions, glucose solutions, mixtures of saline and glucose solutions, and various buffer solutions. Alternatively, the composition may be formulated in powder form, which can be mixed with the liquid carrier at the time of use to prepare an injectable solution.

Other administration methods can also be selected appropriately as the formulation is developed. For example, oral administration can be performed in the form of oral liquids, powders, pills, capsules, and tablets. Oral liquids, such as suspensions and syrups, can be prepared using water, sugars such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol, oils such as sesame oil and soybean oil, preservatives such as alkyl parahydroxybenzoate, and flavors such as strawberry flavor and peppermint. Powders, pills, capsules, and tablets can be formulated using excipients such as lactose, glucose, sucrose, and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin. Tablets and capsules are preferred unit dosage forms for the compositions of this invention due to their ease of administration. When tablets or capsules are produced, solid manufacturing carriers are used.

The amount of antibody or binding fragment effective for treatment varies depending on the nature of the condition being treated and the age and condition of the patient, and is ultimately determined by a physician. For example, it may be 0.0001 mg to 100 mg per kg of body weight per dose. A given dose may be administered once every 1 to 180 days, or may be administered in divided doses two, three, four, or more times per day at appropriate intervals.

Pharmaceutical compositions containing the anti-IL-22BP antibody or binding fragment thereof of the present invention as an active ingredient can be used in combination with other pharmaceutical compositions. Examples of other pharmaceuticals that can be used in combination with the pharmaceutical compositions of the present invention include anti-TNFα antibodies, anti-integrin α4β7 antibodies, 5-aminosalicylic acid preparations, corticosteroids, thiopurine preparations, calcineurin inhibitors, JAK inhibitors, and antibacterial agents. Examples of these other pharmaceuticals include the anti-TNFα antibody adalimumab, the anti-integrin α4β7 antibody vedolizumab, the 5-aminosalicylic acid preparation mesalazine, the corticosteroid prednisolone, the thiopurine preparation azathioprine, the calcineurin inhibitor tacrolimus, the JAK inhibitor tofacitinib citrate, and the antibacterial agent metronidazole. Furthermore, the pharmaceutical compositions of the present invention can also be used in combination with elemental nutrition therapy, leukocyte apheresis therapy, and the like to treat or prevent inflammatory bowel disease. These other pharmaceutical agents and therapies may be one type, or two, three, or more types may be administered or received. These are collectively referred to as the pharmaceutical composition of the present invention and "combined with other pharmaceutical agents" or "combined with other pharmaceutical agents." Pharmaceutical compositions of the present invention that contain other pharmaceutical agents in addition to the antibody, binding fragment thereof, or modified form thereof of the present invention, or that are used in combination with both, are also included in the present invention as embodiments of "combined with other pharmaceutical agents" or "combined with other pharmaceutical agents."

The present invention encompasses the anti-IL-22BP antibody or binding fragment thereof of the present invention, which is used in combination with other pharmaceuticals, as well as pharmaceutical compositions containing the anti-IL-22BP antibody or binding fragment thereof of the present invention as an active ingredient, which is used in combination with other pharmaceuticals.

In addition, pharmaceutical compositions containing a polynucleotide comprising a base sequence encoding an amino acid sequence contained in the antibody or binding fragment thereof of the present invention, a vector containing the polynucleotide, or a cell (also referred to as a host cell) containing the polynucleotide or the vector are also useful for treating or preventing the above-mentioned diseases, such as inflammatory bowel disease, and are included in the present invention.

6. Test Agents, Diagnostic Agents, and Reagents The antibody or binding fragment thereof of the present invention can also be used as a test agent, diagnostic agent, reagent, etc. The antibody or binding fragment thereof of the present invention can be used to measure the amount of IL-22BP in a sample. Furthermore, it can also be used to detect whether or not the function of IL-22 is inhibited in a sample. For example, the amount of IL-22BP or inhibition of IL-22 function can be used as an indicator to determine whether or not a subject has or is at risk of developing an infectious disease, autoimmune hepatitis, alcoholic liver disease, autoimmune cardiomyopathy, bronchitis, uveitis, ulcerative colitis, inflammatory bowel disease including Crohn's disease, etc.

Example 1. Preparation of IL-22BP 1)-1 Preparation of Expression Vector 1)-1-1 Preparation of Expression Vector p3.3nIL2ss_FLAGHis The neomycin expression unit of the vector pcDNA3.3-TOPO/LacZ (ThermoFisher SCIENTIFIC) was deleted using KOD-Plus-Mutagenesis Kit (TOYOBO). The primers used were 5'-CAAATAAAAGCAATAGCATCACAAATTTC-3' (Dneo-F) (SEQ ID NO: 1) and 5'-CCACAGAATTAATTCGCGTTAAATTTTTG-3' (Dneo-R) (SEQ ID NO: 2).

The expression vector p3.3IL2ss_FLAGHis was created by inserting DNA (SEQ ID NO: 4) encoding a polypeptide containing amino acid residues 1 to 20 of the human IL-2 (ACCESSION number: NP_000577) and the FLAGHis tag DYKDDDDKHHHHHH (SEQ ID NO: 3) between the restriction enzyme XbaI and PmeI cleavage sites of the vector.

1)-1-2 Preparation of human IL-22BP-His expression vector A DNA (SEQ ID NO: 5) encoding a sequence comprising a polypeptide in which ENLYFQG is linked to the C-terminus of amino acids 1 to 263 of the amino acid sequence of human IL-22BP (ACCESSION No.: NP_443194) was inserted between the restriction enzyme XbaI and BamI cleavage sites of the expression vector p3.3IL2ss_FLAGHis using In-Fusion HD Cloning Kit (manufactured by CLONTECH), thereby preparing a human IL-22BP (Isoform 1)-His expression vector. A human IL-22BP (Isoform 2)-His expression vector was constructed by deleting the DNA encoding the polypeptide from amino acid 67 to 98 of the human IL-22BP (Isoform 1)-His expression vector using inverse PCR. Hereinafter, human IL-22BP (Isoform 2)-His is 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 A DNA (SEQ ID NO: 8) encoding a sequence comprising a polypeptide in which ENLYFQG is linked to the C-terminus of amino acid residues 1 to 21 of the amino acid sequence of human IL-22BP (ACCESSION No.: NP_443194) and amino acid residues 21 to 231 of the amino acid sequence of monkey IL-22BP (ACCESSION No.: XP_005552009) was inserted between the restriction enzyme XbaI and BamI sites of the expression vector p3.3IL2ss_FLAGHis using an In-Fusion HD Cloning Kit (CLONTECH), thereby preparing a monkey IL-22BP-His expression vector. 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 A mouse IL-22BP-His expression vector was prepared by inserting DNA (SEQ ID NO: 11) encoding a sequence comprising a polypeptide in which ENLYFQG is linked to the C-terminus of the amino acid sequence from positions 1 to 21 of the amino acid sequence of human IL-22BP (ACCESSION No.: NP_443194) and from positions 21 to 230 of the amino acid sequence of mouse IL-22BP (ACCESSION No.: NP_839989), in which Cys106 has been substituted with Ser, into the expression vector p3.3IL2ss_FLAGHis between the restriction enzyme XbaI and BamI cleavage sites using In-Fusion HD Cloning Kit (manufactured by CLONTECH). 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 transfected into FreeStyle 293F cells (ThermoFisher SCIENTIFIC) to allow transient expression. The culture supernatant was loaded onto a HisTrap Excel column (GE Healthcare Japan) equilibrated with 3x PBS(-) (PBS), and the column was washed with 3x PBS. Elution was then performed with 3x PBS and 500 mM imidazole. IL-22BP-His was purified from the collected IL-22BP fraction using HiLoad 26/600 Superdex 200 pg (GE Healthcare Japan) equilibrated with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl. The final buffer was adjusted to 16 mM Tris-HCl (pH 7.5), 120 mM NaCl, and 16% (v/v) glycerol.

