NZ621320B2

NZ621320B2 - Anti-human xcr1 antibodies

Info

Publication number: NZ621320B2
Application number: NZ621320A
Authority: NZ
Inventors: Toshio Imai; Tetsu Kawano; Miyuki Nishimura; Yoshimasa Sakamoto; Yukihisa Sawa
Original assignee: Eisai R&D Management Co Ltd
Priority date: 2011-09-01
Filing date: 2012-08-30
Publication date: 2016-03-01

Abstract

Disclosed are antibodies binding to human XCR1 comprising the CDRs of the sequences as defined in the specification. Also disclosed is their use for treating an immune disease.

Description

DESCRIPTION Title of Invention: ANTI-HUMAN XCRl ANTIBODIES Technical Field The present invention s to an antibody that binds to human XCRl.

Background Art Chemokine is a collective term for basic heparin— binding proteins that have s on leukocyte chemotaxis and leukocyte activation. Based on a comparison of the primary structures of various chemokines, the chemokines are classified into CXC, CC, C, and CX3C subfamilies according to the positions of conserved cysteine residues. XCLl (also referred to as lymphotactin (Ltn) or lymphotactin d (Ltn-a)) and XCL2 (also referred to as lymphotactin B (Ltn—B)) are chemokines classified into the subfamily C described above. XCRl (also referred to as GPRS, SCM-lc, or ATAC) is a G protein-coupled chemokine or, which specifically binds to xcm and XCL2.

Expression of XCRl in various human tissues has been examined at the mRNA level. Expression of XCRl is reportedly high in ta, but low in spleen and thymus gland (NON PATENT LITERATURE 1). Further, XCRl is mainly expressed in dendritic cells. In mice, XCRl is highly expressed, particularly in CD8a+ dendritic cells (NON PATENT LITERATURE 2; and NON PATENT LITERATURE 3). The CD8c+ tic cells are normally t in secondary lymphoid tissues such as spleen and lymph nodes, and are known to perform —presentation," which serves an important role in ons against infection and immunological responses to tumor cells. XCRl is also known to be highly expressed in human CD141+ dendritic cells, which are considered to be homologues of mouse CD8c+ dendritic cells (NON PATENT LITERATURE 4).

Antigen taken up from the outside of cells into antigen—presenting cells is usually degraded into peptide, ted on class II major histocompatibility antigen (MHC class II), and recognized by CD4+ T—cells. In contrast, there is a case» where the antigen taken up from the outside of cells is presented on class I major histocompatibility antigen (MHC class I) via a pathway different from the usual pathway described above. This antigen presentation process is referred to as "cross- presentation." In this process, V the antigen ted on MHC class I is recognized by the CD8+ T—cells, and then. differentiated into cytotoxic T—cells (CTL) that play a role in phylaxis and the elimination of tumor cells in the host (Non Patent ture 5).

Migration of various immune—related cells occurs during inflammation reaction. In particular, migration of dendritic cells to a local inflammatory site occurs for phagocytosis of antigens. Chemokines and chemokine receptors play ant roles in causing such ion of dendritic cells. After migration to a local inflammatory site, the dendritic cells present antigens to T—cells, and activate T-cells. Subsequently, the information is transmitted from T—cells to many more immune-related cells; amplifying the immune reaction (Non Patent Literature 6).

Among antigen presenting cells, the dendritic cells have particularly excellent antigen-presenting ability, and play a very important role in the activation of the T-cells. It has been suggested that because T—cells are involved in the development and exacerbation of s immune diseases including mune es, to control dendritic cells is to control the activation of T—cells, which may lead to the amelioration of various immune diseases (Non Patent Literature 6; and Non Patent Literature 7). r, it has been shown that a rabbit-derived onal antibody t human XCRl has an effect of inhibiting XCL-induced migration of normal oral keratinocytes and oral cancer cells (Non Patent Literature 8).

Citation List Non Patent Literature NFL 1: Yoshida T, Imai T, Kakizaki M, ura M, Takagi S, Yoshie 0. "Identification of Single C motif~ l/lymphotactin receptor XCRl," J. Biol. Chem. 273: 16551-16554 (1998) NFL 2: Crozat K, Guiton R, Contreras V, Feuillet V, Dutertre CA, Ventre E, Vt Manh TP, Baranek T, t AK, Marvel J, Boudinot P, Hosmalin A, Schwartz—Cornil I, Dalod M "The XC chemokine.receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8a+ dendritic cells," J Exp Med, 207: 1283-1292 (2010) NFL 3: Dorner BG, Dorner MB, Zhou X, Opitz C, Mora A, r S, Hutloff A, Mages HW, Ranke K, Schaefer-M, Jack RS, Henn V, Kroczek RA "Selective expression of the ine receptor XCRl on cross—presenting dendritic cells determines cooperation with CD8+ s," Immunity, 31: 823—833 (2009) NPL 4: Bachem A, Gﬁttler s, Hartung E, Ebstein F, Schaefer M, Tannert A, Salama A, Mbvassaghi K, Opitz C, Mages HW, Henn V, Kloetzel PM, Gurka S, Kroczek RA, "Superior antigen cross—presentation and XCRl expression define human CD11L9CD141‘ cells as homologues of mouse CD8+ tic cells," J Exp Med, 207: 281 (2010) NPL 5: Kurts C, Robinson BW, Knolle PA, "Cross—priming in health and disease," Nat Rev Immunol, 10: 403-414 (2010) NPL 6: Kurts C, Robinson BW, Knolle PA, "Cross—priming in health and disease," Nat Rev Immunol, 10: 403—414 (2010) NPL 7: Waldner H, “The role of innate immune responses in autoimmune disease development,“ Autoimmun, Rev 8: 400-404 (2009) NFL 8: Khurram SA, Whawell SA, Bingle L, Murdoch C, McCabe BM, Farthing PM, "Functional sion of the chemokine receptor XCRl on oral epithelial cells," J Pathol, 221: 153—63 (2010) 'Summary of Invention Technical Problem Knowledge that dendritic cells are involved in the development, exacerbation, and the like of immune diseases has been thus far accumulated using e animal models. However. at present, neither an effective treatment method nor a prevention method has been developed for many immune diseases. r, although an anti—human XCRl antibody having an effect of inhibiting cell migration is known (Khurram SA, Whawell SA, Bingle L, Murdoch C, MCCabe BM, Farthing PM, "Functional expression of the chemokine receptor XCRl on oral epithelial cells," J Pathol, 221: 153-63 (2010)), because such an dy is a rabbit-derived polyclonal antibody, it is unlikely to be immediately clinically applicable as a pharmaceutical product. In addition, the above document does not suggest that such an antibody ts cell migration of dendritic cells, and it is impossible to even predict that such an antibody will be effective in the treatment or prevention of immune diseases.

An object of the present invention is to provide a monoclonal antibody that selectively binds to human XCRl; preferably, a onal antibody that selectively binds to human XCRl and ts cell migration: further preferably an antibody that is ive in the treatment or prevention of immune diseases, in particular, immune diseases of the skin, based on the above—described effect.

Solution to Problem The present ors conducted ive studies in an attempt to solve the above problem. As a result, they developed antibodies that bind to human XCRl, and found that such antibodies have an effect of inhibiting cell ion as well as a significant effect in the treatment or prevention of immune es, such as immune diseases of the skin, associated with migration of dendritic cells.

Hereinafter, in the present specification, the abovedescribed antibodies are sometimes simply referred to as the "antibodies," "antibodies of the present invention," or "anti- human XCRl antibodies." Advantageous Effects of Invention The antibodies of the present invention bind to human XCRl. The antibodies of the t invention include an antibody that inhibits binding between human XCRl and human XCLl. Such an antibody has potential as an active ingredient to be added to a human uman XCLl g inhibitor.

The antibodies of the present invention also include an 1b antibody that inhibits cell migration, particularly that of dendritic cells. Such an antibody has potential as an active ingredient to be added to a cell migration inhibitor, particularly a dendritic cell migration inhibitor. r, the antibodies of the t invention also include an antibody that specifically recognizes BDCA3 (also referred to as CD141) ve cells. Therefore, a pharmaceutical composition comprising the antibodies of the present invention has potential as a therapeutic agent for the treatment of immune es associated with cell migration, particularly dendritic cell migration. In particular, the pharmaceutical composition has potential as a therapeutic agent for the treatment of immune diseases of the skin such as delayed—type hypersensitivity, psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis, omyositis, polymyositis, inclusion body is. autoimmune blistering disease (e.g., pemphigus, pemphigoid, or acquired epidermolysis bullosa), pustulosis, systemic scleroderma, herpes ionis, linear IgA bullous dermatosis, ia areata, vitiligo vulgaris, skin diseases associated with enosis (e.g;, systemic lupus erythematosus, Sjogren syndrome, or mixed connective tissue disease), skin diseases associated with Addison's e, skin diseases associated with graft—versus-host disease (GVHD), eczema, and urticaria.

In addition to these immune diseases of the skin, the antibodies of the present invention also has potential as therapeutic agents for the treatment of immune diseases such as diabetes mellitus type 1, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, ankylosing spondylitis, thyroiditis, graft rejection, Crohn’s disease, rheumatoid tis, inflammatory bowel disease, anterior uveitis, wegener's granulomatosis, or Behget’s disease.

Brief Description of Drawings ~[Fig. 1] Fig. 1 shows the s of FACS analysis of the reactivity of mouse anti—human XCRl antibodies (2H6, 5G7, and 11H2) to human XCRl—EGFPwexpressing 9 cells.

[Fig. 2] Fig. 2 shows the is s of a chemotaxis assay of the neutralizing activity of the mouse anti— human XCRl antibodies (2H6, 567, and 11H2) on human lymphotactin— induced migration of human XCRl-EGFP—expressing 3300.19 cells.

[Fig. 3] Fig. 3 shows the results of FACS is of the reactivity of humanized anti-human XCRl dies (HKlLZ and HK5L5) to human XCRl-EGFP-expressing 8300.19 cells, in parallel with the vity of their parent mouse anti—human XCRl antibody (5G7) and its chimeric antibody.

[Fig. 4] Fig. 4 shows the results of FACS analysis of the reactivity of mouse anti-human XCRl antibody (5G7) and humanized anti—human XCRl antibodies (HKlLZ and HKSLS) to human BDCA3+ dendritic cells.

[Fig. 5] Fig. 5 Shows the analysis results of a chemotaxis assay_of the neutralizing activity of the zed anti—human XCRl antibodies (HK1L2 and HKSLS) on human lymphotactin-induced ion of human XCRl—EGFP-expressing B300.l9 cells, in parallel with the neutralizing activity of their parent mouse anti-human XCRl antibody (5G7) and its 3O chimeric antibody.

[Fig. 6] Fig. 6 shows the analysis results of a transendothelial migration assay of the neutralizing activity of the humanized anti-human XCRl antibodies (HK1L2 and HK5L5) and a chimeric antibody on human lymphotactin-induced migration of human BDCA3+ dendritic cells, in parallel with an isotype control antibody (human 1962, K).

[Fig. 7] Fig. 7 shows the comparison of amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the antibodies of the t invention.

The figure also shows the generalized amino acid sequences of, heavy chain CDRs l to 3 and light chain CDRs 1 to 3.

[Fig. 8] Fig. 8 shows a pharmacological effect of the mouse anti-human XCRl antibody (567) of the present invention on a mouse model of delayed—type contact dermatitis (DTH).

Figs. 8A and BB respectively show the results obtained by comparing the degree of ear swelling (mm) 24 hours and 48 hours after ion by DNFB between the mouse anti—human XCRl antibody (5G7) of the present invention and the control antibody.

[Fig. 9] Fig. 9 shows binding specificity of the mouse anti-human XCRl antibody (5G7) of the t ion to various human chemokine receptors. The abscissa axis of the graph in the figure indicates the fluorescence intensity of phycoerythrin (PE) .

[Fig. 10] Fig. 10 shows amino acid sequences of human XCRl to which the antibodies of the present invention bind.

[Fig. 11] Fig. 11 shows cytotoxity to human XCRl expressing cells, using the antibodies of t invention.

[Fig. 12] Fig. 12 shows the analysis of the result of cytotoxic T cyte assay of the mouse anti-human XCRl antibody (567) of the present invention.

[Fig. 13] Fig. 13 shows reactivity of mouse anti—human XCRl antibodies (2H6, 567, and 11H2) of the present invention to the chimeric human/mouse XCRl—expressing cells.

[Fig. 14] Fig. 14 shows the analysis of result of mapping of mouse anti-human XCRl antibodies (2H6, and 5G7)- binding sites on human XCRl extracellular domains by e ELISA.

[Fig. 15] Fig. 15 shows the analysis of the result of mapping of anti—human XCRl polyclonal antibody—binding sites on human XCRl extracellular domains by using alanine mutants.

PCT/JPZOlZ/072667 [Fig. 16] Fig. 16 shows the analysis of the result of g of mouse anti-human XCRl antibody (2H6) —binding sites on human XCRl extracellular s by using alanine mutants.

[Fig. 17] Fig. 17 shows the analysis of the result of mapping of mouse anti—human XCRl antibody (5G7) -binding sites on human XCRl extracellular domains by using alanine s.

[Fig. 18] Fig. 18 shows the analysis of the result of mapping of mouse anti-human XCRl antibody (11H2)-binding sites on human EXRl extracellular domains by using alanine mutants.

[Fig. 19] Fig. 19 shows the analysis of the result of mapping of humanized uman XCRl antibody (HK1L2)-binding sites on human XCRl extracellular domains by using alanine ‘mutants.

[Fig. 20] Fig. 20 shows the analysis of the result of mapping‘of humanized anti-human XCRl antibody )-binding sites on human XCRl extracellular domains by using alanine mutants.

[Fig. 21] Fig. 21 shows the analysis of the result of the competition among mouse anti-human XCRl antibodies (2H6, 567, and 11H2) for binding to human XCRl-expressing cells.

[Fig. 22] Fig. 22 shows binding specificity of the mouse anti-human XCRl monoclonal antibody (5G7) and commercial goat anti—human XCRl onal dy to s human chemokine receptors. The abscissa axis of the graph in the figure indicates the fluorescence intensity of phycoerythrin (PE).

[Fig. 23] Fig. 23 shows binding specificity of the mouse anti-human XCRl monoclonal antibody (5G7) and humanized anti—human XCRl monoclonal antibodies (HK1L2 and HKSLS) to various human chemokine ors. The abscissa axis of the graph in the figure indicates the fluorescence intensity of phycoerythrin (PE).

[Fig. 24] Fig. 24 shows a pharmacological effect of the mouse anti-human XCRl antibody (5G7) of the present invention on a mouse model of delayed-type contact dermatitis (DTH) induced by cterium butyricum.

[Fig. 25] Fig. 25 shows a pharmacological effect of the mouse anti-human XCRl antibody (567) of the present ion on a mouse model of multiple sclerosis (MS) by experimental mune encephalomyelitis (EAE)4 [Fig. 26] Fig.26 shows the analysis of the result of the competitive ligand binding assay of mouse anti-human XCRl antibodies of the present invention. ption of Embodiments various techniques used to practice the t invention are easily and reliably enabled for a person skilled in the art based on known documents and the like, except for those techniques whose sourCes are clearly fied herein. For example, in regard to genetic engineering and molecular biological techniques, reference may be made to documents such as Sambrook and l, "Molecular g: A Laboratory Manual," Cold Spring Harbor Laboratory Press, New York, (2001); and Ausubel, F M et al., "Current Protocols in Molecular y," John Wiley & Sons, New York, NY.

Further, in regard to antibody engineering techniques, reference may be made to documents such as Kabat et al., "Sequences of Proteins of Immunological Interest.“ U.S.

Department of Health and Human Services, (1983); and man and Dﬁbel, “Antibody Engineering," Springer.

Explanation of the Terms The term “nucleic acid" encompasses, for example. ribonucleotides, deoxyribonucleotides, and their modified forms.

The nucleic acid may be either single- or double-stranded, and 3O either polynucleotide or oligonucleotide.

The term "protein" refers to a compound in which two or more amino acids are linked by peptide bonds.

The term "monoclonal antibody" refers to an antibody~ obtained from a population of substantially homogeneous antibodies. In other words, the individual antibodies included in WO 32032 the population are identical except for naturally occurring mutations that may be present in minor amounts. Monodlonal dies are highly specific, and directed to a single nic site. Further, in contrast to polyclonal antibody preparations comprising different dies directed to different inants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their icities, monoclonal antibodies are also advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal“ refers to a teristic of an antibody obtained from a population of substantially homogeneous antibodies, and should not be interpreted to mean that antibodies must be produced by any specific .

For example, a monoclonal antibody that should be used in accordance with the present invention can be prepared by the hybridoma method first described by thler G and Milstein C.

"Continuous cultures of fused cells secreting antibody of predefined icity," Nature, 256: 495-7 , or by a recombinant DNA method (see U.S. Patent No. 7).

Further, “monoclonal antibodies" can be isolated from phage antibody library by using a que, for example, described by Clackson T, Hoogenboom HR, Griffiths AD, and Winter G, "Making antibody fragments using phage display libraries,“ Nature, 352: 624~8 (1991); or Marks JD, Hoogenboom HR, and Bonnert TP, McCafferty J, Griffiths AD, Winter G, “By-passing immunization: Human antibodies from V—gene libraries displayed on phage,“ J Mol Biol, 222: 581-97? (1991).

The "identity" between amino acid sequences or nucleotide sequences refers to the degree of identical amino acid sequences or nucleotide sequences between two or more comparable amino acid sequences or nucleotide sequences. Accordingly, when the identity between two amino acid sequences or nucleotide sequences is high, the identity or similarity of these sequences is highs The level of identity between amino acid sequences or nucleotide sequences is determined, for example, using PASTA, which is a sequence analysis tool, based on default parameters.

Alternatively, it can be determined using the algorithm BLAST by Karlin and Altschul (Karlin S, Altschul SF, “Methods for assessing the statistical significance of lar sequence features by using general scoring schemes," Proc Natl Acad Sci USA, 87: 2264-2268 (1990): and Karlin S, Altschul SF, "Applications and statistics for multiple coring segments in lar ces," Proc Natl Acad Sci USA, 90: 5873-7 (1993)). Programs such as BLASTN and BLASTX based on the above- described BLAST algorithm have been developed (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, “Basic local alignment search tool," J Mol Biol, 215: 403-10 (1990)). For example, BLASTN may be used when analyzing the tide sequence, by setting, for example, the score to 100 and the word length to 12, as ters.

In addition, BLASTX may be used when analyzing the amino acid sequence, by setting, for example, the score to 50 and the word length to 3, as parameters.

When BLAST and Gapped BLAST programs are used, default parameters of each program may be used. Specific techniques of these analySis methods are known. Reference may be made to the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/).

Anti—Human XCRl Antibody The dies of the present invention are isolated antibodies.

The antibodies of the present invention bind to human XCRl. The amino acid sequence of human XCRl is an amino acid 3O sequence shown by NCBI Reference Sequence: NP_001019815.1 or NP_005274.1. In regard to these amino acid sequences, reference may be made to the NCBI websites (respectively, http://www.ncbi.n1m.nih.gov/protein/NP_001019815.1 and http://www.ncbi.nlm.nih.gov/protein/NP_005274.1).

A specific antibody of a first embodiment of the present invention is an antibody comprising a heavy chain le region sing a heavy chain CDR 1 described in (A) or (a) below, a heavy chain CDR 2 described in (B) or (b) below, and a heavy chain CDR 3 described in (C) or (c) below; and a light chain variable region comprising a light chain CDR 1 described in (D) or (d) below, a light chain CDR 2 described in (E) or (e) below, and a light chain CDR 3 described in (F) or (f) below.

(A) A heavy chain CDR 1 ting of the amino acid sequence of SEQ ID NO: 53, (B) a heavy chain CDR 2 conSisting of the amino acid sequence of SEQ ID NO: 54,- (C) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 55; (D) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 56, (E) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 57, and (F) a light chain CDR 3 consisting of the amino acid ce of SEQ ID NO: 58. (a) A heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 42, (c) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID No: 43; (d) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 44, (e) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 45, and (f) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 46.

The_term "CDR" defined in relation to the dies of the present invention is an iation for 90mplementarity Qetermining Region, and is also referred to as a complementarity determining region. CDRs are present in the le region of immunoglobulin, and are deeply involved in the specific binding of an antibody to an antigen. Further, "light chain CDR“ refers to a CDR that is present in the Variable region of the light chains of immunoglobulin, and "heavy chain CDR" refers to a CDR that is present in the variable region of the heavy chains of immunoglobulin.

In addition, “variable region" refers to a region that includes the above—described CDR 1‘to CDR 3 (hereinafter simply referred to as "CDRs 1 to 3"). Although the order of arrangement of the CDRs 1 to 3 is not particularly limited, preferably, CDR 1, CDR 2, and CDR 3 are arranged in that order or in the te order from N-terminus to C—terminus in a sequential manner or via other amino acid sequences called framework regions (FRs).

Further, the "heavy chain variable region" is a region where the above-described heavy chain CDRs 1 to~3 are located, and the ”light chain variable region” is a region where the above- described light chain CDRs l to 3 are d.

As described above, the region other than the above— bed CDRs l to 3 in the each le region is called a framework region (FR). In particular, the region between the N— terminus and CDR l in each variable region is defined as FR 1, the region between CDR l and CDR 2 is defined as FR 2, the region between CDR 2 and CDR 3 is defined as FR 3, and the region n CDR3 and the C—terminus in each variable region is defined as FR 4.

The FRs also have a function as linker sequences for linking the CDRs 1 to 3 that are particularly important as the antigen ition sequences. The FRs are the regions that contribute to the formation of the three-dimensional structure of the entire variable region.

A preferable antibody of the first ment according to the present invention is an antibody comprising a heavy chain le region comprising a heavy chain CDR 1 of (9) below, (m) below, or (a) above, a heavy chain CDR 2 of (h) below, (n) below, or (b) above, a heavy chain CDR 3 of (1) below, (0) below, or (C) above; and a light chain variable region comprising a light chain CDR 1 of (j) below, (p) below, or (d) above, a light chain CDR 2 of (k) below. (q) below, or (e) above, and a light chain CDR 3 of (1) below, (r) below, or (f) above. (g) A heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 17, (h) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 18, (i) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 19; '(j) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 20, (k) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 21, (l) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 22; (m) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 29. (n) a heavy chain CDR 2 ting of the amino acid sequence of SEQ ID NO: 30, (o) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 31: (p) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 32, (q) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 33. and (r) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 34; The heavy chain CDR 3 described in (i) and the heavy chain CDR 3 described in (o) comprise cal amino acid sequences.

An antibody of a second embodiment of the present invention is an antibody comprising a heavy chain variable region comprising the heavy chain CDRs 1 to 3 bed in (A)—(C) above or a heavy chain le region sing the heavy chain CDRs 1 to 3 described in (a)—(c) abdve; and a light chain variable region comprising the light chain CDRs 1 to 3 described in (D)-(F) above or a light chain variable region comprising the light chain CDRs 1 to 3 described in (d)—(f) above.

A more preferable antibody of the second embodiment is an antibody sing any one of a heavy chain variable region comprising the heavy chain CDRs 1 to 3 described in (g)-(i) above, a heavy chain variable region comprising the heavy chain CDRs 1 to 3 described in (m)—(o) above, and a heavy chain variable region comprising the heavy chain CDRs 1 to 3 described in (a)—(c) above; and any one of a light chain variable region comprising the light chain CDRs 1 to 3 described in (j)-(l) above, a light chain variable region comprising the light chain CDRs 1 to 3 described in (p)-(r) above, and a light chain variable region comprising the light chain CDRs 1 to 3 described in (d)—(f) above.

An antibody of a third embodiment of the present PCT/JPZOlZ/072667 invention is an antibody comprising a heavy chain variable region comprising the heavy chain CDRs to 3 described in (A)-(C) above, and a light chain variable region comprising the light chain CDRs to 3 described in (D)—(F) above, or an antibody comprising a heavy chain variable region comprising the heavy chain CDRs to 3 described in (a)~(c) above, and a light chain variable region comprising the light chain CDRs to 3 described in (d)-(f) above.

A more preferable antibody of the third embodiment is an antibody comprising a heavy chain variable region comprising the heavy chain CDRs to 3 described in (g)—(i) above, and a light chain variable region sing the light chain CDRs to 3 described in (j)-(l) above; an antibody comprising a heavy chain le region sing the heavy chain CDRs to 3 described in ) above, and a light chain variable region comprising the light chain CDRs to 3 described in (p)—(r) above; or an antibody comprising a heavy chain variable region sing the heavy chain CDRs to 3 described in (a)-(c) above; and a light chain variable region comprising the light chain CDRs to 3 described in (d)-(f) above.

The lar structures of the antibodies of the present invention are not limited to that of globulin insofar as the antibodies have the above—deScribed heavy and light chain le regions. Examples of specific structures include molecular structures of F(ab’)2 that does not comprise the Fc region; Fab formed by papain digestion of immunoglobulin and composed Of CH1 and CL domains as well as the heavy and light chain variable regions; Fv that does not comprise the immunoglobulin constant region; and scFv, which is a single-chain Fv antibody.

The antibodies of the t invention may also be multivalent, in which the above molecular structures are combined.

Such a multivalent antibody is formed by a technique of accumulating scFv constructs, as in an scFv—Fc construct formed by the combination of the Fc region and scFv construct described above; and a construct called a minibody, formed by the ation of CH3 domain of the constant region and the scFv construct described above. The term "multivalent" refers to the presence of multiple antigen-binding sites.‘ In regard to the antibodies of the present invention, the term is used in the same meaning as the presence of multiple sites that bind to human XCRl.

The antibodies of the present invention may also have a human constant region in addition to the above-described heavy and light chain variable regions.

In immunoglobulin, the "constant region" of the heavy chains comprises domains called CH1, CH2, and CH3; and the “constant region“ of the light chains comprises a domain called As described above. when the antibodies of the present invention have the constant , it is able that the heavy chain variable region is linked to at least one of the CH1, >CH2, and CH3 s, and that the light chain variable region is linked to CL. r, the heavy chain variable region is preferably directly linked to CH1.

The constant region of the antibodies of the t invention is a constant region derived from human immunoglobulin, preferably, a constant region derived from human immunoglobulin IgG. The e of human immunoglobulin IgG is not particularly limited, and may be suitably selected, for example, according to whether to impart ADCC activity, CDC ty, and the like described below to the dies.

The term "ADCC activity" is an abbreviation for PCT/JPZOlZ/072667 Antibody-Dependent Cellular gytotoxicity ty. It is an activity in which cells such as NK cells expressing ors specific for the antibody Fc region bind to the antibodies and damage cells present in the vicinity of the antibodies.

Additionally, the term "CDC activity“ is an abbreviation for Complement—Dependent Cytotoxicity activity. In the case of humans, the subtype of 196 having a high ADCC and/or CDC activity is 1961, and the subtype of 196 having a low ADCC and/or CDC activity is 21ng or 1964.

Amino acid residues in the Fc region of the antibodies of the present invention may be mutated in order to induce a change in ADCC and/or CDC activity. Mutations to be introduced are not particularly limited, and known mutations may be uced“ For example, the following mutations may be uced into the constant region of IgGl for the purpose of increasing ADCC activity: SZ39D, I332E, SZ39D/1332E, SZ39D/I332E/A330L, and the like (Lazar GA, Dang W, Karki S, Vafa 0, Peng JS, Hyun L, Chan C, Chung HS, Eivazi A, ¥oder SC, Vielmetter J, Carmichael DF, Hayes RJ, Dahiyat BI, "Engineered antibody Fc variants with enhanced effector function," Proc Natl Acad Sci USA, 103: 4005—10 (2006)); and SZ98A, K334A, K334A, Sé98A/E333A/K334A, etc., (Shields RL, Namenuk AK, Hong K, Meng YG, Rae J, Briggs J, Xie D, Lai J, Stadlen A, Li B, Fox JA, Presta LG, "High resolution mapping of the binding site on human IgG1 for F0 gamma RI, Fc gamma RII, Fc gamma R111, and FcRn and design of IgGl variants with ed g to the Fc gamma R," J Biol Chem, 276: 6591-604 (2001)).

Examples of mutations that increase CDC activity include 8267E, H268F, $324T, $267E/H268F, SZ67E/S324T, H268F/S324T, 8267E/H268F/8324T (Moore GL, Chen H, Karki S, Lazar GA, "Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions,“ MAbs, 2:181-9 Additionally, for the e of lowering ADCC activity. - known mutations may be introduced; for example, V234A/G237A (Cole WO 32032 MS, Anasetti C, Tso JY, "Human IgGZ variants of chimeric anti—CD3 are nonmitogenic to T cells,“ J Immunol, 159:3613-21 (1997)), H268Q/V309L/A33OS/P3315 (An Z, Forrest G, Moore R, Cukan M, Haytko P, Huang L, Vitelli S, Zhao JZ, Lu P, Hua J, Gibson CR, Harvey BR, Montgomery D, Zaller D, Wang F, Strohl W, "IgG2m4, an engineered antibody isotype with reduced Fc function," MAbs, 1:572—9 (2009)), and the like.

The numbering of the above-described amino acids to be d is in accordance with the Eu numbering (see Sequences of proteins of immunological interest, NIH Publication No. 91—3242).

Chimeric Antibody Among the antibodies of the present invention, an dy in which the heavy and light chain variable regions comprise amino acid sequenCes derived from non-human species and the constant region ses amino acid sequences derived from human is defined as a "chimeric antibody." A first embodiment of the chimeric dy of the present invention is a chimeric antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 13 and a light chain of SEQ ID NO: 14.

As shown in Table 5, the amino acid ce of SEQ ID NO: 13 comprises the heavy chain CDRs l to 3 of SEQ ID NOS: 17 to 19 among the heavy chain CDRs 1 to 3 bed above in the heavy chain le . Further, as shown in Table 5, the amino . acid sequence of SEQ ID NO: 14 comprises the light chain CDRs 1 to 3 of SEQ ID N05: 20 to 22 among the light chain CDRs 1 to 3 described above in the light chain variable region.

The chimeric antibody of the present invention comprises variants caused by mutations in the heavy chain consisting of the amino acid sequence of SEQ ID NO: 13 and/or the light chain consisting of the amino acid sequence of SEQ ID NO: ~14, insofar as such mutations do not abolish the binding ability of the chimeric antibody to human XCRl.

Such variants in the heavy and light chains are preferably ed by introducing mutations into at least any one of FR 1 to FR 4 (hereinafter simply referred to as "FRs l to 4") of the variable region, or at least one site in the constant region of the respective amino acid sequences of SEQ ID NOS: l3 and 14.

The specific number of mutations introduced into the heavy and light chains is not particularly limited. Mutations are usually introduced to obtain a variant having 85% or higher identity, preferably 90% or higher identity, more preferably 95% or higher identity, and most preferably 99% or higher identity with the amino acid sequence before mutation.

The term "mutation" used herein includes substitution, deletion, insertion, and the like. A known method without specific limitation can be ed as a specific method for introducing mutations. For example, in the case of substitution, conservative substitution may be employed. The term "conservative substitution" refers to a substitution of an amino acid residue with another amino acid residue having a r side chain.

For example, a substitution between amino acid residues with basic side chains such as lysine, arginine, and ine 'corresponds to a conservative substitution. In addition, the following substitutions between the amino acid residues also correspond to conservative substitutions: substitutions between amino acid residues with acid side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues with nonfcharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, ne, and cysteine; tutions between amino acid residues with non—polar side chains such as alanine, valine, leucine, cine, proline, phenylalanine, methionine, and phan; tutions between amino acid residues with B—branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues with aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

WO 32032 Humanized dies Among the antibodies Of the present invention, the antibody comprising the above-described CDRs l to 3 in the heavy and light chain variable regions, in which the FRs 1-4 comprise a human—derived amino acid sequence or a variant f, is defined as a "humanized antibody." Such FRs comprising a human-derived amino acid ce are not ularly limited, and may be determined based on a known technique.

Examples of such FRs include fully human framework regions or sub-regions, with FRs derived from human germline sequences being preferable. Reference may be suitably made to, for example, the NCBI website, which Shows a list of currently known sequences of FRs as examples of fully human framework regions or sub—regions.

Non-limiting examples of the sequences of the human heavy chain variable region include VH1-18, VHl-Z, VH1—24, VH1—3, VH1-45. VH1-46, VH1-58, VH1—69, VH1—8, . VH2~5. VH2—70, VH3-11, VH3-13, VH3—15, VH3-16, VH3—20, VH3-21, , VH3-30, VH3—33, VH3—35, VH3-38, VH3-43, VH3—48, VH3-49, VH3—53, VH3—64, , VH3-7, VH3-72, VH3—73, VH3-74, VH3—9, VH4-28, VH4—31, VH4-34, VH4—39, VH4-4, VH4-59, VH4—61, VH5—51,IVH6-1, and VH7-81.

Non-limiting examples of the sequences of the human light chain variable region include VLl-ll, VL1—13, VL1-16, VL1— 17, VL1-18, VL1~19, VL1~2, VLl—ZO, VLl—22, VL1-3, VL—4, VLl-S, VL1—7, VLl-9, VLZ-l, VL2—ll, VL2~13, VL2~14, , VL2-17, VL2~ 19, VL2-6, VL2-7, VL—8, VL3—2, VL3—3, VL3-4, VL4—l, VL4-2, VL4—3, VL4-4, VL4-6. VLS—l, VL5—2, VL5-4, and VL5—6.

Fully human FRs are ed from these functional germline genes. Each of these FRs is usually ent because of the modification of a limited number of amino acids. These FRs may be used in a combination with the CDRs described in the present specification. Non—limiting additional examples of human ERs to be used in combination with the above-described CDRs include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM. In regard to the examples of these human FRs, reference may be made to thev following documents: Kabat, et al., "Sequences of Proteins of Immunological Interest," US Department of Health and Human Services, NIH (1991) USA; Wu TT, Kabat EA, "An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity," J Exp Med, 132: 211-50 (1970); and the like.

A first embodiment of the humanized antibody of the present invention is a humanized antibody comprising a heavy Chain le region sing the amino acid sequence of either SEQ ID NO: 60 or SEQ ID NO: 64, and a light chain variable region of either SEQ.ID NO: 68 or SEQ ID NO: 72.

A more able embodiment is a humanized antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60 and a light chain variable region sing the amino acid sequence of SEQ ID NO: 68, or a humanized antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 72.

As respectively shown in Tables 11-1 and 12-1, the amino acid sequences of SEQ ID NO: 60 and SEQ ID NO: 64 comprise the heavy chain CDRs 1 to 3 of SEQ ID NOS: 1? to 19 among the above—described heavy chain CDRs 1 to 3 in the heavy chain variable regiOn. As respectively shown in Tables 13—1 and 14-1, the amino acid sequences of SEQ ID NO: 68 and SEQ ID NO: 72 comprise the light chain CDRs 1 to 3 of SEQ ID NOs: 20-22 among the above-described light chain CDRs 1 to>3 in the light chain 3O le region.

The humanized antibody of the present invention comprises variants caused by mutations in the heavy chain le region comprising the amino acid sequence of SEQ ID NO: 60 or 64 and/or the light chain variable region comprising the amino acid ce of SEQ ID NO: 68 or 72, insofar as such mutation do not abolish the g ability to human XCRl. Such variants in the heavy and light chain le regions are preferably obtained by introducing mutations into the respective.

FRs 1 to 4.

The specific number of mutations into the heavy and light chain variable regions is not particularly limited.

Mutations are usually introduced to obtain a t having 85% or higher identity, preferably 90% or higher identity, more preferably 95% or higher identity, and most preferably 99% or higher identity with the amino acid sequence before mutation.

The term "mutation" used herein includes substitution, deletion, insertion, and the like. As is the case with the chimeric antibody described above, vative substitution and the like may be employed as a ic method for introducing mutations.

The second.embodiment of the humanized antibody of the present invention includes an antibody comprising a human constant region. es thereof include a zed antibody comprising a heavy chain comprising the amino acid sequence of either SEQ ID NO: 59 or 63, and a light chain comprising the amino acid sequence of either SEQ ID NO: 67 or 71.

A more preferable embodiment is a humanized antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a light chain comprising the amino acid sequence of SEQ ID NO: 67, or a humanized antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 63 and a light chain comprising the amino acid sequence of SEQ ID NO: 71.

As shown in Table 11-1, the amino acid sequence of SEQ ID NO: 59 comprises an amino acid sequence corresponding to the heavy chain variable region of SEQ ID NO: 60, and therefore comprises the heavy chain CDRs l to 3 of SEQ ID NO: 17—19 among the heavy chain CDRs l to 3 described above. Further, as shown in Table 13-1, the amino acid sequence of SEQ ID NO: 67 comprises an amino acid sequence corresponding to the light chain variable region of SEQ ID NO: 68, and therefore comprises the light chain CDRs 1 to 3 of SEQ ID NO: 20—22 among the light chain CDRs 1 to 3 described above.

The heavy and/or light chain described above comprises variants caused by mutations r as such mutations do not abolish the binding y to human XCRl. Such variants in the heavy and light chains are preferably obtained by introducing mutations into the FRs 1 to 4 or the constant region.

The specific number of ons into the heavy and light chains is not particularly limited. Mutations are usually introduced to obtain a variant having 85% or higher identity, preferably 90% or higher identity, more preferably 95% or higher identity, and most preferably 99% or higher identity with the amino acid sequence before mutation.

The term "mutation" used herein includes substitution, deletion, insertion, and the like. As is the case with the chimeric antibody described above, vative substitution and the like may be employed as a specific method for introducing mutations.

Function of the Antibodies The antibodies of the present invention bind to human XCRl. The g of the term "bind" used herein encompasses, at least, binding through hobic bonds and the like as seen in the case of an interaction between proteins. In other words, antibodies that bind to human XCRl at least by hydrophobic binding are sufficient as the antibodies of the present invention.

Further, the antibodies of the present invention and human XCRl may or may not be dissociated after binding.

The antibodies of the t ion preferably specifically bind to human XCRl. The tern1"specific binding" as used herein refers to ic binding to human XCRl, meaning that.the antibodies preferentially bind to human XCRl when human XCRl is present concurrently with molecules other than human XCRl, in particular, les having a structure similar to that of human XCRl, such as a homologue of human XCRl or an orthologue of human XCRl . p e That the antibodies of the present invention ically bind to human XCRl does not mean that the ability of binding to the above-described homologue or orthologue of human XCRl is excluded.

The degree of binding of the antibodies of the present invention to human XCRl can be evaluated by a reaction rate constant such as a Kd, Koff, or Kon value. A Kd value is a value obtained by dividing a Koff value by a Kon value.

The reaction rate constant between the antibodies of the present invention and human XCRl is not particularly limited.

The antibodies of the present ion bind to the extracellular domain of human XCRl. Specifically, the antibodies bind to one or more of amino acid regions 1 to 31, 90 to 103, 168» to 190, and 251 to 267, which corresponds to the extracellular domain region of the amino acid sequence (SEQ ID NO: 91; Fig. 10) of the above-described NCBI Reference Sequence: NP_001019815.1 or NP_005274.1.

More preferably, in the amino acid sequence of SEQ ID NO: 91, the antibodies bind to at least three amino acids selected from the group consisting of the 8m, 11“, 1232 13ﬂ2 14ﬂﬁ 16th, 17th, 22‘”, 23rd, 1763‘, and 177th amino acids.

Said “at least three amino acids” includes, for example, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, seven or more amino acids, eight or more amino acids, nine or more amino acids, ten or more amino acids, or eleven amino acids.

Note that the pe” in the present invention is also ed to as an “antigenic determinant,” and es r epitope” and “discontinuous epitope.” A “linear epitope” is an epitope that is recognized by antibodies by the primary structure of the amino acid sequence, rather than by its Conformational ure. A “discontinuous epitope” is an epitope that is ized by antibodies by the mational structure of the amino acid sequence, based on the higher-order structure Note that a person skilled in the art can ine the epitope of the antibodies of the present invention by suitably modifying the s described in the Examples of the present invention. For example, the epitope can be determined by synthesizing a protein or peptide consisting of a desirable amino acid sequence that falls within the extracellular domain of the amino acid sequence of human XCRl using a known method, and confirming the binding between the obtained protein or peptide and the antibody by a known method. Alternatively, the e can be determined by preparing a mutant by introducing an riate mutation to desired amino acids in the amino acid sequence of human XCRl by a known method, and confirming whether the binding between the prepared mutant and the antibody is reduced; As described above, because the antibodies of the present invention bind to human XCRl, the antibodies of the present invention also include an dy that inhibits binding between human XCRl and human XCLl. Human XCLl is also referred to as human lymphotactin (Ltn) or human tactin a (Ltn-a). Such an inhibitory activity is sometimes referred to as alizing activity“ induced by the antibodies of the present ion.