1)-3 Preparation of Immunoantigen TEV protease with a His tag attached to the N-terminus was added to purified human IL-22BP-His, and the mixture was allowed to react overnight at 4°C to cleave FLAGHis. Ni Sepharose 6 FF (GE Healthcare Japan) was added to the reaction solution, and 5 M imidazole was further added to a concentration of approximately 20 mM. The supernatant reaction solution was loaded into an empty column, and the flow-through fraction was collected and buffer-exchanged into PBS using PD-10 (GE Healthcare Japan). The amino acid sequence of human IL-22BP from which the tag was cleaved is shown in SEQ ID NO: 14.

Example 2. Production of rat anti-human IL-22BP antibody 2)-1 Immunization Female WKY/Izm rats (manufactured by Japan SLC) were used for immunization. A mixture of the 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 administered to the base of the tail of the rats, and the lymph nodes and spleens of the rats were collected and used to produce hybridomas.

2)-2 Hybridoma Production Lymph node cells or spleen cells were electrofused with mouse myeloma SP2/0-ag14 cells (ATCC: CRL-1581) using an LF301-Cell Fusion Unit (BEX), and the resulting mixture was diluted in ClonaCell-HY Selection Medium D (StemCell Technologies) and cultured. Monoclonal hybridomas were produced by recovering the emerged hybridoma colonies. Each recovered hybridoma colony was cultured, and the resulting hybridoma culture supernatant was used to screen for anti-human IL-22BP antibody-producing hybridomas.

Example 3 Evaluation of Rat Anti-Human IL-22BP Antibodies 3)-1 Antibody Screening 3)-1-1 DIRECT-ELISA
Human IL-22BP-His prepared in Example 1)-2 was diluted to 4 μg/ml with 50 mM Tris-HCl (pH 8.5), and 50 μL of the diluted solution was added to Clear Flat-Bottom Immuno 96-Well Plates (ThermoFisher SCIENTIFIC) and allowed to solidify overnight at 4°C. The next day, the medium was removed from the plates, and the plates were washed twice with 5% FBS-containing PBS. Then, 100 μL of 5% FBS-containing PBS was added to each well and the plates were allowed to stand at room temperature for 1 hour. After washing twice with 5% FBS-containing PBS, hybridoma culture supernatant was added and the plates were allowed to stand at room temperature for 2 hours. After washing twice with 5% FBS-containing PBS, 50 μL of anti-rat IgG peroxidase antibody produced in rabbit (Sigma-Arch) diluted 500-fold with 5% FBS-containing PBS was added to each well and allowed to stand at room temperature for 1 hour. After washing five times with 5% FBS-containing PBS, 50 μL of OPD coloring solution (0.05 M trisodium citrate, 0.1 M disodium hydrogen phosphate 12-water, pH 4.5, dissolved in o-phenylenediamine _{dihydrochloride} (Fujifilm Wako Pure Chemical Industries, Ltd.) and H _O to concentrations of 0.4 mg/mL and 0.6% (v/v), respectively) was added to each well. The color reaction was allowed to proceed with occasional stirring, and 50 μL/well of 1 M HCl was added to stop the color reaction, after which the absorbance at 490 nm was measured using Envision (Perkin Elmer). Hybridomas producing culture supernatants with an OD value of 0.1 or more, which was at least twice as high as that of wells to which the human IL-22BP antigen had not been added, were selected as hybridomas positive for producing anti-human IL-22BP antibodies.

3)-1-2 Competition ELISA
6x-His Tag Polyclonal Antibody (ThermoFisher SCIENTIFIC) was diluted to 2 μg/mL with PBS, and 50 μL of this was added to Clear Flat-Bottom Immuno 96-Well Plates (ThermoFisher SCIENTIFIC) and allowed to solidify overnight at 4°C. The next day, the plates were washed twice with PBS containing 0.05% Tween-20 (PBS-T), and then 200 μL of Block BSA in PBS (ThermoFisher SCIENTIFIC) was added and 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 1% BSA-containing PBS-T (dilution buffer) was added to each well and incubated for 1 hour at 37° C. After washing five times with PBS-T, the culture supernatant of the selected anti-human IL-22BP antibody-producing positive hybridoma was added, followed by the addition of 5 μL of Recombinant Human IL-22 Protein (R&D Systems) diluted to 1 μg/mL with dilution buffer or dilution buffer alone, and the incubation was continued for 1 hour at 37° C. After washing five times with PBS-T, 50 μL of Human IL-22 Antibody (R&D Systems) diluted to 0.5 μg/mL with dilution buffer was added to each well and the plates were incubated at 37°C for 1 hour. After washing five times with PBS-T, 50 μL of Goat Anti-Mouse IgG Antibody, HRP conjugate, Species Adsorbed (Merck) diluted to 0.8 μg/mL with dilution buffer was added to each well and the plates were incubated at 37°C for 1 hour. After washing six times with PBS-T, 100 μL of OPD coloring solution was added to each well. After allowing to stand for 6 minutes, 100 μL of 1 M HCl was added to each well. The absorbance at 490 nm was measured using an Envision (Perkin-Elmer). To select hybridomas that specifically inhibit the binding of recombinant human IL-22 protein to human IL-22BP, hybridomas producing culture supernatants with lower absorbance than those obtained with the control dilution buffer were selected as positive anti-human IL-22BP antibody-producing hybridomas with inhibitory activity against the binding of IL-22 to IL-22BP. As a result, six hybridomas producing rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81 were selected from 2,000 hybridoma clones.

3)-2 Determination of antibody isotype The isotypes of the antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81) produced by the binding inhibitory activity-positive anti-human IL-22BP antibody-producing hybridomas selected in Example 3)-1-2 were determined using a Rapid monoclonal Antibody isotyping kit (manufactured by Antagen Pharmaceuticals). As a result, it was confirmed that only rMAb8 was an IgG2a κ chain isotype, and the others were all IgG1 κ chain isotypes.

3)-3 Preparation of Purified Antibody 3)-3-1 Preparation of Culture Supernatant Anti-human IL-22BP monoclonal antibody was purified from the culture supernatant of a rat hybridoma. The rat anti-human IL-22BP monoclonal antibody-producing hybridoma was grown to a sufficient amount in ClonaCell-HY Selection Medium E (StemCell Technologies). The medium was then replaced with Hybridoma SFM (ThermoFisher SCIENTIFIC) supplemented with 20% Ultra Low IgG FBS (ThermoFisher SCIENTIFIC). 8-9 x 107 ^hybridoma cells were then seeded into a 1272 ^cm2 flask (CORNING) and cultured for 7 days. The culture supernatant was collected by centrifugation, passed through a 0.8 μm filter, and then sterilized by passing through a 0.45 μm filter (CORNING).

3)-3-2 Antibody Purification Rat anti-human IL-22BP antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81) were purified from the hybridoma culture supernatant obtained in Example 3)-3-1 by a single-step Protein G affinity chromatography process at 4 to 6°C. The buffer replacement process after Protein G affinity chromatography purification was carried out at 4 to 6°C. First, the hybridoma culture supernatant was applied to a column packed with Protein G HP (GE Healthcare Japan) equilibrated with PBS. After the culture supernatant was completely loaded onto the column, the column was washed with at least two column volumes of PBS. Next, the column was eluted with 0.1 M glycine/hydrochloric acid aqueous solution, pH 2.3, and the antibody-containing fractions were collected. 1 M Tris-HCl, pH 9.0, was added to adjust the pH to 7.0-7.5, and the buffer was then replaced with PBS while concentrating using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, manufactured by Sartorius) at 4-6°C. After replacing with PBS, the IgG concentration was adjusted to 1 mg/mL to prepare a purified sample.

3)-4 Binding evaluation using purified antibodies 3)-4-1 Measurement of binding activity to human IL-22BP (BLI method)
The binding activity of the rat anti-human IL-22BP monoclonal antibody prepared in Example 3)-3-2 to human IL-22BP-His prepared in Example 1)-2 was measured using Octet RED 384 (MOLECULAR DEVICES), in which an antigen was captured as a ligand on a biosensor Anti-Penta-HIS (HIS1K) (MOLECULAR DEVICES), a rat antibody was used as an analyte, and PBS-T (Takara Bio Inc.) was used as a measurement buffer. The biosensor was reacted with 5 μg/mL human IL-22BP-His for 600 seconds, followed by reaction with a diluted solution of rat anti-human IL-22BP monoclonal antibody (7 concentrations starting from 500 nM with a common ratio of 2) for 600 seconds. The biosensor was reacted with 10 mM glycine pH 1.5 (GE Healthcare Japan) as a regeneration solution for 25 seconds. Data analysis was performed by equilibrium value analysis, and the binding dissociation constant KD was calculated.

3)-4-2 Species cross-reactivity assay for mouse and monkey IL-22BP (DIRECT-ELISA)
The species cross-reactivity of the rat anti-human IL-22BP monoclonal antibody prepared in Example 3)-3-2 with mouse and monkey IL-22BP was measured by DIRECT-ELISA in the same manner as in Example 3)-1-1, using mouse IL-22BP-His and monkey IL-22BP-His prepared in Example 1)-2. The results of the binding activity of each clone to mouse, monkey, and human IL-22BP are shown in Figure 1. All of the clones evaluated bound to monkey and human IL-22BP, while rMAb8 and rMAb14 were clones that also bound to mouse IL-22BP.

Example 4 Determination of the Nucleotide Sequence of cDNA Encoding the Variable Region of Rat Anti-Human IL-22BP Antibody 4)-1 Preparation of Total RNA from Hybridomas Producing Rat Anti-Human IL-22BP Antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) To amplify cDNA containing the variable region, total RNA was prepared from hybridomas producing rat anti-human IL-22BP antibodies using a Direct-zol RNA Miniprep kit (ZYMO RESEARCH).

4)-2 Amplification and Sequencing of cDNA Containing the Light Chain Variable Region of Rat Anti-Human IL-22BP Antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81) by 5'-RACE PCR The following reaction was carried out using SMARTer™ RACE 5'/3' Kit (manufactured by CLONTECH).

First-strand cDNA was synthesized from total RNA. Using the following primer combination and the synthesized first-strand cDNA as a template, cDNA containing the variable region of the light chain of the rat anti-human IL-22BP antibody was amplified by 5'-RACE PCR. The primers used were UPM (included in the kit) and an oligonucleotide with the sequence 5'-TCAGTAACACTGTCCAGGACACCATCTC-3' (RKR5). RKR5 was designed from the sequence of the rat light chain constant region in a database.

The PCR fragment amplified by 5'-RACE PCR was cloned using the Zero Blunt TOPO PCR Cloning Kit for Sequencing (ThermoFisher SCIENTIFIC), and the nucleotide sequence of the cloned cDNA containing the light chain variable region was analyzed. RKR5 and NUP (included in the kit) were used as sequencing primers.

The nucleotide sequences of the light chain variable regions (MAb3_VL, MAb4_VL, MAb8_VL, MAb14_VL, MAb20_VL, MAb81_VL) of rat anti-human IL-22BP antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) are shown in SEQ ID NOs: 15 to 20 of the Sequence Listing, and the amino acid sequences are shown in SEQ ID NOs: 21 to 26 of the Sequence Listing. The amino acid sequences of CDRL1, CDRL2, and CDRL3 of MAb3_VL are shown in SEQ ID NOs: 27 to 29, those of CDRL1, CDRL2, and CDRL3 of MAb4_VL are shown in SEQ ID NOs: 30 to 32, those of CDRL1, CDRL2, and CDRL3 of MAb8_VL are shown in SEQ ID NOs: 33 to 35, those of CDRL1, CDRL2, and CDRL3 of MAb14_VL are shown in SEQ ID NOs: 36 to 38, those of CDRL1, CDRL2, and CDRL3 of MAb20_VL are shown in SEQ ID NOs: 39 to 41, and those of CDRL1, CDRL2, and CDRL3 of MAb81_VL are shown in SEQ ID NOs: 42 to 44, respectively.

4)-3 Amplification and Sequencing of cDNA Containing the Heavy Chain Variable Region of Rat Anti-Human IL-22BP Antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, and rMAb81) by 5'-RACE PCR The following reaction was carried out using SMARTer™ RACE 5'/3' Kit (manufactured by CLONTECH).

First-strand cDNA was synthesized from total RNA. Using the following primer combination and the synthesized first-strand cDNA as a template, cDNA containing the heavy chain variable region of the rat anti-human IL-22BP antibody was amplified by 5'-RACE PCR. PCR was performed using KOD-Plus- (manufactured by TOYOBO) as the polymerase. UPM (included in the kit) and an oligonucleotide with the sequence 5'-CTCCAGAGTTCCAGGTCACGGTGACTGGC-3' (RG2AR3) were used as primers. RG2AR3 was designed from the sequence of the rat heavy chain constant region in a database.

The PCR fragment amplified by 5'-RACE PCR was cloned using the Zero Blunt TOPO PCR Cloning Kit for Sequencing (ThermoFisher SCIENTIFIC), and the nucleotide sequence of the cloned cDNA containing the heavy chain variable region was analyzed. RG2AR3 and NUP (included in the kit) were used as sequencing primers.

The nucleotide sequences of the heavy chain variable regions (MAb3_VH, MAb4_VH, MAb8_VH, MAb14_VH, MAb20_VH, MAb81_VH) of rat anti-human IL-22BP antibodies (rMAb3, rMAb4, rMAb8, rMAb14, rMAb20, rMAb81) are shown in SEQ ID NOs: 45 to 50 of the Sequence Listing, and the amino acid sequences are shown in SEQ ID NOs: 51 to 56 of the Sequence Listing. The amino acid sequences of CDRH1, CDRH2, and CDRH3 of MAb3_VH are shown in SEQ ID NOs: 57 to 59, those of CDRH1, CDRH2, and CDRH3 of MAb4_VH are shown in SEQ ID NOs: 60 to 62, those of CDRH1, CDRH2, and CDRH3 of MAb8_VH are shown in SEQ ID NOs: 63 to 65, those of CDRH1, CDRH2, and CDRH3 of MAb14_VH are shown in SEQ ID NOs: 66 to 68, those of CDRH1, CDRH2, and CDRH3 of MAb20_VH are shown in SEQ ID NOs: 69 to 71, and those of CDRH1, CDRH2, and CDRH3 of MAb81_VH are shown in SEQ ID NOs: 72 to 74, respectively.

Example 5 Production of Human Chimeric Anti-Human IL-22BP Antibody 5)-1 Construction of Light Chain Expression Vector pCMA-LK Plasmid pcDNA3.3-TOPO/LacZ (ThermoFisher SCIENTIFIC) was digested with restriction enzymes XbaI and PmeI to obtain a fragment of approximately 5.4 kbp. This fragment was ligated with a DNA fragment containing a DNA sequence encoding the human κ chain secretion signal and human κ chain constant region shown in SEQ ID NO:75 in the Sequence Listing using an In-Fusion Advantage PCR Cloning Kit (CLONTECH) to produce pcDNA3.3/LK.

Using pcDNA3.3/LK as a template, PCR was performed with the following primer set, and the resulting approximately 3.8 kbp fragment was phosphorylated and then self-ligated to construct the expression vector pCMA-LK, which has a signal sequence, a cloning site, and a human κ chain constant region downstream of the CMV promoter.
Primer set 5'-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3' (primer 3.3-F1: SEQ ID NO: 76)
5'-GCTATGGCAGGGCCTGCCGCCCCCGACGTTG-3' (primer 3.3-R1: SEQ ID NO: 77)

5)-2 Construction of heavy chain expression vector pCMA-G1 [0123] pCMA-LK was digested with XbaI and PmeI to remove the κ-chain secretion signal and human κ-chain constant region. A DNA fragment was then ligated with a DNA fragment containing a DNA sequence encoding the amino acids of the human heavy chain secretion signal and human IgG1 constant region shown in SEQ ID NO:78 using an In-Fusion Advantage PCR cloning kit (CLONTECH) to construct expression vector pCMA-G1, which has a signal sequence, cloning site, and human IgG1 heavy chain constant region downstream of the CMV promoter.