, Because human XCRl is t on the cellular surface as a receptor protein in vivo, inhibition of binding between human XCRl and XCLl by the antibodies of the present invention is preferably performed on the cellular surface. It does not matter whether the antibodies of the present invention have an inhibitory activity against binding between human XCRl and XCLZ r as the antibodies have activity to at least inhibit binding between human XCRl and XCLl. Accordingly, the antibodies of the present invention also include an antibody that inhibits binding not only between human XCRl and XCLl, but also between human XCRl and XCLZ.

Examples of preferred cells include cells associated with an immune system activated by the binding between human XCRl and human XCLl, with dendritic cells being particularly able. In particular, as shown by the described examples, because the antibodies of the present invention specifically recognize BDCA3+ dendritic cells, which are dendritic cells expressing a significant amount of human XCRl proteins, it is preferable that the antibodies have an effect of inhibiting binding between human XCRl and human XCLl on BDCA3+ tic cells.

Binding between human XCRl and human XCLl is inhibited by the antibodies of the present invention. Non—limiting examples of forms of such tion e: (1) The antibodies of the present invention bind to XCRl at a site to which human XCLl originally should bind, g a steric obstruction to binding to human XCLl, and resulting in the inhibition of binding between human XCRl and human XCLl. (2) The antibodies of the present invention bind to human XCRl, causing a change in the three—dimensional ure of human XCRl, which consequently causes a change in the structure of human XCRl to which human XCLl should bind. thus resulting in the inhibition of binding n human XCRl and human XCLl. (3) The antibodies of the present invention bind to XCRl, causing an internalization of the receptor, which leads to the inhibition of binding between human XCRl and human XCLl.

The inhibitory activity of the antibodies of the present invention against binding between human XCRl and human XCLl is evaluated based on ICw or ICm values. These values can be obtained, for example, by performing a itive inhibition experiment or the like of binding of human XCLl to human XCRl, using cells those express human XCRl proteins in the presence of the antibodies of the present invention. A known method may be employed as a specific method of such a competitive inhibition ment.

The antibodies of the present invention include an antibody that has an effect of inhibiting cell migration. The term "cell migration“ refers to the phenomenon in which cells actively migrate as a result of external stimuli given to the - cells and stimulus—induced activation of the intracellular signal transduction mechanism. Effects produced by the active cell migration vary depending on the functions of the cells. For example, in the case of cell iOn of dendritic cells, such cell migration is a phenomenon that serves as one of the mechanisms in the immune system. In the present ion, inhibitory activity against cell migration is sometimes referred to as "neutralizing activity." As described above, because the antibodies of the present ion suitably t binding between human XCRl and human XCLl in dendritic cells, particularly BDCA3+ dendritic cells, the antibodies particularly ably inhibit ion of dendritic cells. particularly BDCA3+ dendritic cells.

Human XCRl is a seven-transmembrane G protein-coupled receptor. When human XCLl binds to human XCRl, the three— dimensional structure of human XCRl changes; and, as a result, a G protein d to the intracellular domain of human XCRl is released, and a signal is transduced into the cells.

G proteins is prevented from e by the antibodies of the present invention inhibiting the binding between human XCRl and XCLl in accordance with the above—described forms (1), (2) or the like. As a result, no signal is transduced, thereby inhibiting the phenomena of cell migration.

Alternatively, the phenomena of cell migration may be ted as a result of a mechanism in which binding of the antibodies of the present invention to human XCRl strengthens the bond between human XCRl and G protein coupled to the intracellular domain of human XCRl, the release of G proteins consequently does not occur, thereby inhibiting ellular signal transduction.

The inhibitory activity of the antibodies of the present invention against cell migration of human cells is evaluated based on an ICw or ICm value. Specific values are not particularly limited” For e, an ICE value is y about 0.36 nM or less, preferably about 0.27 nM or less, and more ably about 0.16 nM or less. For e, an IC% value is usually about 2.38 nM or less, preferably about 1.52 nM or less, and more preferably about 0.86 nM or less.

The antibodies of the present invention include, as an embodiment, an antibody that has an effect of decreasing cytotoxic T lymphocyte (CTL) activity. The ism_of decreasing the CTL activity is, for example, the antibodies of ' the present invention inhibiting the interaction between human XCRl and human XCL1 in dendritic cells. Among the dendritic cells, the above—described BDCA3+ dendritic cells are preferable. method for Preparing the Antibodies of the Present ion The antibodies of the present invention can be prepared by a method comprising the following three steps, although it is not limited thereto. (i) Step 1 of introducing a vector into the host to transform the host, the vector comprising a nucleic acid comprising a nucleotide sequence encoding the antibodies of the present invention; (ii) Step 2 of ing the transformed host obtained 3O in step 1 and collecting a fraction containing antibodies that bind to human XCRl; and (iii) Step 3 of isolating or purifying the above antibodies from the fraction obtained in step 2.

Step 1 The nucleic acid used in step 1 is a nucleic_acid that encodes the antibodies of the present invention. The nucleotide sequence of the above nucleic acid can be determined using the in silico technique based on the amino acid sequence information of the antibodies of the present ion. At that time, it is preferable to ine the nucleotide sequence with reference to the codon frequency in the host employed in step 2. Specific examples of nucleotide sequences e the nucleotide sequence of SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 61, 62, 65, 66, 69, 70, 73, or 74; or a variant thereof.

The above variant is preferably generated by introducing mutations (deletion, substitution, insertion, or the like) in the FR or nt region of the antibodies.

The ic number of mutations uced into the variant is not particularly limited. Mutations are usually introduced to obtain a variant having 85% or higher identity, preferably 90% or higher identity, more preferably 95% or higher identity, and most preferably 99% or higher identity with the amino acid ce before mutation.

Further, the above nucleic acid may comprise a nucleotide sequence that encodes a secretion signal peptide at the 5'—terminus. A specific nucleotide sequence encoding a secretion signal peptide is preferably a tide ce that effectively functions as a secretion signal peptide in the host cells employed in step 2. The term "secretion signal peptide“ refers to a peptide comprising an amino acid sequence that acts as a recognition sequence for introducing proteins or es produced in the host into a pathway for ion of the proteins or peptides to the outside of the host.

Examples of nucleotide sequences encoding a secretion signal peptide include: ATGGGATTCAGCAGGATCTTTCTCTTCCTCCTGTCAGTAACTACAGGTGTCCACTCC (SEQ ID NO: 75), ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGCTGTTCTGGTTTCCTGCTTCCAACACT (SEQ ID NO: 76), WO 32032 ATGGAATGGTCATGGGTCTTTCTGTTCTTTCTGAGTGTCACAACCGGGGTGCATAGC (SEQ ID NO: 77), ATGGAATGGTCTTGGGTCTTTCTGTTCTTTCTGTCCGTCACTACCGGGGTCCACTCT (SEQ ID NO: 78), ATGTCCGTGCCTACTCAGGTGCTGGGGCTGCTGCTGCTGTGGCTGACCGATGCTCGTTGC (SEQ ID NO: 79), and ATGTCCGTGCCTACTCAGGTGCTGGGGCTGCTGCTGCTGTGGCTGACCGA'I'GCTCGTTGT (SEQ ID NO: 80).

The vector used in step 1 comprises at least one of the above nucleic acids.

Such a vector may be one of the following vectors: (I) a vector comprising a nucleic acid comprising a nucleotide sequence encoding at least one member selected from the group consisting of heavy chains, heavy chain variable region, and heavy chain CDRs 1 to 3 of the antibodies of the present invention; (II) a vector comprising a nucleic acid comprising a tide ce ng at least one member ed from the group consisting of light chains, light chain variable region, and light chain CDRs 1 to 3 of the antibodies of the present invention: or (III) a vector comprising a nucleic acid comprising a nucleotide sequence encoding at least one member selected from the group consisting of heavy chains, heavy chain variable region, and heavy chain CDRs 1 to 3 of the antibodies of the present invention, and a nucleic acid comprising a nucleotide sequence encoding at least one member selected from the group consisting of light chains, light chain le region, and light chain CDRs 1 to 3 the antibodies of the present invention.

The above vector may be a gene expression . The "gene expression vector" is a vector having a function to cause sion of the nucleotide sequence of the above nucleic acid.

The gene expression vector may contain a promoter sequence, enhancer sequence, repressor sequence, insulator sequence, and ' othe like to control the expression of the nucleOtide sequence.

These sequences are not ularly limited insofar as they function in the above-described host.

The host used in step 1 is not particularly limited insofar as the above gene is expressed. Examples thereof include insect cells, eukaryotic cells. and mammalian cells. Of these cells, HEK cells, CHO cells, NSO cells or SP2/O cells, which are mammalian cells, are particularly preferable in terms of more efficient expression of the tide sequence that encodes antibodies.

A technique for introducing the above vector into the host in step 1 is not particularly limited. A known technique may be used. The s shown in (I) to (III) above may be introduced singly or in a combination of two or more into the host.

A host with the above vector can be ed by such a technique. The vector may be maintained as is in the host, or in such a manner that the nucleic acid comprising the nucleotide sequence ng antibodies in the vector is incorporated into the genome of the host. The prepared host may be maintained using a known technique, and can be stored for a long period of time, if necessary.

Step 2 Step 2 is a step of culturing the above—described host obtained in step 1 and collecting a fraction containing the antibodies of the present invention, which bind to human XCRl.

Culturing the host maintaining the above-described vector allows the host to express the nucleotide sequence encoding the antibodies of the present invention based on the c acid in the vector, resulting in the production of the antibodies of the t invention. The produced antibodies are stored in the host or in the medium used for culturing the host.

In step 2, a known method may be employed as a technique for collecting a on containing the antibodies of the present invention. For example, for collecting a fraction PCT/JPZOIZ/072667 containing the antibodies of the present ion from the host, the host is ted by physical or al means, and the solution obtained by tion is subjected to solid-liquid - separation treatment, thereby obtaining a liquid on. The obtained liquid fraction may be used as the fraction containing the antibodies of the present invention.

On the other hand, for collecting a fraction containing the antibodies of the present invention from the medium used for culturing the host, the medium, i.e., the culture solution of the host obtained in step 1, is subjected to solid—liquid separation treatment, thereby obtaining a liquid fraction. The obtained liquid fraction may be used as the fraction containing the antibodies of the present invention.

In view of simplification of the ion or purification step in the subsequent step 3, it is preferable to collect a fraction containing the antibodies of the present invention from the culture on of the host.

The medium used for cultivation in step 2 is not particularly limited insofar as the medium allows the host to express the nucleotide sequence encoding the antibodies of the present invention, thereby producing the dies of the present invention. However, when ting a fraction containing the antibodies of the present ion from the culture solution of the host as described above, it is preferable to employ serum- free medium in view of simplifying the isolation or purification step as much as possible in the subsequent step 3., In regard to various conditions employed during ation of the host, such as container, temperature, time, host concentration in the medium, and culture conditions, the conditions used in a known method for producing antibodies may be employed.

Step 3 Step 3 is a step of isolating or purifying the antibodies of the present invention, which bind to human XCRl, from the fraction obtained in step 2. The method for isolating and purifying the antibodies of the present invention is not ularly limited. A generally used method for isolating or purifying protein is widely applicable. nal Use of the dies of the Present Invention (1) Use as Therapgutic Agents for Immune Diseases As described above, the antibodies of the present invention have an effect of inhibiting the phenomena of cell migration of tic cells ated with the immune system.

Based on this effect, the antibodies of the present ion, in particular, the humanized antibody, have potential as an active ingredient of a pharmaceutical composition that is clinically applicable to human.

Diseases to which the antibodies of the present invention are applicable are explained below.

Applicable DiSeases (Immune Diseases) XCRl is highly expressed in CD141+ dendritic cells in the case of humans, and in CD8a+ tic cells in the case of mice. These dendritic cells activate T~cells using the above- described antigen presentation method called cross-presentation (Bachem A, Gﬁttler S, Hartung E, Ebstein F, Schaefer M, Tannert A, Salama A, Movassaghi K, Opitz C, Mages HW, Henn V, Kloetzel PM, Gurka S, Kroczek RA, “Superior antigen cross-presentation and XCRl expression define human CD11c+CD141+ cells as homologues of ' mouse CD8+ dendritic cells,“ J Exp Med, 207: 1273—1281 (2010)). r, because the source of production of XCLl. which is a ligand for human XCRl, comprises T-cells, in particular, CD8+ T-cells, the chemokine system in which XCLl-XCRl is involved controls dendritic cell-induced tion of CD8+ T- cells (Crozat K, Guiton R, Contreras V, Feuillet V, Dutertre CA, ventre E, vu Manh TP, Baranek T, Storset AK, Marvel J, Boudinot P, Hosmalin A, tz-Cornil I, Dalod M, "The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8a+ dendritic cells," J Exp Med, 207: 1283- 1292 (2010); and Dorner BG, Dorner MB, Zhou X, Opitz C, Mora A, Gﬁttler S, f A, Mages HW, Ranke K, Schaefer M, Jack RS, Henn V, Kroczek RA, "Selective sion of the chemokine receptor XCRl on cross—presenting dendritic cells determines cooperation with CD8+ T—cells," Immunity, 31:833 (2009)).

As described above, the antibodies of the present invention include, as an ment, an antibody that exhibits an effect of inhibiting binding n human XCLl and human XCRl in dendritic cells, in particular, BDCA3+ dendritic cells.

Accordingly, the antibodies of the present invention has potential as therapeutic agents for the treatment of immune diseases in which s that are activated by migration of the dendritic cells are involved. In particular, the antibodies have potential as therapeutic agents for the ent of diseases associated with the control of the activation of CD8+ T-cells.

As bed above, the antibodies of the present ion include, as an embodiment, an antibody that exhibits an effect of decreasing CTL activity. CTL has a mechanism to activate the immune system by attacking cells or tissues. Various immunological diseases are known to have rated CTL activity: therefore, the antibodies of the present invention have potential as a therapeutic agent for the treatment of immunological diseases by decreasing CTL activity.

Non-limiting es of such diseases include es mellitus type 1, psoriasis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, ankylosing spondylitis, ethyroiditis, graft ion, delayed-type hypersensitivity, Crohn’s disease, dermatomyositis, polymyositis, inclusion body myositis, rheumatoid arthritis, inflammatory bowel disease, anterior uveitis, wegener's granulomatosis, graft—versus—host disease, and Behget’s disease (Kurts C, Robinson BW. Knolle PA, “Cross—priming in health and disease," Nat Rev Immunol, 10: 403— 414 (2010); Kehren J, Desvignes C. Krasteva M, Ducluzeau MT, Assossou O, Horand F, Hahne M, Kagi D, Kaiserlian D, Nicolas JF. oxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity." J Exp. Med. 189: 779-786 (1999); Middel P, Thelen P, Blaschke S, Polzien F, Reich K, Blaschke V, Wrede A, Hummel KM, Gunawan B, Radzun HJ, "Expression of the T—cell chemOattractant ine lymphotactin in Crohn's disease," Am J Pathol, 159: 1751—1761 (2001); Sugihara T, Sekine C, Nakae T, Kohyama K, Harigai M, a Y, Matsumoto Y, Miyasaka N, Kohsaka H, "A new murine model to define the critical pathologic and therapeutic mediators of polymyositis,“ Arthritis Rheum. 56: 1304—1314 (2007); Wang CR, Liu MF, Huang YH, Chen HC,-“Up- regulation of XCRl expression in rheumatoid joints," Rheumatology (Oxford) 43: 569-573 (2004); Muroi E, Ogawa F, Shimizu K, Komura K, Hasegawa M, Fujimoto M, Sato S, "Elevation of serum lymphotactin levels in patients with systemic sclerosis,“ J Rheumatol, 35: 834-838 (2008); Torrence AE, Brabb T, Viney JL, Bielefeldt—Ohmann H, Treuting P, Seamons A, Drivdahl R, Zeng W, Maggio—Price L, "Serum biomarkers in a mouse model of bacterial- induced inflammatory bowel disease," Inflamm Bowel Dis, 14: 480— 490 (2008); Yeh PT, Lin FA, Lin CP, Yang CM, Chen MS, Yang CH, fExpressions of lymphotactin and its receptor, XCR, in Lewis rats with experimental autoimmune or s," Graefes Arch Clin Exp Ophthalmol, 248: 1737-1747 (2010); Blaschke S, Brandt P. wessels JT, Mﬁller GA, "Expression and function of the C-class chemokine lymphotactin (XCLl) in Wegener's granulomatosis," J Rheumatol, 36: 500 (2009); Asuka H, Okazaki Y, Kawakami Y, Hirakata M, Inoko H, Ikeda Y, Kuwana M, "Autoreactive CD8+ cytotoxic T lymphocytes to major histocompatibility x class 3O I related gene A in patients with Behcet's disease,“ tis Rheum, 50: 3658-3662 (2004); Serody JS, Burkett SE, Panoskaltsis-Mortari A, Ng-Cashin J, McMahon E, Matsushima GK, Lira SA, Cook DN, Blazar BR, "T—lymphocyte production of macrophage inflammatory n-lalpha is critical to the recruitment of CD8(+) T cells to the liver, lung, and spleen during graft-versus—host disease," Blood, 96: 2973-2980 (2000); ra T, Sekine C, Nakae T, Kohyama K, Harigai M, Iwakura Y, oto Y, Miyasaka N, Kohsaka H, "A new murine model to define the clinical pathologic and therapeutic mediators of polymyositis," Arthritis & Rheumatism. 56: 1304—1314 (2007)): and Dalakas MC, "Review: An update on inflammatory and autoimmune myopathies," Neuropathol Appl Neurobiol, 37: 226—242 (2011).

It was also revealed that the antibody (anti—human XCRl mouse onal antibody (5G7)) of the present invention significantly inhibits the DTH reaction in the later—described ment that used a mouse model of delayed—type hypersensitivity (hereinafter sometimes referred to as "DTH"). As described above, delayed-type hypersensitivity is a disease known as one of the immune diseases in which CD8+ s that are activated by migration of the dendritic cells are involved. The fact that the antibodies of the present invention are effective in the treatment of delayed—type ensitivity provides evidence that the antibodies of the present invention have , activity of inhibiting cell migration, in ular, dendritic cell migration, because the antibodies of the present invention affect cna+ T—cells.

Further, in addition to delayed—type hypersensitivity, atopic dermatitis and contact dermatitis are also known as immune ‘25 diseases of the skin in which the DTH reaction is ed (Fabrizi G, Romano A, VUltaggio P, Bellegrandi S, lli R, venuti A, "Heterogeneity of atopic dermatitis defined by the immune response to nt and food allergy," Eur J Dermatol, 9: 380-384 (1999); and Fonacier LS, Dreskin SC, Leung DYM, "Allergic skin diseases," J Allergy Clin Immunol, 125: 8138-149 (2010)).

Based on the above, the antibodies of the present ion have potential as therapeutic agents for the ent of immune diseases of the skin such as atopic dermatitis or contact dermatitis.

It has been pointed out that the activation of CD8+ T- cells may also be involved in the DTH reaction (Mbdy CH, Pain III R, Jackson C, Chen G-H, Toews GB, "CD8 Cells play a critical role' in delayed—type hypersensitivity to intact Cryptococcus neoformans,“ J Immunol, 152: 3970—3979 (1994), etc.).

Invasion of CD8+ T—cells into the epidermis is observed in psoriasis, which is an autoimmune skin disease affecting a large number of patients, in ular, in c psoriasis lesions. These cells are ered to be the main effector cells that cause psoriasis lesions nsson JE. Johnston A, Sigmundsdottir H, valdimarsson H, “Immunopathogenic mechanisms in psoriasis," Clin Exp Immunol, 135: 1-8 (2004)).

Based on the above, the antibodies of the present invention have ial as therapeutic agents for the treatment of immune diseases of the skin in which the activation of CD8+ T- cells is involved.