5)-3 Construction of light chain expression vector for human chimeric anti-human IL-22BP antibody DNA fragments containing DNA sequences encoding the light chain variable regions of each clone (MAb3_VL, MAb4_VL, MAb8_VL, MAb14_VL, MAb20_VL, MAb81_VL) were synthesized (GENEART, Artificial Gene Synthesis Service). Using the synthesized DNA fragment as a template, DNA fragments containing DNA sequences encoding MAb3_VL, MAb4_VL, MAb8_VL, MAb14_VL, MAb20_VL, and MAb81_VL were amplified using KOD-Plus- (manufactured by TOYOBO) and the following primer set. The light chain expression vector pCMA-hLK was cleaved with restriction enzymes BsiWI and SacII, and the amplified DNA fragments were inserted into the site using In-Fusion HD Cloning Kit (manufactured by CLONTECH) to construct vectors expressing the light chains of cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81. The nucleotide sequences of the light chains of cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81 are shown in SEQ ID NOs:79 to 84, and the amino acid sequences are shown in SEQ ID NOs:85 to 90 in the Sequence Listing.
Primer set 5'-CTGTGGATCTCCGGCGCGTACGGC-3' (primer CM-LKF: SEQ ID NO: 91)
5'-GGAGGGGGGCGGCCACCGTACG-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 was designated MAb4'_VH, in which Ser66 (IMGT numbering; Immunol Today 18(11), 509 (1997)) was mutated to Ala and Asn106 to Arg to avoid N-glycosylation.

DNA fragments containing DNA sequences encoding the heavy chain variable regions of each clone (MAb3_VH, MAb4'_VH, MAb8_VH, MAb14_VH, MAb20_VH, MAb81_VH) were synthesized (GENEART, Artificial Gene Synthesis Service). Using the synthesized DNA fragment as a template, DNA fragments containing DNA sequences encoding MAb3_VH, MAb4'_VH, MAb8_VH, MAb14_VH, MAb20_VH, and MAb81_VH were amplified with KOD-Plus- (manufactured by TOYOBO) and the following primer set. The amplified DNA fragments were then inserted into the heavy chain expression vector pCMA-G1 cleaved with the restriction enzyme BlpI using In-Fusion HD Cloning Kit (manufactured by CLONTECH) to construct expression vectors expressing the heavy chains of cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81. The nucleotide sequences of the heavy chains of cMAb3, cMAb4, cMAb8, cMAb14, cMAb20, and cMAb81 are shown in SEQ ID NOs: 93 to 98, their amino acid sequences are shown in SEQ ID NOs: 99 to 104, and CDRH1, CDRH2, and CDRH3 of MAb4'_VH are shown in SEQ ID NOs: 105 to 107, respectively.
Primer set 5'-AGCTCCCAGATGGGTGCTGAGC-3' (primer EG-Inf-F: SEQ ID NO: 108)
5'-GGGCCCTTGGTGGAGGCTGAGC-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 ThermoFisher SCIENTIFIC) were passaged and cultured according to the manual.

1.2 × ¹⁰⁹ FreeStyle 293F cells (ThermoFisher SCIENTIFIC) in the logarithmic growth phase were seeded into a 3 L Fernbach Erlenmeyer Flask (CORNING), diluted with FreeStyle 293 expression medium (ThermoFisher SCIENTIFIC) to a volume of 560 mL, and then cultured with shaking at 95 rpm for 1 hour in an 8% _CO2 incubator at 37°C. 1.8 mg of polyethyleneimine (Polyscience) was dissolved in Opti-Pro SFM (ThermoFisher SCIENTIFIC) to make 20 mL, and then 0.24 mg of a light chain expression vector and 0.36 mg of a heavy chain expression vector prepared using a PureLink HiPure Plasmid kit (ThermoFisher SCIENTIFIC) were suspended in Opti-Pro SFM (ThermoFisher SCIENTIFIC) to make 20 mL. 20 mL of the expression vector/Opti-Pro SFM mixture was added to 20 mL of the polyethyleneimine/Opti-Pro SFM mixture, gently stirred, and then allowed to stand for another 5 minutes before being added to the FreeStyle 293F cells. After culturing for 4 hours in _an incubator at 37°C and 8% CO2 with shaking at 95 rpm, 600 mL of EX-CELL VPRO medium (SAFC Biosciences) and 30 mL of BD Recharge CD (BD Bioscience) were added, and the cells were cultured for 6 days in _an incubator at 37°C and 8% CO2 with shaking at 95 rpm. The resulting culture supernatant was filtered through a Disposable Capsule Filter (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 single-step rProtein A affinity chromatography process at 4 to 6°C. The buffer replacement process after rProtein A affinity chromatography purification was carried out at 4 to 6°C. First, the culture supernatant was applied to a column packed with MabSelectSuRe (GE Healthcare Japan) equilibrated with PBS. After the culture medium was completely loaded onto the column, the column was washed with at least two column volumes of PBS. Next, the antibody was eluted with 2 M arginine hydrochloride solution (pH 4.0), and the antibody-containing fractions were collected. The fraction was dialyzed using a Slide-A-Lyzer Dialysis Cassette (ThermoFisher SCIENTIFIC) and the solution was replaced with HBSor (25 mM histidine, 5% sorbitol, pH 6.0). Finally, the fraction was concentrated at 4°C using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 10 mg/mL, and the purified sample was prepared.

Example 6. In vitro evaluation of human chimeric anti-human IL-22BP antibodies 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 using a Biacore T200 (GE Healthcare Japan) by capturing the human chimeric antibody as a ligand onto an anti-human IgG (Fc) antibody immobilized using a Human Antibody Capture Kit (GE Healthcare Japan), and measuring the antigen as an analyte. HBS-EP+ (GE Healthcare Japan) was used as the running buffer. A human chimeric anti-human IL-22BP antibody diluted to 2 μg/mL was added to the sensor chip at a flow rate of 10 μL/min for 30 seconds, and then a dilution series of human IL-22BP-His (5 concentrations starting from 243 nM with a common ratio of 3) was added as an antigen at a flow rate of 30 μL/min for 300 seconds, followed by measurement of the dissociation phase for 600 seconds. 3 M magnesium chloride (GE Healthcare Japan) was added as a regeneration solution at a flow rate of 20 μL/min for 30 seconds. A 1:1 binding model was used to analyze the data, and the association rate constant (ka), dissociation rate constant (kd), and binding dissociation constant (KD) were calculated.

6)-2 Species cross-reactivity assessment (monkeys, mice):
6)-2-1 Binding activity evaluation (monkeys): BLI
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 was measured using Octet RED 384 (MOLECULAR DEVICES), in which the human chimeric antibody was captured as a ligand on a biosensor Protein A (ProA) (MOLECULAR DEVICES), the antigen was used as an analyte, and PBS-T (Takara Bio Inc.) was used as a measurement buffer. The biosensor was reacted with the human chimeric anti-human IL-22BP antibody diluted to 3 μg/mL for 120 seconds, and then reacted with serially diluted solutions of monkey IL-22BP-His (7.81, 15.6, 31.3, 62.5, 125, 250, and 500 nM) for 900 seconds. The biosensor was reacted with 10 mM glycine pH 1.5 (GE Healthcare Japan) as a regeneration solution for 25 seconds. Data analysis was performed 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 using a Biacore T200 (GE Healthcare Japan). The antigen was captured as a ligand onto Anti-6xHis tag antibody [AD1.1.10] (Abcam) immobilized using Amine Coupling Kit, type 2 (GE Healthcare Japan), and the human chimeric antibody was measured as an analyte. HBS-EP+ (GE Healthcare Japan) was used as the running buffer. Mouse IL-22BP-His diluted to 1 μg/mL was added to the sensor chip at 10 μL/min for 60 seconds, and then a dilution series of human chimeric anti-human IL-22BP antibody (five concentration series starting from 1 μM with a common ratio of 2) was added at a flow rate of 30 μL/min for 300 seconds. 3 M magnesium chloride (GE Healthcare Japan) was added as a regeneration solution at a flow rate of 20 μL/min for 45 seconds. Data were 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 and L chain 7)-1-1 Molecular modeling of variable region A known method known as homology modeling (Methods in Enzymology, 203, 121-153 (1991)) was used. Using the commercially available protein three-dimensional structure analysis program Discovery Studio (Dassault Systèmes), structures registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) that have high sequence homology to the variable region were searched. Three-dimensional model structures were created using the hit heavy chain, light chain, and interface structures between the heavy chain and light chain as templates.

7)-1-2 Method for Designing Humanized Antibody A humanized anti-human IL-22BP antibody was constructed by a method commonly known as CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The framework regions of MAb3 were derived from the human κ chain IGKV2D-30*01 and IGKJ4*01 defined in IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM®) and the human κ chain IGKV2D-30*01 and IGKJ4*01 defined in KABAT et al. Because they have high homology to the consensus sequence of human γ-chain subgroup 1 defined in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)), they were selected as the acceptors for the light and heavy chains of MAb3. Because the framework regions of MAb4 have high homology to the consensus sequences of human κ-chain subgroup 1, human κ-chain subgroup 2, human κ-chain subgroup 4, and human γ-chain subgroup 3 defined by Kabat et al., they were selected as the acceptors for the light and heavy chains of MAb4. The donor residues to be transferred onto the acceptor were selected from the consensus sequences defined by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)) and the like, three-dimensional models were analyzed and designed independently for each sequence.

7)-1-3 Humanization of MAb3 Light Chain Humanized antibody light chains were designed by connecting a human IgG1 κ chain constant region to the designed humanized antibody light chain variable region of MAb3, and were named hMAb3_L01, hMAb3_L02, hMAb3_L03, hMAb3_L04, hMAb3_L05, hMAb3_L06, and hMAb3_L07, respectively. 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 Humanized antibody heavy chains were designed by connecting the gamma chain constant region of human IgG1 to the designed MAb3 humanized antibody heavy chain variable region, and were designated hMAb3_H01, hMAb3_H02, hMAb3_H03, and hMAb3_H04, respectively. 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 Humanized antibody light chains were designed by connecting a human IgG1 κ chain constant region to the designed humanized antibody light chain variable region of MAb4, and were designated hMAb4_L01, hMAb4_L02, hMAb4_L03, hMAb4_L04, hMAb4_L05, and hMAb4_L06, respectively. The full-length amino acid sequence of hMAb4_L01 is set forth in SEQ ID NO: 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 set forth in SEQ ID NO: 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 set forth in SEQ ID NO: 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 set forth 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 set forth 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 set forth 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 Humanized antibody heavy chains were designed by connecting a human IgG1 gamma chain constant region to the designed humanized antibody heavy chain variable region of MAb4, and were designated hMAb4_H01, hMAb4_H02, and hMAb4_H03, respectively. The full-length amino acid sequence of hMAb4_H01 is set forth in SEQ ID NO: 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 set forth in SEQ ID NO: 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 set forth in SEQ ID NO: 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 Expression Vector for Light Chain of Humanized Anti-Human IL-22BP Antibody A DNA fragment containing a DNA sequence encoding the variable region in the nucleotide sequence of the light chain of humanized anti-human IL-22BP antibody shown in SEQ ID NOs:110 to 120 in the Sequence Listing was synthesized (gene synthesis service, GENEART). Using the synthesized DNA fragment as a template, a DNA fragment containing the DNA sequence encoding the variable region was amplified with KOD-Plus- (TOYOBO) and the primer set below. The amplified DNA fragment was then inserted into the light chain expression vector pCMA-LK cleaved with the restriction enzyme BsiWI using In-Fusion HD Cloning Kit (CLONTECH), thereby constructing a vector for expressing the light chain of humanized anti-human IL-22BP antibody. The amino acid sequences of the light chains of the humanized anti-human IL-22BP antibodies are shown in SEQ ID NOs: 121 to 131 in the Sequence Listing.
Primer set 5'-CTGTGGATCTCCGGCGCGTACGGC-3' (primer CM-LKF: SEQ ID NO: 91)
5'-GGAGGGGGGCGGCCACCGTACG-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 the variable region in the nucleotide sequence of the heavy chain shown in SEQ ID NOS: 132 to 137 in the Sequence Listing was synthesized (gene synthesis service, GENEART). Using the synthesized DNA fragment as a template, a DNA fragment containing the DNA sequence encoding the variable region was amplified using KOD-Plus- (TOYOBO) and the following primer set. The amplified DNA fragment was then inserted into the heavy chain expression vector pCMA-G1 cleaved with the restriction enzyme BlpI using In-Fusion HD Cloning Kit (CLONTECH), thereby constructing an expression vector for expressing the heavy chain of the humanized anti-human IL-22BP antibody. The amino acid sequence of the humanized anti-human IL-22BP antibody heavy chain is shown in SEQ ID NOS: 138 to 143 in the Sequence Listing.
Primer set 5'-AGCTCCCAGATGGGTGCTGAGC-3' (primer EG-Inf-F: SEQ ID NO: 108)
5'-GGGCCCTTGGTGGAGGCTGAGC-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 ThermoFisher SCIENTIFIC) were passaged and cultured according to the manual.

1 × ¹⁰⁷ FreeStyle 293F cells (ThermoFisher SCIENTIFIC) in the logarithmic growth phase were diluted to 9.6 mL with FreeStyle293 expression medium (ThermoFisher SCIENTIFIC), then inoculated into a 30 mL Square Storage Bottle (Nalgene) and cultured with shaking at 90 rpm for 1 hour in _an incubator at 37°C and 8% CO2. 30 μg of polyethyleneimine (Polyscience) was dissolved in 200 μL of Opti-Pro SFM (ThermoFisher SCIENTIFIC), and then 6 μg of a light chain expression vector and 4 μg of a heavy chain expression vector prepared using a PureLink HiPure Plasmid kit (ThermoFisher SCIENTIFIC) were added to 200 μL of Opti-Pro SFM (ThermoFisher SCIENTIFIC). 200 μL of the expression vector/Opti-Pro SFM mixture was added to 200 μL of the polyethyleneimine/Opti-Pro SFM mixture, gently stirred, and then allowed to stand for another 5 minutes before being added to FreeStyle 293F cells. The cells were cultured at 37°C in an 8% _CO2 incubator with shaking at 90 rpm for 7 days, and the resulting culture supernatant was filtered through a Minisart-Plus filter (Sartorius) to prepare a sample for evaluation.