In addition to delayed—type hypersensitivity, atopic dermatitis, and contact dermatitis, non—limiting examples of immune diseases Of the skin in which CD8+ T—cells are involved also include dermatomyositis, polymyositis, inclusion body myositis, psoriasis, parapsoriasis, autoimmune blistering diseases (e.g., pemphigus, pemphigoid, and acquired epidermolysis bullosa), osis, herpes gestationis, linear IgA bullous dermatosis, alopecia areata, vitiligo vulgaris, skin diseases associated with collagenosis (e.g., systemic lupus erythematosus, Sjogren syndrome, and mixed connective tissue disease), skin diseases associated with Addison's e, skin diseases associated with graft-versus-host disease , eczema, and urticaria.

Herein below, the relationship n the antibodies of the present invention and various immune diseases (multiple sclerosis, human type 1 es us, glomerulonephritis, mune hepatitis, thyroiditis, graft-versus—host disease, dermatomyositis, polymyositis, and inclusion body myositis) is ZOIZ/072667 bed. Diseases to which the antibodies of the present invention are effective are not limited to the following specific diseases.

Multiple Sclerosis Invasion of CD8+ T—cells in addition to CD4+ T-cells into the central lesions in multiple sclerosis in humans has been recently reported. Further, it has been reported that, in the experiment using mice, implantation of CD8+ T-cells activated by antigen d from the myelin sheath in central nerves induce experimental autoimmune encephalomeningitis, which is a model of human multiple sclerosis. Compared to the conventional model, the above-mentioned model more closely mimics the ogy of human multiple sclerosis (repeated bations and remissions, icant demyelination, invasion of many CD8+ T-cells and macrophages/microglial cells in demyelinated lesions). As described above, it has been suggested that CD8+ T-cells play an important role in human le sis and its mouse model (Friese MA, Fugger L, “Autoreactive CD8+ cells in multiple ' sclerosis: a new target for therapy?" Brain, 128: 1747-1763 (2005)).

‘Accordingly, the antibodies of the present invention that control the activation of CD8+ T—cells has potential as therapeutic agents for the treatment of multiple sclerosis.

Human $222 1 Diabetes Hellitus Noneobese diabetic (NOD) mice enting a model of human type 1 diabetes mellitus have shown that depletion of CD8+ T—cells results in inhibition of the onset of diabetes mellitus (Wang B, Gonzales A, t C, Mathis D, "The role CD8+ T-Cells in the initiation of insulin-dependent diabetes mellitus," Eur J Immunol, 26: 1762—1769 (1996)). This ts that CD8+ T-cells are also involved in the development of the pathology of diabetes mellitus type 1.

Accordingly, the antibodies of the present invention that control the activation of CD8+ T-cells has potential as therapeutic agents for the ent of human type 1 diabetes Glomerulonephritis In a mouse model of glomerulonephritis, it has been shown that CD8+ T-cells are involved in the process of the formation of renal s (Heymann F, Meyer-Schwesinger C, Hamilton—Williams EE, Hammerich L, Panzer w, Kaden s, n SE, Floege J, Grdne H—J, Kurts C, “Kidney dendritic cell activation is required for progression of renal disease on a mouse model of glomerular ,“ J Clin , 119: 1286-1297 ).

Invasion of many CD8+ T-cells into the kidney is observed in patients with severe autoimmune lupus tis. The correlation between the number of these CD8+ T—cells and an increase in the renal activity score and the serum creatinine level, which indicate aggravation of the renal function, has been reported (Couzi L, Merville P, Deminiére C, Moreau J-F, Combe C, Pellegrin J-L, Viallard J-F, Blanco P, "Predominance of CD8+ T lymphocytes among periglomerular infiltrating cells and link to the prognosis of class III and class IV lupus nephritis," Arthritis Rheum, 56: 2362-2370 (2007)). As described above, CD8+ T—cells are considered to be involved in the onset of autoimmune glomerulonephritis or progression of the pathology thereof in '25 human and mouse models.

Accordingly, the antibodies of the present invention, which control the activation of CD8+ s, have potential as therapeutic agents for the treatment of glomerulonephritis.

Autoimmune tis It has been suggested that infection with hepatitis C virus (HCV) is involved in the process of the development of autoimmune hepatitis. It has also been suggested that CD8+ CTLs induced with respect to HCV are involved in the development of autoimmune hepatitis by eliminating HCV and damaging the infected liver cells (Kammer AR, van der Burg SH, Grabscheid B, Hunziker IP, Kwappenberg KMC, Reichen J, Melief CJM, Cerny A, I’Molecular mimicity of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T cell recognition,“ J Exp Med, 190: 169-175 (1999)).

Accordingly, the antibodies of the present ion. which control the tion of CD8+ T—cells, have potential as therapeutic agents for the treatment of autoimmune hepatitis. ditis CD8+ CTLs are known to be involved in the development of experimental autoimmune thyroiditis (EAT), which is a mouse model of human thyroiditis (for e, Hashimoto's disease). It has been reported that the mice in the model show lesions similar to human thyroiditis (antithyroglobulin antibodies are found in Lthe peripheral blood, and the invasion of CD8+ T-cells and CD4+ T-cells into the thyroid gland is observed). As described above, it has been suggested that CD8+ Tecells are involved in the development of thyroiditis in human and mouse models (Brazillet M—P, Batteux F, Abehsira—Amar O, Nicoletti F, Charreire J, tion of experimental autoimmune thyroiditis by heat- denatured porcine thyroglobulin: a Tcl—mediated disease," Eur J Immunol, 29: 1342-1352 (1999)).

Accordingly, the antibodies of the t invention, iwhich control the activation of CD8+ T~cells, have potential as eutic agents for the ent of human thyroiditis.

Rheumatoid arthritis As described in the Examples below, 567, which is one of the antibodies of the present invention, exhibits a significant effect in ng rheumatoid tis in the experiment of DTH induced by MYcobacterium butyricum. Therefore, the antibodies of the present invention have potential as a therapeutic agent for the treatment of rheumatoid arthritis.

Graft Rejection CD8+ T—cells play an ant role in the graft rejection after human organ transplantation. A graft is rejected by CD8+ T—cells in the host that recognizes MHC class I being ‘expressed in the cells in the graft. Further, on of many CD8+ T-cells into the kidney has been reported in renal lant patients experiencing ion. As described above, it has been ted that CD8+ T—cells also play a central role in the graft rejection after human organ transplantation (Bueno V, Pestana-JOM, "The role of CD8+ T-cells during allograft rejection," Braz J Med Biol Res, 35: 1247-1258 (2002)).' ingly, the antibodies of the present invention. which control the tion of CD8+ T-cells, have potential as therapeutic agents for the treatment of graft-versus-host disease.

Dermat ositis, Pol ositis, and Inclusion Bod ositis When lymphocytes that invade the lesion site of patients with dermatomyositis and polymyositis were established as cell lines, CD8+ T—cell lines showed cytotoxicity against their own cultured muscle cells. This indicates that muscle cell damage in the patients with the above-described myositis is caused by CD8+ T—cells with antigen-specific cytotoxicity (Hohlfeld R, Engel AG, "Coculture with autologous myotubes of cytotoxic T cells isolated from muscle in inflammatory myopathies," Ann , 29: 498—507 (1991)). Further, invasion of CD8+ T-cells into the lesion site has been observed in the patients with inclusion body myositis (Dalakas MC, "Review: An update on inflammatory and autoimmune hies,“ Neuropathol Appl Neurobiol, 37: 2 (2011)). Accordingly, the antibodies of the present invention, which control the activation of CD8+ T- cells, have potential as therapeutic agents for the treatment of dermatomyositis, polymyositis, or inclusion body myositis.

As described above, because the antibodies of the present invention have potential as therapeutic agents for the treatment of immune diseases, in particular, immune diseases of the skin, the present invention provides a pharmaceutical composition comprising the dies of the present invention.

Such a pharmaceutical ition has potential as a therapeutic agent for the treatment of immune disease, for the purpose of treating immune diseases, in particular, immune diseases of the skin.

The term "treatment" used herein means attainment of desired pharmacological and/or physiological effects. The effects include an effect of partially or completely curing disease and/or adverse effects caused by the disease (pathologies and symptoms). The above effects also include an effect of inhibiting or ng the ssion of the e and/or adverse effects caused by the disease (pathologies and symptoms); an effect of alleviating pathologies and symptoms (i.e., ameliorating the disease or symptoms, or causing reversal of the progression of symptoms); and an effect of preventing recurrence of the disease.

The above effects also include an effect of partially or completely preventing the onset of the disease and/or adverse effects caused by the disease (pathologies and symptoms) in the duals who may possess a predisposition to the disease and/or adverse effects caused by the disease (pathologies and symptoms) but who have not been sed as having the predisposition. Accordingly, the term “treatment" also means "relief," "prevention of recurrence," and ntion of disease." In the present invention, a ceutical ition comprising the antibodies of the present inVention can be suitably used for the treatment of human immune es, in particular, immune diseases of the skin. It is understood that the above pharmaceutical composition is capable of ing, for example, an effect of partially or completely curing various symptoms of immune diseases; an effect of partially or completely inhibiting various symptoms of immune diseases (i.e., inhibiting or delaying the progression); an effect of alleviating various PCT/JPZOlZ/072667 symptoms of immune diseases (i.e.. ameliorating the disease or symptoms, or causing reversal of the ssion of symptoms): or an effect of preventing recurrence of various symptoms of immune diseases.

Specific examples of target diseases are as described above, with immune diseases of the skin being preferable.

The content of the antibodies of the t invention in the above pharmaceutical composition is not ularly ’10 limited insofar as the pharmaceutical composition comprises an effective amount of the antibodies of the present invention. The content can be ly determined, for example, in such a manner the antibodies of the present invention are contained in the pharmaceutical composition in an amount of 0.001 to 99.99 wt% ‘ relative to 100 wt% of the composition, by taking into account the type of the target immune disease, dosage form, adminiStration method, and the like.

The term "effective amount" used herein refers to an amount that allows the antibodies of the present invention to trate an effect of ting cell migration of dendritic cells, or an amount that allows the antibodies to demonstrate the above-described desired pharmacological and/or physiological effects (treatment effect for immune diseases).

Pharmaceutically acceptable carriers or ves may be added in combination with the antibodies of the present invention to the pharmaceutical composition. The term "pharmaceutically acceptable carriers or additives" used herein refers to optional carriers, diluents, excipients, ding agents, lubricants, adjuvants, vehicles, ry systems, emulsifiers, egrants, absorbents, preservatives, surfactants, colorants, fragrances, or sweeteners. Known carriers or additives may be used.

Non-limiting examples of dosage forms of the pharmaceutical composition include tablets, syrups, liniments, injections, and infusions, with injections or infusions being preferable. Such injections and infusions may be in s, non—’ aqueous, or suspension form. Additionally, the pharmaceutical composition may have a dosage form that is prepared just before stration.

The pharmaceutical composition of the present invention, specifically, a therapeutic agent for an immune disease, has potential in methods of treating an immune disease, comprising a step of administering the composition to a human t with an immune disease, in particular, an immune disease of the skin. As described above, the ceutical composition also has potential in methods of preventing an immune disease, comprising administering the composition to a human subject who has not developed pathologies or symptoms of an immune disease, in ular, an immune disease of the skin, but who may possess a . predisposition to the immune disease.

The dosage amount and administration method of the pharmaceutical composition peutic agent for immune ‘ diseases) can be suitably ined within a range of 0.001 to 100 mg/kg/day, according to the type of immune disease, the human subject’s sex, race, age, and general ion, the severity of the disease, and the like.

The antibodies of the present invention may be administered at the above—described dosage once a day, or in divided dosage l times per day. Further, in the range that the antibodies have a ent effect on the above-described diseases, the administration interval may be every day, every other day, every week, every other week, every 2 to 3 weeks, every month, or every 2 to 3 months. Non-limiting examples of administration methods include oral, intramuscular, intravenous, 3O intraarterial, intrathecal, intradermal, intraperitoneal, intranasal, intrapulmonary, intraocular, intravaginal, intracervical, intrarectal, and subcutaneous administrations. (2) Application as Immunotoxin The antibodies of the present invention may have been conjugated to cytotoxic molecules. Because such antibodies bind to human XCRl protein that is expressed in a significant amount in dendritic cells associated with the immune system, the antibodies may be used as immunotoxins that target dendritic cells.

The term "cytotoxic molecules" used herein refers to molecules that demonstrate s, such as apoptosis and/or necrosis, which cause the death of cells.

Examples of such molecules include n, ricin, Pseudomonas exotoxin, diphtheria toxin, and chemotherapeutic agents. Binding between the antibody and a toxic substance may be performed by a method used for the preparation of tional immunotoxins. (3) Other Applications of the dies of the Present Invention Because the antibodies of the present invention also e, as an embodiment, an antibody that binds to XCRl that is expressed in a significant amount in dendritic cells, the antibodies has ial in a method for detecting tic cells. In this case, it is preferable to label the antibodies of’ the t invention for the use. The term "label" used herein refers to binding the antibodies to labeled molecules such as fluorescent molecules, luminescent molecules, chromogenic molecules and radioisotope molecules.

The binding pattern is not limited insofar as the bond is not dissociated in a detection step. A known method may be employed as a specific detection method. For example, a flow cytometry technique may be employed.

Further, the antibodies of the present invention may 3O also be ly applicable in methods of isolating and/or removing dendritic cells after the detection of dendritic cells.

Known s may also be employed for these s. For example, a known cell-sorting device may be suitably used in a combination with a flow cytometry technique.

WO 32032 - The present invention s to the antibodies ned above, and widely encompasses the inventions of the embodiments described below.

Item 1 An antibody binding to human XCRl, wherein the antibody binds to linear or discontinuous epitopes which comprise at least three amino acids selected from the group consisting of the 8th, 11th, 12th, 13th, 14th, 16th, 17th, 22nd, 23rd, 176th, and 177th amino acids in the amino acid sequence of SEQ ID NO: 91.

Item 2 The antibody according to above item 1 , wherein the antibody is: the antibody comprising a heavy chain variable region comprising heavy chain CDRs 1 to 3 described in (g) to (1) below and a light chain variable region comprising light chain CDRs 1 to 3 described in (j) to (1) below; the antibody sing a heavy chain variable region comprising heavy chain CDRs lato 3 described in (m) to (0) below and a light chain variable region comprising light chain CDRs 1 to 3 described in (p) to (r) below; or the antibody comprising a heavy chain variable region compriSing heavy chain CDRs l to 3 described in (a) to (c) below and a light chain variable region comprising light chain CDRs 1 to 3 described in (d) to (f) below: (a) a heavy chain CDR 1 consisting of the amino acid ce of SEQ ID NO: 41, (b) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 42, (c) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 43; (d) a light chain CDR 1 ting of the amino acid sequence of SEQ ID NO: 44, (e) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO; 45, and (f) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 46; (g) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 17, (h) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 18, (i) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 19: (j) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 20, (k) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 21, (l) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 22; (m) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 29, (n) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 30, (o) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 31; .20 (p)-a light chain CDR 1 ting of the amino acid ce of SEQ ID NO: 32, (q) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 33, and (r) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 34.

Item 3 The antibody according to above item 1 or 2, wherein the antibody ses a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60 or 64, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 68 or 72.

Item 4' The antibody according to any one of above items 1 to 3, PCT/JPZOlZ/072667 wherein the dy comprises a heavy chain variable region comprising an amino acid ce of SEQ ID NO: 60, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 68.

Item 5 The antibody according to any one of above items 1 to 3, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 64, and a light chain variable region sing an amino acid sequence of SEQ ID NO: 72.

Item 6 The antibody according to any one of above items 1 to 5. wherein the antibody comprises a human constant region.

Item 7 The antibody according to any one of above items 1 to 6, wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 59, and a light chain comprising an amino acid sequence of SEQ ID NO: 67.

Item 8 The antibody according to any one of above items 1 to 6, wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 63, and a light chain comprising an amino acid sequence of SEQ ID NO: 71.

Item 9 The antibody according to any one of above items 1 to 8 comprising an Fc region, wherein the Fc region is mutated to induce a change in ADCC activity.

Item 10 The dy according to above item 9, wherein the Fc region is mutated to lower ADCC activity.

Item 11 The antibody according to any one of above items 1 to 10, wherein the antibody is conjugated to a cytotoxic molecule.

Item 12 The antibody according to any one of above items 1 to 11, wherein the antibody inhibits interaction n human XCR1 and human XCLl.

Item 13 The antibody according to any one of above items 1 to 12, wherein the antibody ts cell migration of dendritic cells.

Item 14 The antibody ing to any one of above items 1 to 13, wherein the antibody suppresses the activity of cytotoxic T lymphocytes.

Item 15 A pharmaceutical composition comprising the antibody according to any one of above items 1 to 14 and a pharmaceutically acceptable carrier or additive; Item 16 The pharmaceutical composition according to above item , wherein the pharmaceutical composition is a therapeutic agent for an immune disease.

Item 17 The pharmaceutical ition ing to above item 16, wherein the immune disease is an immune disease of the skin.

Item 18 The pharmaceutical composition according to above item 17, wherein the immune disease of the skin is psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease (pemphigus, pemphigoid, or acquired epidermolysis bullosa), pustulosis, herpes gestationis, linear IgA bullous dermatosis, alopecia areata. vitiligo vulgaris, skin disease ated with collagenosis mic lupus erythematosus, degren Syndrome, or mixed connective tissue disease), skin disease associated with Addison's e, skin disease associated with versus—host disease (GVHD), eczema, or urticaria.

Item 19 The pharmaceutical composition according to above item 17, wherein the immune disease of the skin is sis, atopic dermatitis, contact dermatitis, dermatomyositis, ositis, or inclusion body myositis.

Item 20 The pharmaceutical ition according to above item 1719, wherein the immune disease of the skin is atopic dermatitis or contact dermatitis.

'Item 21 The pharmaceutical composition according to above item 16, wherein the immune disease is thyroiditis, rheumatoid arthritis, type 1 diabetes, or le sclerosis.

Item 22 A nucleic acid comprising a nuCleotide sequence encoding the antibody according to any one of above items 1 to 14.

Item 23 PCT/JPZOlZ/072667 A method of treating an immune disease comprising administering an effeCtive amount of the antibody according to any one of above items 1 to 14 or the pharmaceutical composition according to above item 15 to a human affected by an immune disease.

Item 24 The method ing to above item 23, wherein the immune e is an immune disease of the skin.

Item 25 The method according to above item 24, wherein the immune disease of the skin is psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease (pemphigus, pemphigoid, or acquired epidermolysis bullosa). DUStulosis, .herpes gestationis, linear IgA s dermatosis, ia areata, vitiligo vulgaris, skin disease associated with collagenosis (systemic lupus erythematosus, degren syndrome, or 'mixed connective tissue disease), skin disease ated with Addison's disease, skin e associated with graft-versus4host disease (GVHD), eczema, or urticaria.

Item 26 The method according to above item 24, wherein the immune disease of the skin is psoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, or inclusion body myositis.

Item 27 The method according to above item 23, wherein the immune disease is thyroiditis, rheumatoid arthritis, type 1 diabetes, or le sclerosis.

The present invention also encompasses the embodiments described below Item l-A An antibody comprising a heavy chain variable region comprising heavy chain CDRs l to 3 described in (g) to (1) below and a light chain variable region comprising light chain CDRs 1 to 3 described in (j) to (1) below; an dy comprising a heavy chain variable region comprising heavy chain CDRs l to 3 described in (m) to (0) below and a light chain variable region sing light chain CDRs l to 3 described in (p) to (r) below; or an antibody comprising a heavy chain variable region comprising heavy chain CDRs 1 to 3 described in (a) to (c) below and a light chain variable region comprising light chain CDRs 1 to 3 described in (d) to (f) below: is (a) a heavy chain CDR 1 ting of the amino acid sequence of SEQ ID NO: 41, (b) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 42, (C) a heavy chain CDR 3 consisting of the amino acid sequence of .20 SEQ ID NO: 43; (d) a light chain CDR 1 consisting of the amino acid sequence of‘ SEQ ID NO: 44. (e) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 45, and (f) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 46: (g) a heavy chain CDR 1 ting of the amino acid sequence of SEQ ID NO: 17, (h) a heavy chain CDR 2 consisting of the amino aCid‘sequence at SEQ ID NO: 18, (1)1 a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 19; (j) a light chain CDR 1 consisting of the amino acid sequence of ‘SEQ ID NO: 20, (k) a light chain CDR 2 consisting of the amino acid sequence of 2012/072667 SEQ ID NO: 21, (1) a light chain CDR 3 consisting of the amino acid ce of SEQ ID NO: 22; (m) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 29, (n) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 30, (o) a heavy chain CDR 3 consisting of the amino acid ce of SEQ ID NO: 31; (p) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 32, (q) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 33. and (r) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 34.