The humanized antibody obtained by combining hMAb3_H01 and hMAb3_L03 is designated "hMAb3_H01L03", the humanized antibody obtained by combining hMAb3_H01 and hMAb3_L04 is designated "hMAb3_H01L04", the humanized antibody obtained by combining hMAb3_H03 and hMAb3_L01 is designated "hMAb3_H03L01", the humanized antibody obtained by combining hMAb3_H03 and hMAb3_L03 is designated "hMAb3_H03L03", the humanized antibody obtained by combining hMAb3_H03 and hMAb3_L05 is designated "hMAb3_H03L05", the humanized antibody obtained by combining hMAb3_H04 and hMAb3_L03 is designated "hMAb3_H04L03", and The humanized antibody obtained by combining MAb3_H04 and hMAb3_L07 is designated "hMAb3_H04L07", the humanized antibody obtained by combining hMAb4_H01 and hMAb4_L01 is designated "hMAb4_H01L01", the humanized antibody obtained by combining hMAb4_H01 and hMAb4_L02 is designated "hMAb4_H01L02", the humanized antibody obtained by combining hMAb4_H01 and hMAb4_L04 is designated "hMAb4_H01L04", the humanized antibody obtained by combining hMAb4_H01 and hMAb4_L05 is designated "hMAb4_H01L05", the humanized antibody obtained by combining hMAb4_H01 and hMAb4_L06 is designated "hMAb4_H01L06", and the humanized antibody obtained by combining hMAb4_H01 and hMAb4_L06 is designated "hMAb4_H01L07". The humanized antibody obtained by combining hMAb4_H02 and hMAb4_L01 is designated "hMAb4_H02L01", the humanized antibody obtained by combining hMAb4_H02 and hMAb4_L02 is designated "hMAb4_H02L02", the humanized antibody obtained by combining hMAb4_H02 and hMAb4_L03 is designated "hMAb4_H02L03", the humanized antibody obtained by combining hMAb4_H02 and hMAb4_L04 is designated "hMAb4_H02L04", the humanized antibody obtained by combining hMAb4_H02 and hMAb4_L05 is designated "hMAb4_H02L05", the humanized antibody obtained by combining hMAb4_H02 and hMAb4_L06 is designated "hMAb4_H02L06", and the humanized antibody obtained by combining hMAb4_H02 and hMAb4_L06 is designated "hMAb4_H02L07". The humanized antibody obtained by combining hMAb4_H03 and hMAb4_L01 was named "hMAb4_H03L01," the humanized antibody obtained by combining hMAb4_H03 and hMAb4_L02 was named "hMAb4_H03L02," the humanized antibody obtained by combining hMAb4_H03 and hMAb4_L03 was named "hMAb4_H03L03," the humanized antibody obtained by combining hMAb4_H03 and hMAb4_L04 was named "hMAb4_H03L04," the humanized antibody obtained by combining hMAb4_H03 and hMAb4_L05 was named "hMAb4_H03L05," and the humanized antibody obtained by combining hMAb4_H03 and hMAb4_L06 was named "hMAb4_H03L06."

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

FreeStyle 293F cells (ThermoFisher SCIENTIFIC) were passaged and cultured according to the manual.

1.2 × ¹⁰⁹ FreeStyle 293F cells (ThermoFisher SCIENTIFIC) in the logarithmic growth phase were seeded into a 3 L Fernbach Erlenmeyer Flask (CORNING), diluted with FreeStyle 293 expression medium (ThermoFisher SCIENTIFIC) to a volume of 560 mL, and then cultured with shaking at 95 rpm for 1 hour in an 8% _CO2 incubator at 37°C. 1.8 mg of polyethyleneimine (Polyscience) was dissolved in Opti-Pro SFM (ThermoFisher SCIENTIFIC) to make 20 mL, and then 0.24 mg of a light chain expression vector and 0.36 mg of a heavy chain expression vector prepared using a PureLink HiPure Plasmid kit (ThermoFisher SCIENTIFIC) were suspended in Opti-Pro SFM (ThermoFisher SCIENTIFIC) to make 20 mL. 20 mL of the expression vector/Opti-Pro SFM mixture was added to 20 mL of the polyethyleneimine/Opti-Pro SFM mixture, gently stirred, and then allowed to stand for another 5 minutes before being added to the FreeStyle 293F cells. After culturing for 4 hours in _an incubator at 37°C and 8% CO2 with shaking at 95 rpm, 600 mL of EX-CELL VPRO medium (SAFC Biosciences) and 30 mL of BD Recharge CD (BD Bioscience) were added, and the cells were cultured for 6 days in _an incubator at 37°C and 8% CO2 with shaking at 95 rpm. The resulting culture supernatant was filtered through a Disposable Capsule Filter (ADVANTEC).

8)-5 Purification of humanized anti-human IL-22BP antibody The culture supernatant obtained in Example 8)-4 was purified in two steps: rProtein A affinity chromatography at 4 to 6°C and ceramic hydroxylapatite at room temperature. The buffer exchange steps after rProtein A affinity chromatography and ceramic hydroxylapatite purification were carried out at 4 to 6°C. First, the culture supernatant was applied to MabSelect SuRe (GE Healthcare Japan) equilibrated with PBS. After the culture medium was completely loaded onto the column, the column was washed with at least two column volumes of PBS. Next, the column was eluted with 2 M arginine hydrochloride solution, pH 4.0, and the antibody-containing fractions were collected. The fraction was dialyzed using a Slide-A-Lyzer Dialysis Cassette (ThermoFisher SCIENTIFIC) and replaced with PBS. The antibody solution was then diluted 5-fold with a buffer of 5 mM sodium phosphate, 50 mM MES, pH 7.0, and applied to a ceramic hydroxylapatite column, Bio-Scale CHT Type-I Hydroxyapatite Column (Japan Bio-Rad), equilibrated with a buffer of 5 mM NaPi, 50 mM MES, 30 mM NaCl, pH 7.0. Linear gradient elution with sodium chloride was performed, and fractions containing the antibody were collected. The fraction was dialyzed using a Slide-A-Lyzer Dialysis Cassette (ThermoFisher SCIENTIFIC) and the solution was replaced with HBSor (25 mM histidine, 5% sorbitol, pH 6.0). Finally, the fraction was concentrated at 4°C using a Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF10K, Sartorius) to adjust the IgG concentration to 30 or 50 mg/mL or higher, and used as a purified sample.

Example 9. In vitro evaluation of humanized anti-human IL-22BP antibodies 9)-1 Binding activity evaluation (human): SPR
The binding dissociation constants between the humanized anti-human IL-22BP antibody prepared in Example 8)-5 and human IL-22BP-His prepared in Example 1)-2 were measured and calculated by the SPR method in the same manner as in Example 6)-1. As shown in Figure 2, the binding dissociation constants of the humanized antibodies of the hMAb3 series ranged widely from 13.8 nM to 513 nM, while the binding dissociation constants of the humanized antibodies of the hMAb4 series ranged from 8.14 nM to 12.5 nM, generally showing stronger binding than the hMAb3 series.

9)-2 Species cross-reactivity evaluation (monkeys): 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 using Octet RED 384 (MOLECULAR DEVICES), in which the humanized antibody was captured as a ligand on a biosensor Protein A (ProA) (MOLECULAR DEVICES), the antigen was used as an analyte, and PBS-T (Takara Bio Inc.) was used as a measurement buffer. The biosensor was reacted with the humanized anti-human IL-22BP antibody diluted to 3 μg/mL for 120 seconds, and then reacted with serially diluted solutions of monkey IL-22BP-His (7.0, 20.0, 40.0, 75.0, 150, 270, and 500 nM) for 900 seconds. The biosensor was reacted with 10 mM glycine pH 1.5 (GE Healthcare Japan) as a regeneration solution for 25 seconds. Data analysis was performed using equilibrium value analysis, and the binding dissociation constant (KD) was calculated. Figure 3 shows a list of binding dissociation constants.

9)-3 Competitive inhibition experiment using Competition ELISA 6x-His Tag Polyclonal Antibody (ThermoFisher SCIENTIFIC) was diluted to 2 μg/mL with PBS, added in 50 μL aliquots to a 96-well Maxi-Sorp plate (Nunc), and immobilized overnight at 4°C. The next day, the plate was washed twice with PBS containing 0.05% Tween-20 (PBS-T), and then 200 μL of Block BSA in PBS (ThermoFisher SCIENTIFIC) was added to each well and 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 (dilution buffer) containing 1% BSA (manufactured by JACKSON IMMUNORESEARCH) was added to each well and allowed to stand at 37°C for 1 hour. After washing five times with PBS-T, 50 μL of a dilution series of humanized anti-human IL-22BP antibody prepared in dilution buffer (5 concentrations starting from 75 μg/mL with a common ratio of 5) was added to each well. Subsequently, recombinant IL-22BP diluted to 1 μg/mL with dilution buffer was added to each well.

Five μL of human IL-22 protein (R&D Systems) or dilution buffer alone was added to each well and incubated at 37°C for 1 hour. After washing five times with PBS-T, 50 μL of human IL-22 antibody (R&D Systems) diluted to 0.5 μg/mL with dilution buffer was added to each well and incubated at 37°C for 1 hour. After washing five times with PBS-T, 50 μL of goat anti-mouse IgG antibody, HRP conjugate, Species Adsorbed (Merck) diluted to 0.8 μg/mL with dilution buffer was added to each well and incubated at 37°C for 1 hour. After washing six times with PBS-T, 100 μL of OPD coloring solution was added to each well, and after leaving the wells to stand for 6 minutes, 100 μL of 1 M HCl was added to each well, and the absorbance at 490 nm was measured using an Emspire (Perkin Elmer). The inhibition rate was calculated using the following formula:
Inhibition rate (%) = (1 - absorbance Abs490 when antibody was added / maximum absorbance Abs490 when IL-22 alone was added) x 100

Control IgG (ctrlIgG) and HBS or pH 6.0 were used as controls. As shown in Figure 4, all antibodies exhibited concentration-dependent inhibitory activity, with the human chimeric antibody cMAb3 and its derived humanized antibodies hMAb3H01L03, hMAb3H01L04, and hMAb3H04L03 demonstrating stronger inhibitory activity than the human chimeric antibody cMAb4 and its derived humanized 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 region
Extraction of the crypt region of human colonic epithelial cells was performed with reference to Mizoguchi E, et al. (Gastroenterology 125:148-161 (2003)). 2 cm x 2 cm tissue samples were collected from surgically excised human colon specimens and washed with PBS. To increase the penetration efficiency of EDTA, the lamina propria was detached from the muscularis layer and then washed again with PBS. The isolated lamina propria was immersed in 30 mL of 30 mM EDTA/HANKS solution and left at room temperature for 15 minutes. It was then vigorously shaken by hand for approximately 30 seconds to confirm that the epithelium had been detached in the form of crypt structures.

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

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

After harvesting, cells were washed twice with PBS and lysed in lysis buffer [50 mM Tris (pH 8.0), 0.5% NP-40, 1 mM EDTA, 150 mM NaCl, 1 mM sodium vanadate, 50 mM sodium pyrophosphate, 1 mM PMSF, 1 tablet of protease inhibitor cocktail] to homogenize, then flash-frozen on dry ice. Western blotting for detecting phosphorylated STAT3 was performed according to Mizoguchi A, et al. (Immunity. 16(2):219-230(2002)). The frozen samples were rapidly thawed at 37°C, and the cell membranes were completely disrupted by sonication. The supernatants were then collected by centrifugation at 12,000 rpm at 4°C for 30 minutes. Each supernatant was then run on SDS-PAGE and transferred to a nitrocellulose membrane. Bands specific to phosphorylated STAT3 were detected using ECL detection reagents (GE Healthcare Japan). An anti-phosphorylated STAT3 antibody (Cell Signaling Technology) was used for detection.

Figure 5 shows the detection results for phosphorylated STAT3 after pretreatment with the antibody being evaluated. Compared to the blank group, a strong STAT3 phosphorylation signal was observed in cells derived from the crypt region of human colon epithelial cells in the IL-22-added group, whereas the phosphorylation signal band disappeared when an equal amount of IL-22BP was added. In contrast, the phosphorylation signal was confirmed to be restored when each humanized anti-human IL-22BP antibody was added. The total STAT3 band indicates the signal for the total amount of STAT3 protein.

The antibodies of the present invention are useful for treating inflammatory bowel disease, etc.