Item 2-A The antibody ing to above item l—A, comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60 or 64, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 68 or 72.

Item 3—A The antibody according to above item l-A or 2-A, comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 68.

Item 4—A, The antibody according to above item 1-A or z—A, comprising a heavy chain-variable region comprising an amino acid sequence of SEQ ID NO: 64, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72.

Item 5—A 2012/072667 The dy according to any one of above items l—A to 4—A, comprising a human constant region.

Item 6-A The antibody according to any one of above items l~A to —A, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 59, and a light chain comprising an amino acid sequence of SEQ ID NO: 67.

Item 7-A The antibody according to any one of above items l—A to —A, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 63, and a light chain comprising an amino acid sequence of SEQ ID NO: 71.

Item 8—A The antibody according to any one of above items 1-A to 7-A comprising an Fc region, wherein the Fc region is mutated to induce a change in ADCC activity.

Item 9—A The antibody according to above item 8—A, wherein the Fc region is mutated to lower ADCC activity.

Item 10—A The antibbdy ing to any one of above items l—A to 9—A, wherein the antibody inhibits interaction between human XCRl and human XCLI. 3O Item ll-A The antibody according to any one of above items l—A to ~A, wherein the antibody inhibits cell ion of dendritic cells.

Item 12-A A pharmaceutical composition comprising the antibody according to any one of above items 1—A to ll-A and a pharmaceutically able carrier or additive.

Item 13—A The pharmaceutical composition according to above item 12-A, wherein the pharmaceutical composition is a therapeutic agent for an immune disease.

Item 14-A The ceutical composition according to above item l3—A, wherein the immune e is an immune disease of the skin.

Item 15-A The pharmaceutical composition ing to above item 14-A, wherein the immune e of the skin is psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease (pemphigus, pemphigoid, or acquired epidermolysis bullosa), pustulosis, herpes ionis, linear IgA s dermatosis, alopecia areata, vitiligo vulgaris, skin disease associated with collagenosis (systemic lupus erythematosus, Sjogren syndrome, or mixed tive tissue disease), skin diSease associated with Addison's e, skin disease associated with graft-versus~host disease (GVHD), eczema, or urticaria.

Item 16-A iThe pharmaceutical composition according to above 3O item14—A, wherein the immune disease of the skin is psoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, or inclusion body myositis.

Item 17~A The pharmaceutical compoSition according to above item 2012/072667 14-A, wherein the immune disease of the skin is atopic dermatitis or contact dermatitis.

Item 18-A A nucleic acid comprising a nucleotide sequence encoding the antibody according to any one of above items 1—A to ll-A.

Item 19—A An immune e treatment method comprising a step of administering an effective amount of the antibody according to any one of above items l—A to ll-A to a human affected by an immune disease.

The present invention r encompasses the embOdiments described below.

Item l—B An antibody comprising a heavy chain variable region comprising heavy chain CDRs l to 3 described in (g) to (1) below and a light chain variable region comprising light chain CDRs 1 to 3 described in (j) to (1) below; an dy comprising a heavy chain le region comprising heavy chain CDRs 1 to 3 described in (m) to (0) below and a light chain variable region comprising light chain CDRs l to 3 described in (p) to (r) below; or an antibody comprising a heavy Chain variable region comprising heavy chain CDRs 1 to 3 described in (a) to (c) below and a light chain variable region comprising light chain CDRs l to 3 described in (d) to (f) below: .(a) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) a heavy chain CDR 2 consiSting of the amino acid sequence of SEQ ID NO: 42, (c) a heavy chain CDR 3 consisting of the amino acid-sequence of SEQ ID NO: 43; 2012/072667 (d) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 44, (e) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 45, and (f) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 46; (g) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 17, (h) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 18, (i) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 19; (j) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 20, (k) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 21, (l) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 22; (m) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 29, (n) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 30, (o) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 31: (p) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 32, (q) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 33, and (r) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 34.

Item 2—3 The antibody according to above item 1—B, sing a heavy chain variable region comprising an amino acid ce of SEQ ID NO: 60 or 64, and a light chain variable region comprising? WO 32032 an amino acid sequence of SEQ ID NO: 68 or 72.

Item 3-3 The antibody ing to above item 1—8 or 2-B, comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 68.

Item 44B The antibody according to above item 1~B or 2—B, comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 64, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72.

Item 5—B The antibody according to any one of above items 1-3 to 4—3, comprising a human nt region.

Item 6-B The antibody according to any one of above items 1—3 to -B, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 59, and a light chain comprising an amino acid sequence of SEQ ID NO: 67.

Item 7-B The antibody according to any one of above items 1-3 to —B, comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 63, and a light chain sing an amino acid sequence of SEQ ID NO: 71.

Item 8-B The antibody according to any one of above items 1-3 to 7-B comprising an Fc region, wherein the Fc region is mutated to induce a change in ADCC activity.

Item 9-B The antibody according to above item 8—3, wherein the Fc region is mutated to lower ADCC activity.

Item 10-B The antibody according to any one of above items l-B to 9-3, n the antibody is conjugated to a cytotoxic molecule.

Item 11-B The antibody according to any one of above items 1-B to —8, wherein the antibody inhibits interaction n human XCRl and human XCLl.

Item 12—B The antibody according to any one of above items 1-B to ll-B, wherein the antibody inhibits cell migration of dendritic cells.

Item 13—8 A pharmaceutical composition comprising the antibody ing to any one of above items l-B to 12-B and a pharmaceutically acceptable carrier or additive, Item 14—3 The pharmaceutical composition ing to above item 13-B, wherein the pharmaceutical composition is~a therapeutic agent for an immune disease.

Item 15-B The pharmaceutical composition ing to above item 14—B, wherein the immune disease is an immune disease of the skin.

Item 16-B The pharmaceutical composition according to above item 15-B, wherein the immune disease of the skin is psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease (pemphigus, pemphigoid, or acquired epidermolysis bullosa). osis, herpes gestationis, linear IgA bullous dermatosis, ia areata, vitiligo vulgaris, skin disease associated with collagenosis (systemic lupus matosus, degren syndrome, or mixed connective tissue disease), skin disease associated with Addison's disease, skin disease associated with graft-versus-host disease (GVHD), eczema, or urticaria.

Item 17—3 The pharmaceutical composition ing to above itemlS-B, n the immune disease of the skin is psoriasis, atopic dermatitis, contact dermatitis, omyositis, polymyositis, or inclusion body myositis.

Item 18-3 The pharmaceutical composition according to aboVe item 15—B, wherein the immune e of the skin is atopic dermatitis or contact dermatitis.

Item 19-B A nucleic acid comprising a nucleotide sequence .encoding the antibody according to any one of above items 1~B to 12—8.

Item 20-8 A method of treating an immune disease comprising 3O administering an effective amount of the antibody according to any one of above items l-B to 12—3 or the pharmaceutical composition ing to above item 13-B to a human affected by an immune disease.

Item 21~B The method according to above item 20-B, wherein the immune disease is an immune disease of the skin.

Item 22-B The method according to above item Zl—B, wherein the immune disease of the skin is sis, parapsoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease igus, pemphigoid, or acquired epidermolysis bullosa), osis, herpes ionis, linear IgA bullous dermatosis, alopecia areata, vitiligo vulgaris, skin disease associated with collagenosis (systemic lupus erythematosus, degren syndrome, or mixed connective tissue disease), skin disease associated with Addison's e, skin disease associated with graft-versus—host disease (GVHD), , or urticaria.

Item 23-8 The method according to above item Zl-B, wherein the immune disease of the skin is psoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, or inclusion body myositis.

Item 24-B The method according to above item Zl-B, wherein the immune disease of the skin is atopic dermatitis or contact dermatitis.

Examples Herein belbw, the present invention is described in '30 more detail based on Examples. Needless to say, the present invention is not limited to the Examples. (1) Preparation of Mouse uman XCRI Mbnoclonal dies To obtain monoclonal antibodies against human XCRl, membrane on of human XCRl-expressing B300.19 cells was immunized to XCRl knockout mice. The membrane on was prepared as the following procedure: first, human xpressing B300.19 cells, being suspended in a Ho buffer (0.25 M Sucrose, 10 mM Hepes (pH 7.4), 1 mM EGTA, 0.5 mM M9012, 1x Complete mini EDTA— free (Roche Applied Science)), were disrupted (800 psi, for 30 minutes on ice) by a nitrogen gas cell disruption vessel (Parr Instrument Company) and then centrifuged (2,000 g, 10 minutes).

The supernatant was collected and re-centrifuged (100,000 g, 30 minutes). The pellet was suspended in a 50 mM Hepes (pH 7.4) , and ated as a membrane fraction. 160 pg or 260 pg of this ne fraction was mixed with equal volume of GERBU adjuvant (GERBU Biotechnik GmbH), and then injected subcutaneously into the footpads of the XCRl knockout mice (Deltagen). Five or six additional injections were then administered every other week. Three or four days after the final immunization, the mice were sacrificed, and the peripheral lymph-node cells were fused with P3Ul myeloma cells at a 2:1 or :1 ratio in the presence of GenomeONE-CF (Ishihara Sangyo Kaisha, Ltd.). The fused cells were then ed in 96-well plastic plates.

FACS analysis was performed for primary screening.

Parent CHO cells and human XCRl~EGFP-expressing CHO cells were mixed at a 1:1 ratio, and suspended in a FACS buffer (1 mM EDTA, 1% PBS-containing PBS' (Sigma)). The cells were incubated for 20 minutes on ice with culture supernatants from each hybridoma. The cells were washed with the FACS buffer three times, and then incubated for 20 minutes on ice with PE—labeled ouse IgG polyclonal dy (Jackson, #715—116-151, diluted at 1:100 in the FACS buffer). The cells were washed with the FACS buffer three times, and then suspended in the FACS buffer. The fluorescence intensity was measured using a FACSCanto II Cell analyzer (BD Bioscience). As results, supernatants collected from three wells showed high reactivity to human XCRl-EGFP—expressing CHO cells. 2012/072667 A standard ng dilution method was used to obtain clones from these three positive wells (2H6, 567, and 11H2). The reactivity of each clone was confirmed by the FACS analysis described above.

Subsequently, an in vitro chemotaxis assay was performed to evaluate the neutralizing activity of these three clones on human lymphotactin—induced migration of human XCRl- expressing BaFBcells or B300.19 cells. The chemotaxis assay was performed in 24-well transwell culture supports (pore 3 pm, Costar, #3399) or 96~well transwell culture plates (MultiScreen, pore 5 pm, Millipore, #IvIAMIC 5810).

In the case of the 24-well transwell culture supports, human xpressing BaF3 cells (1 x 106 cells) were suspended in a mixture of .50 uL of a taxis buffer (RPMIl 640 medium (Invitrogen) containing 0.5% BSA, 0.5% FBS, and 20 NM HEPES (pH 7.4)) and 50 pL of each culture supernatant, and incubated at room temperature for 30 minutes. Subsequently, recombinant human lymphotactin (Genzyme, #2695) dissolved in the chen'otaxis buffer at a concentration of 1 pg/mL was added to the lower wells at 600 uL/well, and the incubated cells were added to the upper wells.

After 4 hours of incubation in a 5% (102 incubator at 37°C, the transwells were centrifuged at 1,350 rpm for 5 minutes, and migrated cells were collected into the lower wells. The collected cells were fixed with paraformaldehyde (final concentration: 1%) , and 30 pL of each sample was applied to the FACSCanto II cell analyzer to count the number of the cells.

In the case of the 96—well transwell e plates, human XCRl-expressing B300.1é cells (2 x 105 cells) were suspended in a mixture of 25 pL of a chemotaxis buffer (RPMIl 640 (Invitrogen) ning 0.5% BSA, 0.5% FBS, and 20 KM HEPES (pH 7.4), and 50 3.1M 2-mercaptoethanol) and 50 pl. of each culture supernatant, and incubated at room temperature for 30 minutes. Subsequently, recombinant human tactin (Genzyme, #2695) ved in chemotaxisybuffer at a concentration of 1 ug/mL was added to the lower wells at 150 l, and the incubated cells were added to the upper wells. After 4 hours of incubation in a.5% oozincubator at 37°C, the transwells were centrifuged at 1,350 rpm for 5 s, and migrated cells were ted in the lower wells. 30 uL of each sample was applied to the FACSCanto II cell analyzer to ‘count the number of the cells.

The culture supernatants produced by three hybridoma clones (2H6, 5G7, and llHZ) demonstrated neutralizing activity against human tactin-induced migration of human XCRl- sing BaF3 cells and B300.l9 cells. (2) Reactivity of Mouse Anti-Human XORl Antibodies (2H6, 567, and 11H2) to Human XCR1~§§pressing Cells In order to evaluate the reactivity and neutralizing activity of purified antibodies from these three clones, the antibodies were purified with recombinant protein A (GE Healthcare, #17-5080—01) from culture supernatants of each clone.

The isotype of each clone was ined using monoclonal antibody isotyping kit (Serotec, #MMTl). 2H6 and SS? were IgGZb, K and 11H2 was IgGZa, K.

The reactivity of the purified dies to human XCRl was evaluated by FACS analysis. Parent 8300.19 cells and human GFP-expressing 3300.19 cells were mixed at a 1:1 ratio and suspended in a FACS buffer (1% PBS-containing PBS" (Sigma)). The cells were blocked for 10 minutes on ice with the FACS buffer containing 100 ug/mL of human immunoglobulin. The cells were then ‘incubated for 20 minutes on ice with the purified antibodies (2H6, 567, and 11H2) at various concentrations from 0 to 10 pglmL or with mouse isotype control antibody, IgG2a (eBioscience, #14- 4724-82) or IgG2b (eBioscience, #14-4732—82), at a tration of 10 ug/mL. The cells were washed with the FACS buffer three times, and then incubated for 20 minutes on ice with PE-labeled anti-mouse IgG polyclonal antibody (Jackson, #715—116-151, diluted at 1:50 in the FACS buffer). The cells were washed with the FACS buffer three times, and then suspended in the FACS buffer. The fluorescence ity was measured by a FACSCanto II 2012/072667 cell analyzer.

These three purified antibodies (2H6, 567, and 11H2) showed the reactivity to human XCRl-EGFP-expressing B300.19 cells, but not to parent B300.19 cells (Fig. 1). In contrast, the mouse isotype control antibody did not react to either human XCRl-EGFP- expressing 3300.19 cells, or to parent cells (data not shown). (3) Neutralizing Activity of HOuse Anti-Human XCRl Antibodies (2H6, 567, and 11H2) against Human Lymphotactin-Induced ion of Human XCRl—Egpressing Cells The neutralizing activity of purified antibodies from these clones was evaluated by in vitrc chemotaxis assay. The axis assay was performed using l transwell culture plates (MUltiScreen, pore 5 pm, Millipore, #MAMIC 5S10). Human XCRl- expressing B300.19 cells (2 x ldscells) were suspended in a 75 pL of axis buffer (RPMI 1640 medium (Invitrogen) containing 0.5% BSA, 0.5% FBS, 20 HM HEPES (pH 7.4), and 50 pM 2—mercaptoethanol) containing each of the purified antibodies (2H6, 5G7, and 11H2) , at various concentrations from 0 to 10 ug/mL; and incubated at room temperature for 3h minutes. r, inant human lymphotactin (R&D, #695-LT/CF) was dissolved in the chemotaxis buffer at a final concentration of 1 ug/mL, in which the purified antibodies were dissolved at various concentrations from 0 to 10 ug/mL. The mixture of lymphotactin and purified antibodies were added to the lower wells at 150 pL/well, and incubated at room temperature for 30 minutes. 30 s later, the incubated cells were added to the upper wells, and incubated in a 5%(Xh hmnﬂator at 37°C for 4 hours. Subsequently, 30 pL of each sample was applied to the nto II cell analyzer to count the number of the cells. 2H6, 567, and 11H2 mAbs completely inhibited cell migration at a concentration of about 3 pg/mL. Fig. 2 shows the typical pattern of the concentration-dependent inhibition. ICm and 10% values were calculated from three independent experiments.

Table 1 shows these values as the mean 1 standard error.

' PCT/JPZOlZ/072667 Table 1 ICm and IC% values of 2H6, 5G7, and 11H2 by Chemotaxis Assay —m—11H2 1050mm 1..28:t0172 0.63i0.139 .168 ICgo(nM) 7.60i2.331 1.5210645 6..32:t1830 (4) Sgggence Analygis of Mbuse Anti-Human XCRl Antibodies (2H6, 567, and 11H2) ' A cleotide comprising a gene sequence encoding the heavy and light chains of the clones (2H6, 5G7, and 11H2) were amplified by 5’-RACE (5'-rapid amplification of cDNA ends) method. The total RNA was ed from the hybridoma of these three clones using TRIZOL (Invitrogen) and treated with DNase (QIAGEN, RNase free DNase set). Double—stranded cDNA was prepared from the total RNA, using a cDNA synthesis kit (TAKARA). 5' adaptor obtained by annealing ad29S; ACATCACTCCGT (SEQ ID NO: 81) and asZ9AS; TGATGTCCGTCGACGTATCTCTGCGTTGATACTTCAGCGTAGCT (SEQ ID NO: 82) was added to the cDNA. The obtained cDNA was amplified using ’ d primer (5'—PCR4 primer, AGCTACGCTGAAGTATCAACGCAGAG: SEQ ID NO: 83) 3’ reverse primer AGGGGTTGATTGTTGA: SEQ ID NO: 84, or CTCAAGTTTTTTGTCCACCGTGGTGC: SEQ ID NO: 85 was used to amplify IgGZb heavy chain; CTCAATTTTCTTGTCCACCTTGGTGC: SEQ ID NO: 86, or GCCAGTGGATAGACTGATG: SEQ ID NO: 87 was used to amplify IgGZa heavy chain; and CTCATTCCTGTTGAAGCTCTTGACAAT: SEQ ID NO: 88.

GATGGATACAGTTGGTGCAGC: SEQ ID NO: 89, or CAGATCCTCAGCCTCCACTCTGCT: SEQ ID NO: 90 304 was used to amplify ng light chain). The amplified cDNA was inserted into pCR2.1 vector (Invitrogen). The gene sequences were analyzed using ABI313OXL. Tables 2-1 to 4-2 show amino acid PCT/JPZOlZ/072667 sequences encoded by the gene sequences identified by the analysis.

Table 2-1 ‘ Amino Acid Sequences of Mouse Anti-Human XCRl dy (2H6) Heavy Chain QAYLQQSGAELVRPGASVKMSCKASGYTFSSHNMHWIKQTLRQGLE variable region WIGAIYPGKGNTSYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSA (SEQ ID NO: 1) VYFCARWGSVVGDWYFDVWGTGTTVTVSS Light chain DVVVTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPG variable region QSPKLLIYRVSNRFSGVPDRFSGSGLGRDFTLKISRVEAEDLGVYF (SEQ ID NO: 2) CSQSTFVPWTFGGGTKLEIK Table 2-2 CAGGCTTATCTACAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGG CCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTAGCAG TATGCACTGGATAAAGCAGACACTTAGACAGGGCCTGGAA Heavy chain TGGATAGGAGCTATTTATCCAGGAAAAGGTAATACTTCCTACAATC variable region AGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACAAATCCTCCAG (SEQ ID NO: 3) CACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAAGACTCTGCG GTCTATTTCTGTGCAAGATGGGGTTCGGTAGTAGGAGACTGGTACT TCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCTTCA GATGTTGTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTG GAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACA CAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGC Light chain CCAAAGCTCCTGATCTACAGAGTTTCCAATCGATTTTCTG variable region GGGTCCCAGACAGGTTCAGTGGCAGTGGATTAGGGAGAGATTTCAC (SEQ ID NO: 4) GATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTC TGCTCTCAAAGTACATTTGTTCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAA Table 3-1 Amino Acid Sequences of Mouse Anti—Human XCRl Antibody (5G7) Heavy chain QAYLQQSGAELVRPGASVKMSCKASGYTFTSHNLHWVKQTPRQGLQ variable region WIGAIYPGNGNTAYNQKFKGKATLTVDKSSSTAYMQLSSLTSDDSA (SEQ ID NO: 5) VYFCARWGSVVGDWYFDVWGTGTTVTVSS Light chain DVVMTQTPLSLPVTLGNQASIFCRSSLGLVHRNGNTYLHWYLQKPG ' variable region QSPKLLIYKVSHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYF (SEQ ID NO: 6) CSQSTHVPWTFGGGTKLEIK Table 3-2 Nucleic Acid Sequences of Mbuse Anti-Human XCRl Antibody (567) CAGGCTTATCTTCAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGG CCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTCACCAG TCACAATTTGCACTGGGTAAAGCAGACACCTAGACAGGGCCTGCAA Heavy chain GGAGCTATTTATCCAGGAAATGGTAATACTGCCTACAATC variable region AGAAGTTCAAGGGCAAGGCCACGCTGACTGTAGACAAATCCTCCAG (SEQ ID NO: 7) TACAGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCG GTCTACTTCTGTGCAAGATGGGGTTCGGTTGTAGGAGACTGGTACT TCGACGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCACTCTTG GAAATCAAGCCTCCATTTTTTGTAGATCTAGTCTGGGCCTTGTACA TGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGC Light chain CAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCCACCGATTTTCTG variable region GGGTCCCAGACAGGTTCAGTGGCAGTGGCTCAGGGACAGATTTCAC (SEQ ID NO: 8) ACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGGGTTTATTTC TGCTCTCAAAGTACCCATGTTCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAA Table 4-1 Amino Acid Sequences of Mouse Anti—Human XCRl Antibody (11H2) Heavy chain EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYVNWVKQSHGASLE variable region WIGVSNPKNGDKSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSA (SEQ ID NO: 9) VYYCARGLYYAGTYGYFDVWGTGTTVTVSS Light chain ATSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKL variable region LIYYTSRLHSGVPSRFRGSGSGTDFSLTISNLEQEDIATYFCQQGK (SEQ ID NO: 10) TLPRTLGGGTKLEIK Table 4—2 c Acid Sequences of Mouse Anti-Human XCRl Antibody (11H2) GAGGTCCAGCTTCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGG Heavy chain CTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGA variable region CTACTATGTGAACTGGGTGAAACAGAGCCATGGAGCGAGCCTTGAG (SEQ ID NO: 11) TGGATTGGAGTTAGTAATCCTAAGAACGGTGATAAAAGTTACAACC AGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAG TACAGCCTACATGGAGCTCAACAGCCTGACATCTGAGGACTCTGCT GTCTATTACTGTGCAAGAGGGCTTTACTACGCTGGTACCTACGGGT ACTTCGATGTCTGGGGCACGGGGACCACGGTCACCGTCTCCTCA GATATCCAGATGACACAGGCTACATCCTCCCTGTCTGCCTCTCTGG GAGACAGAGTCACCATCAGTTGTAGGGCAAGTCAGGACATTAGCAA Light chain TTATTTAAACTGGTATCAGCAGAAGCCAGATGGAACTGTTAAACTC variable region CTGATCTACTACACATCAAGATTACACTCAGGTGTCCCATCAAGGT (SEQ ID NO: 12) TCAGAGGCAGTGGGTCTGGGACAGATTTCTCTCTCACCATTAGCAA CCTGGAGCAAGAAGATATTGCCACTTATTTTTGCCAACAGGGTAAA ACGCTTCCTCGGACGCTCGGTGGAGGCACCAAGCTGGAAATCAAA (1) Preparation of Chimeric Anti-Human XCRl Antibody and Humanized Anti-Human XCRl Antibodies 5G7, which demonstrated the highest neutralizing activity among 2H6, 567, and 11H2, was used to produce a chimeric antibody and humanized antibodies.

The chimeric antibody was prepared by combining, by pping extension PCR, the gene sequence of the 567 heavy chain variable region and the gene sequence of the human IgG2 constant region into which V234A/GZ37A mutation was inserted for heavy chain and the sequence of the 5G7 light chain variable region and the gene ce of human IgK constant region, and by ing the ing sequence into expression vectors (pEEd.4 or pEE12.4). Tables 5 and 6 tively show amino acid sequences and nucleotide sequences of the specific chimeric antibody.

Table 5 Amino Acid Sequences of Chimeric Anti-Human XCRl Antibody Sequence (The variable region is indicated in bold, and CDRs in the variable region are underlined,) , QAYLQQSGAELVRPGASVKMSCKASGYTFTSHNLHWVKQTPRQGLQ PGNGNTAYNQKFKGKATLTVDKSSSTAXMQLSSLTSDDSA VYPCARWGSVVGDWYFDVWGTGTTVTVSSASTKGPSVFPLAPCSRS Heavy chain TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY (SEQ ID NO: l3) SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL 2012/072667 VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DVVMTQTPLSLPVTLGNQASIFCRSSLGLVHRNGNTYLHWYLQKPG QSPKLLIYKVSHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYF Light chain CSQSTHVPWTFGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVV (SEQ ID NO: l4) CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 6 Nucleic Acid Sequences of Chimeric Anti-Human XCRl Antibody wSequence(The variable region is ted in bold.) CAGGCTTATCTTCAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGG CCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTCACCAG TCACAATTTGCACTGGGTAAAGCAGACACCTAGACAGGGCCTGCAA TGGATTGGAGCTATTTATCCAGGAAATGGTAATACTGCCTACAATC AGAAGTTCAAGGGCAAGGCCACGCTGACTGTAGACAAATCCTCGAG TACAGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCG GTCTACTTCTGTGCAAGATGGGGTTCGGTTGTAGGAGACTGGTACT TCGACGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAGCTAG CACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGC ACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAG CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCC AGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT Heavy chain GACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGC (SEQ ID NO: 15) CCAGCACCACCTGCCGCAGCCCCGTCAGTCTTCCTGTTCCCCCCAA AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCAC GGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCAC CGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAG AACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCA AAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG GTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCACTCTTG GAAATCAAGCCTCCATTTTTTGTAGATCTAGTCTGGGCCTTGTACA Light chain CAGAAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGC (SEQ ID N0: 16) CAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCCACCGATTTTCTG GGGTCCCAGACAGGTTCAGTGGCAGTGGCTCAGGGACAGAITTCAC ACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGGGTTTATTTC TGCTCTCAAAGTACCCATGTTCCGTGGACGTTCGGTGGAGGCACCA AGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTT CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG AAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGTTAG The antibody was humanized by grafting the complementary ining region of mouse antibody 5G7 into the human antibody variable region. The complementary determining region was determined according to the Kabat numbering system and a method for identifying the complementary determining region (for example, Kabat et al., (1991) Sequences of ns of Immunological Interest: US Department of Health and Human Services, NIH, USA). Further, the complementary determining regions of 2H6 and 11H2 were also determined in a r .' Tables 7-1 to 9-2 Show amino acid sequences and nucleotide' sequences of the complementary determining regions of these three clones.

Table 7—1 Amino Acid Sequences of Complementary Determining Region of 5G7 Heavy chain CDR 1 (SEQ ID NO: 17) HeaVY Chain CDR 2 GNTAYNQKFKG (SEQ ID NO: 18) Heavy chain CDR 3 WGSVVGDWYFDV (SEQ ID NO: 19) ' Light chain CDR 1 l RSSLGLVHRNGNTYLH (SEQ ID NO: 20) ' Light chain CDR 2 -‘ KVSHRFS ' (SEQ ID No: 21) Light chain CDR 3 , SQSTHVPWT .

(SEQ ID NO: 22) ' Table 7-2 Nucleic Acid Sequences of Complementary Determining Region of 567 HeaVY chain CDR 1 AGTCACAATTTGCAC ' (SEQ ID NO: 23) Heavy chain CDR 2 GCTATTTATCCAGGAAATGGTAATACTGCCTACAATCAGAAGTT (SEQ ID NO: 24) CAAGGGC Heavy chain CDR 3 TGGGGTTCGGTTGTAGGAGACTGGTACTTCGACGTC (SEQ ID NO: 25) Light chain CDR 1 AGATCTAGTCTGGGCCTTGTACACAGAAATGGAAACACCTATTT (SEQ ID NO: 26) ACAT Light chain CDR 2 AAAGTTTCCCACCGATTTTCT (SEQ ID NO: 27) Li ht chaing CDR 3 TCTCAAAGTACCCATGTTCCGTGGACG (SEQ ID NO: 28) Table 8-1 Amino Acid Sequences of Complementary ining Region of 2H6 Heavy chain CDR l (SEQ ID NO: 29) Heavy chain CDR 2 AIYPGKGNTSYNQKFKG (SEQ ID NO: 30) Heavy chain CDR 3 WG GD V (SEQ ID NO: 31) Light chain CDR 1 .

RSSQSLVHSNGNTYLH (SEQ ID NO: 32) Light chain CDR 2 RVSNRFS (SEQ ID NO: 33) Li ht Chain CDRg 3 SQSTFVPWT ' (SEQ ID NO: 34) Table 8-2 Nucleic Acid Sequences of Complementary Determining Region of 2H6 Heavy chain CDR 1 . AGTCACAATATGCAC (SEQ ID NO: 35) .

Heavy chain CDR 2 GCTATTTATCCAGGAAAAGGTAATACTTCCTACAATCAGAAGTT (SEQ ID NO: 36) C Heavy chain CDR 3 TGGGGTTCGGTAGTAGGAGACTGGTACTTCGATGTC (SEQ ID NO: 37) Light chain CDR 1 AGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTT (SEQ ID No: 38) Light chain CDR 2 AGAGTTTCCAATCGATTTTCT .

(SEQ ID NO: 39) .

L‘ ' 19ht 6 a1“ CDRh 3 TCTCAAAGTACATTTGTTCCGTGGACG (SEQ ID NO: 40) Table 9-1 Amino Acid Sequences of Complementary Determining Region of 11H2 Heavy-chain CDR 1 DYYVN (SEQ ID NO: 41) Heavy chain CDR 2 ‘ VSNPKNGDKSYNQKFKG (SEQ ID NO: 42) .

‘ Heavy chain CDR 3 GLYYAGTYGYFDV (SEQ ID NO: 43) Light chain CDR 1 RASQDISNYLN (SEQ ID NO: 44) Light chain CDR 2 YTSRLHS (SEQ ID NO: 45) A Light chain CDR 3 KTLPRT (SEQ ID NO: 46) ' Table 9—2 Nucleic Acid Sequences of Complementary Determining Region of 11H2 Heavy chain CDR 1 TATGTGAAC (SEQ ID NO; 47) Heavy chain CDR 2 GTTAGTAATCCTAAGAACGGTGATAAAAGTTACAACCAGAAGTT (SEQ ID NO: 48) C Heavy chain CDR 3 GGGCTTTACTACGCTGGTACCTACGGGTACTTCGATGTC (SEQ ID NO: 49) Light chain CDR l AGGGCAAGTCAGGACATTAGCAATTATTTAAAC (SEQ ID NO: 50) Light chain CDR 2 TACACATCAAGATTACACTCA (SEQ ID NO: 51) Light chain CDR 3 CA T CGCTTCCTC CG (SEQ ID NO: 52) As is clear from Tables 7-1 and 8-1, the identity of.

WO 32032 2012/072667 the amino acid sequences of the CDRs between SG7 and 2H6 is high; in particular, the heavy chain CDR 3 amino acid sequences were completely cal. Accordingly, in regard to 5G7 and 2H6, the amino acid sequences can be generalized as shown in Table 10 below. Additionally, Fig. 7 shows the comparison of amino acid sequences of the CDRs 1‘to 3 of these clones.

Table 10 Generalized Amino Acid Sequences of Complementary Determining . Regions of 5G7 and 2H6 Heavy chain CDR 1 SHNXH (SEQ ID NO: 53) I Heavy chain CDR 2 AIYPGXGNTXXNQKFKG (SEQ ID NO: 54) Heavy chain CDR 3 WG GD DV ‘ (SEQ ID NO: 55) Light chain CDR 1 - VHXNGNTYLH ' (SEQ ID NO: 56) ‘ Light chain CDR 2 ‘ xvs s (SEQ ID NO: 57) Light chain CDR 3 SQST WT (SEQ ID NO: 58) , The "X" in the table may be any of the following: alanine (Ala: A), arginine (Arg: R), asparagine (Asn: N), aspartic acid (Asp: D), ne (Cys: C), glutamine (Gln: Q), glutamic acid (Glu: E), glycine (Gly: G), histidine (His: H), isoleucine.(Ile: I), leucine (Leu: L), lysine (Lys: K), methionine (Met: M), phenylalanine (Phe: F), proline (Pro: P), ,serine (Ser: S), threonine (Thr: T), tryptophan (Trp: W), tyrosine (Tyr: Y), and valine (Val: V).

The FRs of a human antibody with high identity to the FR of 5G7 were selected as the FRs of the humanized antibodies.

Subsequently, the amino acids in the FRs, which interact with the CDRs of 567, were predicted using the 3D model of the resulting ~ 76 antibody, and grafted with the CDRs. The human IgG2 nt region into which V234A/G237A mutation was ed was used as the constant region. HKl and HKS were designed as the humanized antibody heavy chains, and L2 and L5 were designed as the humanized dy light chains. Tables 11—1 to 14-2 show amino acid sequences and nucleotide sequences of the specific humanized antibodies.