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 DYKDDDDKHHHHHH, 1st to 20th amino acids of the amino acid sequence of human IL2
SEQ ID NO: 5: DNA encoding a sequence comprising 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: Nucleotide sequence of human IL-22BP-His SEQ ID NO: 7: Amino acid sequence of human IL-22BP-His SEQ ID NO: 8: DNA encoding a sequence comprising a polypeptide in which ENLYFQG is linked to the C-terminal side of the amino acid sequence from positions 1 to 21 of human IL-22BP and the amino acid sequence from positions 21 to 231 of monkey IL-22BP.
SEQ ID NO: 9: Nucleotide sequence of monkey IL-22BP-His SEQ ID NO: 10: Amino acid sequence of monkey IL-22BP-His SEQ ID NO: 11: DNA encoding a sequence comprising a polypeptide in which ENLYFQG is linked to the C-terminal side of the amino acid sequence of mouse IL-22BP from positions 1 to 21 of the amino acid sequence of human IL-22BP and positions 21 to 230 of the amino acid sequence of mouse IL-22BP in which Cys106 is substituted with Ser.
SEQ ID NO:12: nucleotide 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 from which the tag has been cleaved SEQ ID NO:15: nucleotide sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb3 SEQ ID NO:16: nucleotide sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb4 SEQ ID NO:17: nucleotide sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb8 SEQ ID NO:18: nucleotide sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb14 SEQ ID NO:19: nucleotide sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb20 SEQ ID NO:20: nucleotide sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb81 SEQ ID NO:22: Amino acid sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb3: Amino acid sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb4: Amino acid sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb8: Amino acid sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb14: Amino acid sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb20: Amino acid sequence of the variable region of the light chain of rat anti-human IL-22BP antibody rMAb81: Amino acid sequence of CDRL1 of MAb3_VL: SEQ ID NO:28: Amino acid sequence of CDRL2 of MAb3_VL: SEQ ID NO:29: Amino acid sequence of CDRL3 of MAb3_VL: SEQ ID NO:30: Amino acid sequence of CDRL1 of MAb4_VL: Amino acid sequence of CDRL2 of MAb4_VL SEQ ID NO:32: Amino acid sequence of CDRL3 of MAb4_VL SEQ ID NO:33: Amino acid sequence of CDRL1 of MAb8_VL SEQ ID NO:34: Amino acid sequence of CDRL2 of MAb8_VL SEQ ID NO:35: Amino acid sequence of CDRL3 of MAb8_VL SEQ ID NO:36: Amino acid sequence of CDRL1 of MAb14_VL SEQ ID NO:37: Amino acid sequence of CDRL2 of MAb14_VL SEQ ID NO:38: Amino acid sequence of CDRL3 of MAb14_VL SEQ ID NO:39: Amino acid sequence of CDRL1 of MAb20_VL SEQ ID NO:40: Amino acid sequence of CDRL2 of MAb20_VL SEQ ID NO:41: Amino acid sequence of CDRL3 of MAb20_VL SEQ ID NO:42: SEQ ID NO:43: Amino acid sequence of CDRL1 of MAb81_VL: SEQ ID NO:44: Amino acid sequence of CDRL2 of MAb81_VL: SEQ ID NO:45: Nucleotide sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb3: SEQ ID NO:46: Nucleotide sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb4: SEQ ID NO:47: Nucleotide sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb8: SEQ ID NO:48: Nucleotide sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb14: SEQ ID NO:49: Nucleotide sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb20: SEQ ID NO:50: Nucleotide sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb81: SEQ ID NO:52: Amino acid sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb3: SEQ ID NO:53: Amino acid sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb4: SEQ ID NO:54: Amino acid sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb8: SEQ ID NO:55: Amino acid sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb14: SEQ ID NO:56: Amino acid sequence of the variable region of the heavy chain of rat anti-human IL-22BP antibody rMAb20: SEQ ID NO:57: Amino acid sequence of CDRH1 of MAb3_VH SEQ ID NO:58: Amino acid sequence of CDRH2 of MAb3_VH SEQ ID NO:59: Amino acid sequence of CDRH3 of MAb3_VH SEQ ID NO:60: Amino acid sequence of CDRH1 of MAb4_VH SEQ ID NO:61: Amino acid sequence of CDRH2 of MAb4_VH SEQ ID NO:62: Amino acid sequence of CDRH3 of MAb4_VH SEQ ID NO:63: Amino acid sequence of CDRH1 of MAb8_VH SEQ ID NO:64: Amino acid sequence of CDRH2 of MAb8_VH SEQ ID NO:65: Amino acid sequence of CDRH3 of MAb8_VH SEQ ID NO:66: Amino acid sequence of CDRH1 of MAb14_VH SEQ ID NO:67: Amino acid sequence of CDRH2 of MAb14_VH SEQ ID NO:68: Amino acid sequence of CDRH3 of MAb14_VH SEQ ID NO:69: Amino acid sequence of CDRH1 of MAb20_VH SEQ ID NO:70: Amino acid sequence of CDRH2 of MAb20_VH SEQ ID NO:71: Amino acid sequence of CDRH3 of MAb20_VH SEQ ID NO:72: SEQ ID NO: 73: Amino acid sequence of CDRH1 of MAb81_VH: SEQ ID NO: 74: Amino acid sequence of CDRH2 of MAb81_VH: SEQ ID NO: 75: Nucleotide sequence containing DNA encoding a human κ chain secretion signal and a human κ chain constant region: SEQ ID NO: 76: Primer 3.3-F1
SEQ ID NO: 77: Primer 3.3-R1
SEQ ID NO:78: Nucleotide sequence comprising the DNA sequence encoding the amino acids of the human heavy chain signal sequence and the human IgG1 constant region SEQ ID NO:79: Human chimeric anti-human IL-22BP antibody cMAb3 light chain nucleotide sequence SEQ ID NO:80: Human chimeric anti-human IL-22BP antibody cMAb4 light chain nucleotide sequence SEQ ID NO:81: Human chimeric anti-human IL-22BP antibody cMAb8 light chain nucleotide sequence SEQ ID NO:82: Human chimeric anti-human IL-22BP antibody cMAb14 light chain nucleotide sequence SEQ ID NO:83: Human chimeric anti-human IL-22BP antibody cMAb20 light chain nucleotide sequence SEQ ID NO:84: Human chimeric anti-human IL-22BP antibody cMAb81 light chain nucleotide sequence SEQ ID NO:85: Human chimeric anti-human IL-22BP antibody cMAb3 light chain amino acid sequence SEQ ID NO:86: Human chimeric 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 sequence SEQ ID NO:88: Human chimeric anti-human IL-22BP antibody cMAb14 light chain amino acid sequence SEQ ID NO:89: Human chimeric anti-human IL-22BP antibody cMAb20 light chain amino acid sequence SEQ ID NO:90: Human chimeric anti-human IL-22BP antibody cMAb81 light chain amino acid sequence SEQ ID NO:91: Primer CM-LKF
SEQ ID NO: 92: Primer KCL-Inf-R
SEQ ID NO:93: Human chimeric anti-human IL-22BP antibody cMAb3 heavy chain nucleotide sequence SEQ ID NO:103: Amino acid sequence of the heavy chain of human chimeric anti-human IL-22BP antibody cMAb14: Amino acid sequence of the heavy chain of human chimeric anti-human IL-22BP antibody cMAb20: Amino acid sequence of the heavy chain of human chimeric anti-human IL-22BP antibody cMAb81: Amino acid sequence of the heavy chain of human chimeric anti-human IL-22BP antibody cMAb81 SEQ ID NO:105: Amino acid version of the sequence of CDRH1 of MAb4'_VH SEQ ID NO:106: Amino acid version of the sequence of CDRH2 of MAb4'_VH SEQ ID NO:107: Amino acid version of the sequence 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 of hMAb3_L01 SEQ ID NO:111: Nucleotide sequence of hMAb3_L03 SEQ ID NO:112: Nucleotide sequence of hMAb3_L04 SEQ ID NO:113: Nucleotide sequence of hMAb3_L05 SEQ ID NO:114: Nucleotide sequence of hMAb3_L07 SEQ ID NO:115: Nucleotide sequence of hMAb4_L01 SEQ ID NO:116: Nucleotide sequence of hMAb4_L02 SEQ ID NO:117: Nucleotide sequence of hMAb4_L03 SEQ ID NO:118: Nucleotide sequence of hMAb4_L04 SEQ ID NO:119: Nucleotide sequence of hMAb4_L05 SEQ ID NO:120: Nucleotide sequence of hMAb4_L06 SEQ ID NO:121: Amino acid sequence of hMAb3_L01 SEQ ID NO:122: Amino acid sequence of hMAb3_L03 SEQ ID NO:123: Amino acid sequence of hMAb3_L04 SEQ ID NO:124: Amino acid sequence SEQ ID NO:125 for hMAb3_L05: Amino acid sequence SEQ ID NO:126 for hMAb3_L07: Amino acid sequence SEQ ID NO:127 for hMAb4_L01: Amino acid sequence SEQ ID NO:128 for hMAb4_L02: Amino acid sequence SEQ ID NO:129 for hMAb4_L03: Amino acid sequence SEQ ID NO:130 for hMAb4_L04: Amino acid sequence SEQ ID NO:131 for hMAb4_L05: Amino acid sequence SEQ ID NO:132 for hMAb4_L06: Nucleotide sequence SEQ ID NO:133 for hMAb3_H01: Nucleotide sequence SEQ ID NO:134 for hMAb3_H03: Nucleotide sequence SEQ ID NO:135 for hMAb3_H04: Nucleotide sequence SEQ ID NO:136 for hMAb4_H01: Nucleotide sequence SEQ ID NO:137 for hMAb4_H02: Nucleotide sequence SEQ ID NO:138 for hMAb4_H03 Amino acid sequence of hMAb3_H01 SEQ ID NO: 139: Amino acid sequence of hMAb3_H03 SEQ ID NO: 140: Amino acid sequence of hMAb3_H04 SEQ ID NO: 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 hereby incorporated by reference in their entirety.

Claims (11)

(a2) a light chain CDRL1 consisting of the amino acid sequence shown in SEQ ID NO: 30;
(b2) a light chain CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 31, and (c2) a light chain CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 32;
and (d2) a heavy chain CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 60 or SEQ ID NO: 105;
(e2) a heavy chain CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 61 or SEQ ID NO: 106, and (f2) a heavy chain CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 62 or SEQ ID NO: 107.
an antibody, or binding fragment thereof, comprising a heavy chain comprising:
a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 21 to 133 of SEQ ID NO: 128 in the Sequence Listing, and a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 20 to 137 of SEQ ID NO: 143 in the Sequence Listing,
An antibody or a binding fragment thereof having the following properties (i) to (iv):
(i) a monoclonal antibody that binds to human IL-22BP;
(ii) binds to simian IL-22BP;
(iii) the binding dissociation constant (KD) for human IL-22BP-His is 1 x ^10-8 M or less; and (iv) in cells derived from the crypt region of human colon epithelial cells, the STAT3 phosphorylation signal observed upon addition of IL-22 and IL-22BP is lost, but the signal is restored by simultaneous or pretreatment with IL-22 and IL-22BP. The antibody or binding fragment thereof of claim 1, wherein the antibody is a chimeric antibody or a humanized antibody . The antibody or binding fragment thereof according to claim 1 or 2, which does not cross-react with rodent IL-22BP. The antibody according to any one of claims 1 to 3, comprising a light chain comprising the amino acid sequence shown in amino acid numbers 21 to 239 of SEQ ID NO: 128 in the sequence listing, and a heavy chain comprising the amino acid sequence shown in amino acid numbers 20 to 467 of SEQ ID NO: 143 in the sequence listing . 5. The antibody or binding fragment thereof of claim 1 , wherein the heavy chain lacks a carboxyl-terminal lysine. A polynucleotide comprising a nucleotide sequence encoding the amino acid sequences contained in the heavy and light chains of the antibody according to any one of claims 1 to 5 . A vector comprising the polynucleotide of claim 6 . A host cell comprising the polynucleotide of claim 6 or the vector of claim 7 . A method for producing an antibody or binding fragment thereof according to any one of claims 1 to 5 , comprising culturing a host cell according to claim 8 and recovering the antibody or binding fragment thereof from the culture. The antibody or binding fragment thereof according to any one of claims 1 to 5 , which is complexed with a drug. 6. An antibody or binding fragment thereof according to any one of claims 1 to 5 , which is comprised in a multispecific molecule.