Table 11—1 Amino Acid Sequences of Humanized Anti—Human XCRl Antibody Heavy Chain (HKl) ‘Sequence (The variable region is indicated in bold, and CDRs in the variable region are underlined.) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHNLHWVRQAPGQRLE PGNGNTAYNQKFKGRVTITRDTSASTAXMELSSLRSEDTA V!YCARWGSVVGDWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY Heavy chain SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC (SEQ ID NO: 59) PAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTTTSHNLHWVRQAPGQRLE le region WMGAIYPGNGNTAYNQKFKGRVTITRDTSASTAYMELSSLRSEDTA (SEQ ID NO: 60) VYYCARWGSVVGDWYFDVWGQGTLVTVSS Table 11-2 Nucleic Acid Sequences of Humanized Anti-Human XCRl Antibbdy Heavy Chain (HKl) Sequence (The variable regiOn is ted in bold, and CDRs in the variable region are underlined.) CAGGTGCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCAGGGG CCTCTGTCAAGGTGAGTTGCAAGGCCTCCGGTTACACTTTCACC29 Heavy chain CCACAACCTGCATTGGGEGAGACAGGCTCCTGGACAGCGACTGGAG (SEQ ID NO: 61) TGGATGGGAGCAATCTACCCAGGCAACGGAAATACTGCCTATAATC AGAAGTTTAAAGGCAGGGTGACAATTACTCGGGACACTTCCGCAAG CACCGCCTACATGGAGCTGTCCAGCCTGAGGAGTGAAGATACCGCT GTGTACTATTGTGCACGATGGGGATCCGTGGTCGGAGACTGGTATT TCGATGTGTGGGGGCAGGGTACCCTGGTCACAGTGTCTAGTGCCTC CACAAAGGGCCCCAGCGTGTTTCCACTGGCTCCCTGCTCTAGGAGT rACATCAGAGTCCACTGCCGCTCTGGGATGTCTGGTGAAGGACTATT AACCAGTCACCGTGAGTTGGAACTCAGGGGCTCTGACATC TGGTGTCCACACTTTTCCTGCAGTGCTGCAGTCATCCGGCCTGTAC TCCCTGAGCTCTGTGGTCACAGTCCCAAGTTCAAATTTCGGAACCC AGACATATACTTGCAACGTGGACCATAAGCCCAGCAATACCAAGGT CGATAAAACAGTGGAGCGAAAGTGCTGTGTCGAATGCCCACCTTGT CCAGCTCCACCAGCAGCAGCTCCTTCTGTGTTCCTGTTTCCTCCAA AGCCAAAAGACACTCTGATGATCAGCCGGACCCCCGAGGTCACATG TGTGGTCGTGGACGTGTCTCACGAGGATCCTGAAGTCCAGTTTAAC TGGTACGTGGATGGGGTCGAAGTGCATAATGCAAAGACAAAACCAC GAGAGGAACAGTTCAACTCTACATTTCGTGTCGTGAGTGTGCTGAC TGTCGTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAA GTGTCCAATAAGGGACTGCCCGCCCCTATCGAGAAAACTATTAGCA AGACCAAAGGCCAGCCTAGAGAACCACAGGTGTACACCCTGCCCCC TAGTCGCGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTG AGGGTTCTATCCCAGCGACATTGCCGTGGAGTGGGAATCTA ATGGTCAGCCTGAGAACAATTACAAGACCACACCACCCATGCTGGA 'CTCCGATGGGAGCTTCTTTCTGTATTCAAAGCTGACCGTGGATAAA TCCAGGTGGCAGCAGGGTAATGTCTTTAGCTGCTCTGTGATGCACG AAGCCCTGCACAACCATTACACTCAGAAGTCCCTGTCCCTGTCACC TGGAAAGTGA ' CAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAACCAGGGG CCTCTGTCAAGGmGAGTTGCAAGGCCTCCGGTTACACTTTCACegg CCACAACCTGCATTGGGTGAGACAGGCTCCTGGACAGCGACTGGAG Heavy chain TGGATGGGAGCAATCTACCCAGGCAACGGAAATACTGCCTATAATC variable region AGAAGTTTAAAGGCAGGGTGACAATTACTCGGGACACTTCCGCAAG (SEQ ID NO: 62) CACCGCCTACATGGAGCTGTCCAGCCTGAGGAGTGAAGATACCGCT GTGTACTATTGTGCACGATGGGGATCCGTGGTCGGAGACTGGTATT TCGATGTGTGGGGGCAGGGTACCCTGGTCACAGEGTCTAGT Table 12-1 Amino Acid ces of Humanized Anti~Human XCRl Antibody Heavy Chain (HKS) Sequence (The-variable region is indicated in bold, and CDRs'in-the variable region are underlined.) ‘ (SEQ ID NO: 63) WMGAIYPGNGNTAYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTA 2012/072667 VYYCARWGSVVGDWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHNLHWVRQAPGQGLE variable region WMGAIYPGNGNTAYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTA (SEQ ID NO: 64) VYYCARWGSVVGDWYFDVWGQGTLVTVSS Table 12-2 Nucleic Acid Sequences of Humanized Anti-Human XCRl Antibody Heavy Chain (HK5) Sequence (The variable region is indicated in. 'bold, and CDRs in the variable region are underlined.)’ ‘ CAGGTGCAGCTGGTGCAGTCTGGGGCCGAAGTGAAGAAACCAGGGG CTTCTGTCAAGGTGAGTTGCAAAGCATCAGGTTACACTTTCACCEQ CCACAACCTGCAETGGGTGCGACAGGCTCCTGGACAGGGACTGGAG TGGATGGGAGCAATCTACCCAGGGAACGGTIATACCGCTTATAATC AGAAGTTTAAAGGCAGGGTCACAATGACTCGGGACACCTCCACAAG ' CACTGTGTACATGGAGCTGTCCAGCCTGCGAAGEGAAGATACAGCA TATTGTGCACGTTGGGGATCCGTGGTCGGTGACTGGTATT TCGATGTGTGGGGCCAGGGAACCCTGGTCACAGTGTCTAGTGCTTC CACTAAGGGGCCCAGCGTGTTTCCACTGGCACCCTGCTCTCGGAGT GAGTCCACCGCCGCTCTGGGCTGTCTGGTGAAGGACTATT TCCCTGAACCAGTCACAGTGAGTTGGAACTCAGGCGCACTGACTTC Heavy chain TGGAGTCCACACCTTTCCTGCCGTGCTGCAGTCATCCGGCCTGTAC (SEQ ID NO: 65) TCCCTGAGCTCTGTGGTCACTGTCCCAAGTTCAAATTTCGGAACCC AGACATATACTTGCAACGTGGACCATAAGCCCAGCAATACAAAGGT CGATAAAACTGTGGAGAGAAAGTGCTGTGTGGAATGCCCACCTTGT CCAGCACCACCAGCAGCAGCTCCTTCTGTGTTCCTGTTTCCTCCAA AGCCAAAAGACACACTGATGATCAGCCGCACACCCGAGGTCACTTG TGTGGTCGTGGACGTGTCTCACGAGGATCCTGAAGTCCAGTTTAAC TGGTACGTGGATGGCGTCGAAGTGCATAATGCCAAGACCAAACCAA GAGAGGAACAGTTCAACTCTACTTTTCGCGTCGTGAGTGTGCTGAC CGTCGTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAA GTGTCCAATAAGGGACTGCCCGCTCCTATCGAGAAAACCATTAGCA AGACAAAAGGACAGCCTAGGGAACCACAGGTGTACACCCTGCCCCC TAGTCGGGAGGAAATGACCAAGAACCAGGTCTCACTGACATGTCTG GTGAAAGGGTTCTATCCCAGCGACATTGCCGTGGAGTGGGAATCTA ATGGTCAGCCTGAGAACAATTACAAGACCACACCACCCATGCTGGA CTCCGATGGCAGCTTCTTTCTGTATTCAAAGCTGACCGTGGATAAA TGGCAGCAGGGAAATGTCTTTAGCTGCTCTGTGATGCACG TGCATAATCACTACACTCAGAAGAGCCTGTCCCTGTCACC TGGTAAATGA CAGGTGCAGCTGGTGCAGTCTGGGGCCGAAGTGAAGAAACCAGGGG CTTCTGTCAAGGTGAGTTGCAAAGCATCAGGTTACACTTTCACC29 CCTGCATTGGGTGCGACAGGCTCCTGGACAGGGACTGGAG Heavy chain TGGATGGGAGCAATCTACCCAGGGAACGGTAATACCGCTTATAATC variable region AGAAGTTTAAAGGCAGGGTCACAATGACTCGGGACACCTCCACAAG (SEQ ID NO: 66) CACTGTGTACATGGAGCTGTCCAGCCTGCGAAGTGAAGATACAGCA GTGTACTATTGTGCACGTTGGGGATCCGTGGTCGGTGACTGGTATT TCGAWGTGTGGGGCCAGGGAACCCTGGTCACAGTGTCTAGT Table 13—1 Amino Acid Sequences of Humanized Anti-Human XCRl Antibody Light Chain (L2) Sequence (The variable region is indicated in. bold; and CDRs in the variable region are underlined.) DVVMTQSPLSLPVTLGQPASISCRSSLGLVHRNGNTYLHWFQQRPG QSPRLLIYKVSHRFSGVPDRFSGSGSGTDPTLKISRVEAEDVGVYY Light chain CSQSTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV (SEQ ID NO: 67) CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTXSLSSTL ‘TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Light chain DVVMTQSPLSLPVTLGQPASISCRSSLGLVHRNGNTYLHWFQQRPG variable region QSPRLLIYKVSHRFSGVPDRFSGSGSGTDFTLKISRVBAEDVGVYY (SEQ ID NO: 68) CSQSTHVPWTFGQGTKVEIK Table 13-2 Nucleic Acid Sequences of Humanized Anti—Human XCRl Antibody Light Chain (L2) Sequence (The variable region is ted in bold, and CDRs in the variable region are underlined.) - GATGTCGTGAIGACCCAGTCTCCTCTGAGCCTGCCTGTGACTCTGG GCCAGCCAGCATCAATCTCCTGCCGATCCAGCCTGGGACTGGTGCA Light chain CCGTAACGGGAATACCTACCTGCATTGGTTCCAGCAGAGGCCTGGT (SEQ ID NO: 69) CCCCGGCTGCTGATCTATAAGGTGTCTCACAGATTCAGTG GCGTCCCAGACCGCTTTAGCGGCTCTGGAAGTGGGACTGAETTCAC 2012/072667 CCTGAAAATTTCCCGAGTGGAGGCAGAAGACGTGGGAGTCTACTAT TGCTCACAGTCCACACATGTGCCCTGGACTTTTGGTCAGGGCACCA AGGTCGAGATCAAACGCACCGTGGCCGCTCCTAGCGTCTTCATTTT TCCCCCTTCTGACGAACAGCTGAAGTCAGGAACAGCTTCCGTGGTC TGTCTGCTGAACAATTTTTACCCCAGAGAGGCAAAGGTGCAGTGGA AAGTCGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGAGTGTGAC GGACTCAAAGGATTCCACTTATAGCCTGTCTAGTACCCTG ACACTGTCTAAAGCTGATTACGAGAAGCACAAAGTGTATGCATGTG AAGTCACCCACCAGGGGCTGTCATCACCCGTCACCAAGTCCTTTAA TAGAGGGGAGTGTTGA GATGTCGTGATGACCCAGTCTCCTCTGAGCCTGCCTGTGACTCTGG GCCAGCCAGCATCAATCTCCTGCCGATCCAGCCTGGGACTGGTGCA CCGTAACGGGAATACCTACCTGCATTGGTTCCAGCAGAGGCCTGGT Light chain CAGAGTCCCCGGCTGCTGATCTATAAGGTGTCTCACAGATTCAGTG variable region GCGTCCCAGACCGCTTTAGCGGCTCTGGABGTGGGACTGATTTCAC (SEQ ID NO: 70) CCTGAAAATTTCCCGAGTGGAGGCAGAAGACGTGGGAGTCTACTAT TGCTCACAGTCCACACATGTGCCCTGGACTTTTGGTCAGGGCACCA AGGTCGAGATCAAA Table 14—1 Amino Acid Sequences of Humanized Anti—Human XCRl dy Light Chain (L5) Sequence (The variable region is indicated in bold, and‘CDRs in the variable region are underlined.) DIVMTQTPLSLPVTPGQPASISCRSSLGLVHRNGNTYLHWYLQKPG QSPQLLIYKVSHRPSGVPDRPSGSGSGTDFTLKISRVEAEDVGVYY Light chain CSQSTHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV (SEQ ID NO: 71) CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL YEKHKVYACEVTHQGLSSPVTKSFNRGEC Light chain DIVMTQTPLSLPVTPGQRASISCRSSLGLVHRNGNTYLHWYLQKPG Variable Region QSPQLLIYKVSHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY (SEQ ID NO: 72) CSQSTHVPWTFGQGTKVEIK Table 14-2 Nucleic Acid Sequences of Humanized Anti-Human XCRl Antibody Light Chain (L5) ce (The variable region is indicated in bold, and CDRs in the variable region are underlined.) Light chain GATATTGTGATGACTCAGACTCCACTGAGCCTGCCAGTGACTCCCG WO 32032 (SEQ ID NO: 73) GCCAGCCTGCATCAAICTCCTGCAGATCCAGCCTGGGACTGGNGCA CCGCAACGGGAATACCTACCTGCATTGGTATCTGCAGAAGCCTGGT CAGAGTCCCCAGCTGCTGATCTACAAAGTGTCTCACAGGTTCAGTG CCGACCGGTTTAGCGGCTCTGGAAGTGGGACTGATTTCAC CCTGAAGATTTCCCGAGTGGAGGCCGAAGACGTGGGCGTCTACTAT TGCTCACAGTCCACACATGTGCCTTGGACTTTTGGTCAGGGCACCA AGGTCGAGATCAAAAGGACCGTGGCCGCTCCAAGCGTCTTCATTTT TCCCCCTTCTGACGAACAGCTGAAGTCAGGAACAGCTTCCGTGGTC TGTCTGCTGAACAATTTCTACCCCAGAGAGGCAAAGGTGCAGTGGA AAGTCGATAACGCCCTGCAGAGCGGCAACTCCCAGGAGAGTGTGAC AGAACAGGACTCAAAGGATTCCACTTATAGCCTGTCTAGTACCCTG ACACTGTCTAAAGCTGATTACGAGAAGCACAAAGTGTATGCATGTG AAGTCACACACCAGGGTCTGAGTTCCCCCGTCACCAAATCCTTTAA TCGTGGAGAGTGCTGA GATATTGTGATGACTCAGACTCCACTGAGCCTGCCAGTGACTCCCG GCCAGCCTGCATCAATCTCCTGCAGATCCAGCCTGGGACTGGTGCA CCGCAACGGGAATACCTACCTGCATTGGTATCTGCAGAAGCCTGGT Light chain CAGAGTCCCCAGCTGCTGATCTACAAAGTGTCTCACAGGTTCAGTG Variable Region CCCGACCGGTTTAGCGGCTCTGGAAGTGGGACTGATTTCAC (SEQ ID NO: 74) ' CCTGAAGATTTCCCGAGTGGAGGCCGAAGACGTGGGCGTCTACTAT TGCTCACAGTCCACACAIGTGCCTTGGACTTTTGGTCAGGGCACCA AGGTCGAGATCAAA Gene sequences of these humanized antibodies were entirely synthesized by GenScript USA ‘Inc. ,_ and inserted into sion vectors (pEE6~.4 or, pEE12.4 sed from Lonza) . produce antibodies, the expression vectors were transfected into IiEK293E cells (Invitrogen) using Lipofectamine 2000 ing to the instructions for Lipofectamine 2000 (Invitrogen) . supernatants were collected and purified using Protein A (GE Healthcare) . The neutralizing activity» was evaluated using these purified humanized dies. zed antibodies having neutralizing activity against human lymphotactin-induced migration of human XCRl- expressing cells were identified by performing in vitro chemotaxis assays using human XCRl-expressing 3300.19 cells. The chemotaxis assay was performed as described above using 96—well transwell culture plates (MultiScreen, pore 5 pm, Millipore, #MAMIC 5810 or Corning #3387 or #3388). However, in the case of using Corning 2012/072667 transwell culture plates, the amount of the recombinant human lymphotactin and purified antibodies to be added to the lower wells is 235 uL per well.

Among humanized antibodies having neutralizing activity, two types of the following antibodies, HK1L2 and HK5L5, were ted in further detail. (2) Reactivity of Humanized Anti-Human XCRl Antibodies (HK1L2 and HK5L5) to Human XCRléggpressing Cells FACS analysis was performed using these two humanized antibodies (HK1L2 and , the parent antibody 5G7, and the chimeric antibody.r Parent B300.19 cells and human XCRl—EGFP- sing B300.19 cells were mixed at a 1:1 ratio and suspended in a FACS buffer (1% PBS—containing PBS' (Sigma)). The cells were incubated for 20 minutes on ice With the purified antibodies at various concentrations from 0 to 10 ug/mL. The cells were washed three times with the FACS bufferJ and then incubated for 20 minutes on ice with PE-labeled ouse IgG polyclonal antibody (Jackson, #715151: used for cells which had been stained with parent antibody 567. diluted at 1:100 in the FACS ) or with PE—labeled anti-human IgG polyclonal antibody (Jackson.- #709149: used for cells which had been stained with chimeric antibody or humanized antibodies (HKlLZ and HKSLS), diluted at 1:100 in FACS buffer. The cells were washed three times with FACS , buffer, and then suspended in FACS buffer. The fluorescence intensity was measured using a FACSCanto II cell analyzer (BD Bioscience).

Humanized antibodies (HK1L2 and HK5L5) showed concentration—dependent reactivity to human GFP-expressing ' B300.19 cells. Parent antibody 5G7 and chimeric dy showed substantially the same reactivity (Fig. 3).

The reactivity of humanized antibodies (HKlLZ and HKSLS) to human XCRl was further examined by FACS is using human eral blood mononuclear cells. Because human XCRl gene is known to be expressed in BDCA3+ dendritic cells, which is a minor population in human peripheral blood mononuclear cells, first, the dendritic cells were concentrated from human peripheral blood mononuclear cells and used for FACS analysis.

Human peripheral blood clear cells were isolated from the blood of healthy human subjects using Ficoll—Paque (GE Healthcare, #1702). CD3, CD14, CD19, and CD56 positive cells from human peripheral blood mononuclear cells were labeled with CD3, C014, CD19, CD56 antibody eads (Miltenyi, #130—050—101, 50~ 201, #130—050—301, #130-050—401), and depleted using auto-MACS nyi). Thereby, human dendritic cells were concentrated.

The concentrated dendritic cells were blocked for 10 minutes on ice with a FACS buffer (1% PBS—containing PBS’ (Sigma)) containing 1% rat serum, 1% mouse serum, 100 ug/mL human immunoglobulin. The cells were then stained for 30 minutes on ice separately using PE-labeled 567, HK1L2, HKSLS, and isotype control antibody mouse IgG2b, K (eBioscience, #14—4732—82) or human Ing, K (Sigma, #I5404) with FITC-labeled anti-BDCA3 antibody (Miltenyi, #130- 090—513), AFC-labeled anti—CD123 antibody (Miltenyi, 90— 901), APC—Cy7-labe1ed anti—HLA—DR antibody (BioLegend, #307617). and Alexa700—labeled anti—CD3, CD14, CD19, CD56 antibodies (BioLegend, #300324, #301822, #302225, and #318316). The cells were washed three times with the FACS buffer, and then suspended in the FACS buffer. The fluorescence intensity was measured using a FACSCanto II cell analyzer.

As is the case with parent antibody 567, the humanized antibodies (HK1L2 5L5) selectively reacted to BDCA3+ dendritic cells sing human XCRl (Fig. 4). (3) NeutraliZing ty of Humanized Anti-Human XCRl Antibodies ,' (HK1L2 and HK5L5) on Human tactin-Induced Migration of Human XCRl-Eﬁpressing Cells The neutralizing activity of these humanized antibodies was evaluated in parallel with parent antibody 5G7 and a chimeric antibody by in Vitro chemotaxis assay as described above.

In ison with parent dy 5G7, both humanized antibodies maintained the neutralizing activity. Fig. 5 shows the l pattern of concentration-dependent inhibition. ICw and IC% values were calculated from three independent experiments.

Table 16 shows these values as the mean 1 standard error.

Table 16 Neutralizing Activity of Humanized Antibodies (HK1L2 and HKSLS) in Chemotaxis Assay Next, the neutralizing activity of humanized antibodies (HKlLZ and HKSLS) was further examined by transendothelial migration assay that used human dendritic cells d of the human XCRl—expressing 8300.19 cells. The transendothelial migration assay was performed using 24-well transwell culture supports (pore 5 pm, Costar, #3421). First, ECV304 cells were suspended in 10% PBS—containing Medium 199 s medium (Invitrogen), and seeded into the upper chamber of the transwell at 2 x 105 cells per well, ed by incubation in a 5% C02 incubator at 37°C for 3 days. On the day of an assay, ECV304 cells were washed with assay buffer (a mixture of Medium 199 Earle’s medium and RPMI 1640 medium at a 1:1 ratio. to which 0.5% BSA and 20 mM HEPES (pH 7.4) were added). Recombinant human lymphotactin dissolved in the assay buffer at a concentration of 1 ug/mL, to which the ic antibody, HKlLZ, HK5L5, or isotype control antibody human IgG2, K (Sigma) was added at a concentration of 10 ug/mL, was added to the lower wells at 600 uL/well. Human dendritic cells were concentrated as bed above, suspended in the assay buffer to which the chimeric antibody, the humanized antibodies (HK1L2 and HK5L5) and isotype control antibody human IgG2, K (Sigma) were added at a concentration of 10 pg/mL, and added to the upper wells containing ECV304 cells. After incubation for 4 hours in a 5% C02 incubator at 37°C, the cells in the transwell were fuged at 1,350 rpm for 5 minutes, and migrated cells were collected; The collected cells were stained for 30 minutes on ice, using cell lineage markers, FITC-labeled DCA3 antibody (Miltenyi, #130-090—513), PE—labeled anti-BDCAI antibody (BioLegend, #331517), AFC—labeled anti-CD123 antibody (Miltenyi, #130 901), and beled anti—HLA—DR dy (BioLegend, #307617). 170 pL of each sample was then applied to a nto II cell analyzer (BD Bioscience) to count the number of cells.

Both humanized antibodies inhibited ion of BDCA3+ dendritic cells, as is the case with the chimeric antibody (Fig. 6).

Pharmacological Effect of House Anti-XCRI Antibody Pharmacological effect of anti—human XCRl mouse monoclonal antibody (5G7) prepared in Example 2 above was confirmed using a mouse model of delayed-type contact dermatitis (DTH). (1) Effect of Mouse Anti—human XCRI Antibody on Bar Swelling of DNPB (Dinitrofluorobenzene)—Sensitized Mice mental Method 1. Sample Mice Human XCRl knock-in mice (mice whose XCRl gene has been 2012/072667 replaced with human XCRl gene) on 6 background between the ages of 7 weeks and 12 weeks were used for the experiment. 2. Method for Preparing DNFB for Sensitization and DNFB for Induction DNFB for sensitization and induction was prepared by mixing DNFB to a 4:1 mixture of acetone and olive oil to obtain a concentration of 0.5%. Further, a 4:1 mixture of acetone and olive oil was used as a control solution for induction. 3. Method for Administering DNFB The abdominal hair of the mice was shaved to expose the skin, and 50 uL of 0.5% DNFB for sensitization was applied thereto. On the following day, 50 uL of 0.5% DNFB was applied again to the same site. 4 days after the application, 25 uL of 0.5% DNFB for induction was applied to the front side of the right ear of the mice. At the same time, as a control, 25 uL of the control solution ed by mixing acetone and olive oil at a 4:1 ratio was applied to the front side of the left ear of the mice. 4. Method for Administering dies Anti-human XCRl mouse monoclonal antibody (5G7) and its control antibody, i.e., mouse IgG (Jackson Laboratory), were prepared in PBS to a final concentration of 2 mg/mL. The day when the first sensitization was conducted was defined as Day 0. Each of the above antibodies was intraperitoneally stered into the mice in an amount of 250 uL/mouse (500 ug/mouse) on Day —1, Day 1, and Day 4.

. Method for measuring the Ear ng of DNFB-Sensitized Mouse, Model On the first day and the following day, the mice were sensitized by applying 50 uL of 0.5% DNFB to the exposed skin of the abdomen. 4 days after the sensitization, the ear thickness was measured using a caliper. After measurement, 25 pL of 0.5% DNFB was applied to the front side of the right ear of the mice for induction. Further, as a control, 25 pL of the control solution formed by mixing acetone and olive oil at a 4:1 ratio was applied to the left ear of the mice. The ear thickness was measured 24 hours and 48 hours after induction. The swelling was determined by converting measured values by the following formula.

Formula Ear thickness d by DNFB (swelling: mm) = ([AJ-[B])—([C]- [D]) [A]: thickness of right ear after induction (mm) [B]: thickneSs of right ear before induction (mm) [C]: ess of left ear after application of control solution (mm) [D]: thickness of left ear before application of contrdl solution (mm) Experimental Results and Analysis Fig. 8 clearly shows a significant suppression of ear swelling 24 after induction by DNFB in the mice administered with anti-human XCR1~mouSe monoclonal antibody (5G7), compared to the mice stered with the l antibody (Fig. 8A). The effect also showed icant suppression in a similar manner 48 hours ' after induction by DNFB (Fig. 88).

Although the antibody was ically administered intraperitoneally, the swelling was suppressed in the ear induced by DNFB. Therefore, it is presumed that the antibody transferred from the abdominal cavity into the blood and, along with the blood flow, reached an inflammatory site or a lymph node, where the dy demonstrates the effect of suppressing ear ng.

This suggests that the antibodies of the present invention have a specific effect on the inflammatory site in a site-specific manner. 2012/072667 vity of Mbuse Anti-Human XCRl Monoclonal Antigggy (567) to Various Human Chemokine Receptors The reactivity of mouse anti—human XCRl monoclonal antibody (5G7) to s human chemokine receptors was evaluated by FACS analysis. Parent B300.l9 cells and human chemokine receptor-EGFP—expressing B300.19 cells (XCRl, CXCRl, CXCR3, CXCR4.

CXCR5, CXCRG, CCRl, CCRZ, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRll, or CX3CR1, and) were suspended in a FACS buffer (PBS‘ (Sigma) containing 1 % fetal bovine serum). The cells were blocked for 20 minutes on ice with a blocking buffer (a FACS buffer containing 100 pg/mL of human immunoglobulin). The cells were then incubated for 50 minutes on ice with the blocking buffer containing 567 or mouse isotype control antibody IgGZb (eBioscience, #1482) at a tration of 10 ug/mL.' The cells were washed three times with the FACS , and then incubated for 20 minutes on ice with PE—labeled anti—mouse IgG polyclonal antibody (Jackson, #715151, diluted at 1:50 in the blocking buffer). The cells were washed with the FACS buffer three times, and then suspended in the FACS buffer. The fluorescence intensity was measured using a FACSCanto II cell analyzer.

Anti—human XCRl antibody 567 showed a high reactivity to human XCRl-EGFP-eXpressing B300.l9 cells. Further, anti—human XCRl antibody 5G7 showed a very low reactivity to human CX3CR1— EGFP-expressing B300.l9 cells, and no reactivity to other human chemokine receptors-EGFP-expressing cells (Fig. 9). 0n the other hand, mouse isotype control antibody did not show reactivity to any 8300.19 cells.

Exgmgle 5 V Extotoxicity of uman XCRI antibodies using sanrin- conjugated Fab ouse Ig§ secondary antibody to hmman XCR1 gggressing cells In order to demonstrate the cytotoxic activity- of anti- human XCRl antibodies to XCRl—expressing cells, cytotoxicity of mouse anti-human XCRl mAbs to cells On which human XCRl is exogenously expressing was examined by using saporin—conjugated Fab anti—mouse IgG secondary antibody. ‘2 x 103 cells of B300.19 parent cells or human XERl—EGFP- expressing 8300.19 cells inr 80 pL of RPM11640 (Invitrogen, #11875-093) containing 10% fetal bovine serum (Cell e Bioscience, 2), 100 ug/ml of cin sulfate (Invitrogen, #15160—054) and 50 pH 2-Mercaptoethanol (2-ME, Invitrogen, #21985-023) were added into each well of a 96 well plate. Mbuse unmu1 XCRlI antibodies (2H6, 5G7, or 11H2), mouse isotype control antibodies, IgGZa, K (eBioscience, #1685) or IgGZb, K (eBioscience, #16-4732—85), were d. with RPM11640 ning 10% fetal bovine serum, 100 pg/ml of kanamycin sulfate and-50 pM z-ME and 10 pl of the diluted antibodies were added to the cells at various concentrations frmn 0 to 0.17 ug/ml. The cells were then incubated in a 5% C02 incubator at 37°C for 20 min.

Then, saporin—conjugated Fab ouse IgG (Advanced Targeting ‘Systems, #IT-48) was diluted to 10 pg/ml with RPM11640 containing % fetal bovine serum, 100 ug/ml of kanamycin sulfate and 50 pM 2-ME, and 10 ul of the diluted saporin-conjugated Fab ouse IgG was added to each well at the final concentration of 1 HQ/ml.

The cells were then incubated in a 5% (lb incubator at 37°C for 72 hrs. Then the number of cells in each well was measured by using Cell Count Reagent SF (Nacalai tesque, 07553-15 or- -44). The reagent was added to each well and the cells were incubated in a % CO2 incubator at 37°C for 2 or 3 hrs. ODﬁo was then measured with a plate reader (Arvo, PerkinElmer).

Mouse anti-human XCRl antibodies (2H6, 567, and 11H2) with saporin-conjugated secondary antibody showed growth suppression of human XCRl-EGFP-expressing 9 cells (Fig. 11). The ICE values of 2H6, 567, and 11H2 calculated by Graphpad Prism software were 0.141 nM, 0.017 nM, and 0.155 nM, respectively. On the other hand, these antibodies with saporin-conjugated secondary antibody did not show cell growth suppression of parent B300.19 cells. l antibodies with saporin-conjugated secondary antibody did not suppress cell growth of human XCRl— EGFP-expressing B300.19 cells nor parent B300.19 cells (Fig. 11).

These findings indicate that mouse uman XCRl dies, 2H6, 5G7, and 11H2, were internalized. with saporin-conjugated secondary antibody and acted as an immunotoxin. mug Effects of 5G7 Mab on otoxitic T l h e assa in vivo In order to investigate the inhibitory activity of 5G7 Mab on the CTL function, CTL assay was performed.

The engineered hXCRl-knocked—in mice, in which human XCRl is expressed instead of mouse XCRl, were immunized subcutaneously with ovalbumin (200 ug/head) emulsified with CFA on day 0. The 567 Mab or the control mouse IgG (Jackson Laboratory). was intraperitoneally injected at a dose of 500 ug/head on day —1, day 2 and day 5. Six days later, splenocytes from naive C57BL/6 mice were incubated for 30 min at 37°C with or without 10 ug/ml OVAuTa“ peptide EKL; MBL).

These peptide-pulsed target and non-target cell populations were labeled with 2.5 and 0.25 uM CFSE (Invitrogen Life Technologies), respectively, then mixed at a 1:1 ratio, and injected intravenously into the immunized mice.

One day after the injection of CFSE-labeled splenocytes, the target cell-killing activity was evaluated using the ratio of CFSE-positive populations in the spleen as follows.

The CFSE—positive cells in the spleen in the immunized mice were detected by flow cytometry, and the CTL activity of each mouse was calculated with the ratio of CFSEmchells and w cells as follows: CTL activity = (% of CFSEmgh/% of CFSEW).

Then, the ve CTL activity was calculated as follows: Relative CTL activity = (CTL ty in each zed mouse)/ (CTL ty in control mouse).

The s showed that the relative CTL activity in the mice treated with 567 Mab showed lower relative CTL activity as compared to that in the mice treated with the control IgG (Fig. 12).

The data indicated the ssion of the in viva CTL activity by the treatment with CRl antibody, and suggested that the treatment with anti—XCRl antibodies may be beneficial for immune diseases, such as graft rejection, GVHD and tissue injury in autoimmune diseases. 32213.7 Reactivity of Mouse Anti-Human XCRI Antibodies (21-16, 567, and 11H2) to the Chimeric Human/MOuse XCRl—Egpressing Cells To determine epitopes of human XCRl recognized by mouse 15_ anti-human XCRl antibodies (2H6, 5G7, and llHZ), reactivity of these antibodies to chimeric Zhuman/mouse AXCRl-expressing' cells ‘was evaluated.

Because mouse anti-hunﬁuz XCRl antibodies (2H6, 567, and llHZ) reacted to human XCRl but not to mouse XCRl, a panel of human/mouse XCRl chimeric receptors was prepared. In this panel, each ellular domain of human XCRl was replaced by the homologous region of mouse XCRl, and vice versa. Expression s of this panel were ucted using an overlapping extension polymerase chain reaction (PCR) method. Each chimeric receptor-EGFP was expressed in TK-l cells and mAb reactivity was determined by FACS analysis. Parent TK—l cells, human XCRl—EGFP—, mouse XCRl—EGFP-, or chimeric XCRl—EGFP-expressing TK—l cells were suspended in a FACS buffer (PBS' (Sigma) containing 1% fetal bovine serum). The cells were blocked for 10 minutes on ice with a FACS buffer containing 100 ug/mL of human immunoglobulin. The cells were then incubated for 20 ndnutes on ice with the anti— human XCRl dies (2H6, 5G7, or 111-12) at s concentrations from 0 to 10 ug/mL, mouse isotype control antibodies, IgGZa (eBioscience, #14-4724—82) or IgGZb (eBioscience, #1482), at a concentration of 10 ug/mL, or a FACS buffer without dy. The cells were washed with the FACS buffer three times, and then incubated for 20 minutes on ice with PE-labeled anti—mouse IgG polyclonal dy (Jackson, #715—116- 151, diluted at 1:50 in the FACS buffer) or PE-labeled anti-human XCRl polyclonal antibody (R&D, #FAB857P, diluted at 2:5 in the FACS buffer, used for cells that had been incubated with the FACS buffer t antibody). The cells were washed with the FACS buffer three_times, and then suspended in the FACS buffer.r The fluorescence intensity was measured by a FACSCanto II cell analyzer.

Mouse uman XCRl antibodies (2H6, 567, and _11H2) showed the reactivity to human GFP—expressing TK—l cells, but not to parent TK-l cells or mouse XCRl—EGFP-expressing TK-l cells (Fig. 13; the origins of the four extracellular domains ~ were designated by four-letter codes (e.g., w is wild—type human XCRl, gmmm has human inal extracellular domain and mouse first, second, and third extracellular loops, etc.)). These three antibodies showed reactivity to chimeric XCRls, having the human XCRl N-terminus, —EGFP-expressing TK—l cells. Reactivity to chimera receptor, mmﬁm, was also examined in another experiment, and reactivity was not observed (data not shown).

In contrast, the mouse isotype l antibodies did not show reactivity to any TK-l cells (data not shown). my Mapping of Mouse Anti-Human XCRI Antibodies (2H6, 567, and 11H2)- Bindin Sites' on the Extracellular s of Human XCRl Pegtide ELISA To define the contact residues of anti-human XERl antibodies (2H6, 5G7, and 11H2) on human XCRl extracellular domains, peptide scan is was performed using sets of lZ-mer peptides covering the extracellular domains of human XCRl.

Two sets of peptides with biotin and spacer GSGS at N— terminal were synthesized by Sigma. The first set of 13 peptides WO 32032 contained all possible lZ-mers from the human XCRl N terminus, each offset by 2 amino acids. The second set of 13 es ned all possible 12-mer from the human XCRl extracellular loops, each offset by 3 amino acids. Peptides were initially reconstituted in 100% dimethyl sulfoxide and subsequently diluted in 30% dimethyl sulfoxide solution to give a final concentration of 50 ug/mL for direct ELISA.

Streptavidin—coated microtiter plates (Perkin Elmer) were coated with 50 ug/mL of peptide per well in a volume of 50 uL, and incubated at room temperature for 1 hour. The peptide solution was removed and PBS’ containing 4% Block-Ace was added to each well and incubated ght at 4° C, Each well was washed three times with an ELISA wash buffer (0.02% Tween20 in PBS—).

Anti-human XCRl antibodies (2H6, 5G7, or 11H2) were added to each well in amount of 10 pg/mL, and incubated for 6 hours at room temperature. Each well was washed three times with the ELISA wash buffer. Horseradish peroxidase—conjugated donkey anti-mouse IgG antibody (Jackson, #7157035-150), diluted 1:5,000 in the ELISA wash buffer, was added to each well and incubated for 1 hour at room temperature. Each well was washed three times with the ELISA wash . TMBZ (3,3’ ,5,5' tetramethyl benzidine; Sigma) was added to each well and incubated at room temperature. The on was d 'with 2N H2304, and A450,,“ was measured by Arvo plate reader (PerkinEJmer) .

Anti—human XCRl antibodies 2H6 and 567 showed strong binding to one peptide containing 7PES'I"I'F1='Y¥DLQ18 (SEQ ID NO: 96) , -and weak binding to ”TFFYYDLQSQPCZZ (SEQ ID NO: 110) (Fig. 14). 5G7 also showed weak binding to three non—sequential peptides containing lngPCENQAWVFAm (SEQ ID NO: 101) ,. ”ZSSGCDYSELTWYIB (SEQ 3O ID NO: 110), and 175CDYSELTWYLT8186 (SEQ ID 'No: 111). On the other hand, 11H2 showed no reactivity to these peptides (data not shown) . 229.122 g of Binding Residues of Mouse Anti-Human XCRl Antibodies (2H6, 567, and 11H2) and Emmanized Anti-Human XCRl Antibodies (HRILZ and HK5L5) on Human XCRl Extracellular Domains vain Alanine Mutants To determine the critical residues of human XCRl recognized by' mouse anti—human XCRl antibodies (2H6, 5G7. and 11H2) and humanized anti~human XCRl antibodies (HK1L2 and , alanine substitution assay was performed.

A panel of alanine substitution mutants of human XCRl was ed. In this panel,‘ each amino acid in 7PESTTFFYYDLQSQPCENQAWVFA3° (SEQ ID NO: 118) and 175CDYSELTWYLT3185 (SEQ ID NO: 119) of human, XCRl extracellular s were replaced by alanine. Expression vectors for alanine substitution mutants were constructed by using site—directed mutagenesis. Each mutant was expressed on 9 cells, and dy reactivity was ined by FACS analysis. Parent 3300.19 cells and human XCRl-EGFP- or each alanine mutant human XCRi-EGFP-expressing B300.19 cells were mixed at a 1:1 ratio and suspended in a FACS buffer (PBS‘ (Sigma) containing 1% fetal bovine serum). The cells were blocked for 10 minutes on ice with a FACS buffer Containing % rat serum. The cells were then incubated for 20 minutes on ice with mouse anti—human XCRl antibodies (2H6, 5G7, or 11H2), humanized antibodies (HKlLZ or‘ HKSLS), mouse e control antibodies, IgG2a (eBioscience, 24—82) or IgGZb (eBioscience, 32-82), or human isotype control antibody IgG2 (Sigma, #15404), at a concentration of 10 ug/mL;. or incubated with.a FECS buffer without antibody.‘ The cells were washed with the FACS buffer three times, and then incubated for minutes on ice with PE-labeled anti-mouse IgG polyclonal ‘30— antibody (Jackson, #715-116—151, diluted at 1:50- in the FACS buffer, used for cells that had been incubated with mouse antibodies), PE-labeled anti-human IgG polyclonal. antibody (Jackson, #709—116-149, diluted at 1:50 in the FACS buffer, used for cells which had been incubated with humanized antibodies or human control IgG), or PE-labeled anti—human XCRl polyclonal antibody (R&D, #FAB857P, diluted at 2:5 in the FACS buffer, used for cells that had been incubated with the FACS buffer without antibody). The cells were washed three times with the FACS buffer, and then suspended in the FACS buffer. The fluorescence intensity was measured using a nto II cell analyzer (BI) Bioscience) .

Each alanine mutant was detected by PE-labeled anti-human XCRl polyclonal antibody, except for C175A mutant (Fig. 15) .

Because expression s of each alanine mutants on cell ce were varied among these mutants as shown in Fig. 15, reactivity of antibodies to each alanine mutant was evaluated by a relative PE mean value (mAb/pAb) , calculated as per the following procedure. At first, a relative PE mean value for each dy was calculated by setting the PE mean value, which was obtained by staining of human XCRl—EGFP—expressing B300.19 cells (wild type) using each antibody, as 1.0. The relative PE mean values (mAb/pAb) were then ated by the following equation: each relative PE mean values for mouse anti-human XCRl antibodies (2H6, 5G7, or 11H2) or humanized antibodies (HK1L2 or HK5L5) was divided by the relative PE mean values for PE-labeled anti—human XCRl polyclonal antibody. The s showed that 2H6 (Fig. 16) , 567 (Fig. 17), HKlLZ (Fig. 19), and HK5L5 (Fig. 20) showed lower reactivity to many alanine mutants in which each residue in N- terminus or 2“Cl loop was replaced with alanine. In particular, no reactivity or weak reactivity to Y14A, D16A, and Ll7A mutants were observed. Additionally, reactivity to E8A, F13A, CZZA, and Y177A were lower among these mutants. Taken er, these results te that 2H6, 5G7, HK1L2, and HK5L5 - recognize E8, F13, Y14, D16, L17, C22 and Yl77 on human XCRl extracellular domain. lle (Fig. 18) showed similar reactivity to other mAbs except for F13A and D16A, indicating that 11H2 binds to E8, Yl4, L17, C22, and Yl77. _E_:__ca__m21e 10 Co_mEtition among Mouse Anti-Human XCRl Antibodies (2H6, 5G7, and 11H2), Recognizing Similar EgitoEs, for Binding to Human XCRl— WO 32032 PCT/JPZOlZ/072667 Egpressing Cells To determine whether anti—human XCRl antibodies, recognizing similar epitopes, compete with each other for binding to human XCRl, a competition assay was performed.

The competition assay was med as per the following procedure. Parent B300.19 .cells and Innmnm XCRl—EGFP—expressing B300.19 cells were mixed at a 1:1 ratio and suspended in a FACS buffer (PBS' (Sigma) containing 1% fetal bovine serum). The cells were blocked for 10 minutes on ice with the FACS buffer ning 10% rat serum. The cells were then incubated with mouse anti-human XCRl antibodies (2H6, 5G7,L or 11H2), mouse isotype control antibodies, IgGZa cience, #1685) or IgGZb (eBioscience, #16~4732—85), at various concentrations from 0 to 10 ug/mL in the FACS buffer for 20 minutes on ice. The cells were then incubated with biotinylated mouse anti-human XCRl antibody (5G7) at a concentration of 0.3 ng/mL in the FACS buffer for 20 minutes on ice. The cells were washed with the FACS buffer three times, and then ted for 20 nﬁnutes on ice with PE— labeled streptavidin (BD Pharmingen, #554061, diluted with the FACS buffer at a dilution factor of 1:50). The cells were washed three times with the FACS buffer, and then.suspended in the FACS buffer. Fluorescence intensity was measured using a FACSCanto II cell analyzer (BD Bioscience).

Binding of biotinylated mouse anti-human XCRl antibody (5G7) to human XCRl-EGFP-expressing B300.19 cells was competed with unlabeled 5G7 , unlabeled 2H6 and 11H2; recognizing similar epitopes on human XCRl (Fig. 21). On the other hand, control 'antibodies did not e with biotinylated antibody .(5G7).for binding to human XCRl—EGFP-expressing B300.19 cells.

Eﬁgmple 11 Reactivity of Mbuse Anti-Human XCRl Monoclonal Antibody, 5G7, and Humanized Anti-Human XCRl Monoclonal Antibodies, HK1L2 and HKSLS, to Various Human ine Receptors The reactivity of mouse anti-human XCRl onal antibody, 5G7 and humanized anti—human XCRl monoclonal dies, HK1L2 and HK5L5 to various human chemokine receptors were evaluated by FACS analysis.

Parent 3300.19 cells and human chemokine receptor—EGFP— expressing 3300.19 cells (XCRl, CXCRl, CXCR3, CXCR4, CXCRS, CXCR6, CCRl, CCRZB, CCR3, CCR4, CCRS, CCR6, CCR7, CCR8, CCR9, CCRll, or CX3CR1) were suspended in a FACS buffer (PBS' (Sigma) containing 1% fetal bovine serum) at a concentration of 1 x 106 cells/mL and ts of 100 pl were dispensed into wells of a 96 well round bottom plate. The cells were then centrifuged, and supernatants were discarded. Mouse anti—human XCRl mAb, 5G7, mouse isotype control antibody IgGZb (eBioscience, #14—4732—82), humanized anti—human XCRl monoclonal antibodies, HK1L2 and HKSLS, and control human IgG bishi, #128-26053—9) were diluted with the FACS buffer at a tration of 5 ug/mL. PE-labeled goat anti-human XCRl polyclonal antibodies (R&D, #FABBS7P, and LifeSpan BioScience, #LS—C76885) were diluted with the FACS buffer at the dilution factors of 2:5 and 1:5, respectively.

Fifty uL of the diluted antibodies were added to each well, and the cells were incubated for 20 minutes on ice. The cells were then washed three times with the FACS buffer. PEslabeled anti— mouse IgG polyclonal antibody (Jackson, #715151, diluted with the FACS buffer at a dilution factor of 1:50) was added to the cells that had been incubated with 5G7 or mouse isotype control antibody. PE-labeled anti-human IgG polyclonal dy (Jackson, 16—149, diluted with the FACS buffer at a dilution factor of 1:50) was added to the cells that had been incubated with HKlLZ, HK5L5 or human control IgG. The FACS buffer was added to the cells that had been ted with anti—hXCRl polyclonal antibodies. The cells were then incubated for 20 minutes on ice. The cells were washed with the FACS.buffer three times, and then suspended in the FACS . Fluorescence intensity was measured using a FACSCanto II cell analyzer, and expressed as a delta PE mean value. The delta PE mean value was calculated by subtracting background PE mean value from each PE mean value, which was obtained by staining each cell line with each antibody; MouSe anti—human XCRl antibody, 5G7 selectively reacted to human XCRl-EGFP—expressing 3300.19 cells except for human CX3CR1—EGFP—expressing cells (Fig. 22). On the other hand, goat anti-human XCRl onal antibodies reacted to various human chemokine or-EGFP—expressing cells in addition to human XCRl-EGFP-expressing cells (Fig. 22). Humanized anti-human XCRl antibodies, HK1L2 and HKSLS showed reduced reactivity to human CX3CR1-EGFP-expressing cells in spite of their high reactivity to human XCRl-EGFP-expressing cells (Fig. 23).

Exgple 12 Effect of 567 Mab on oterium butyEicum-induced DTH response It is known that a delayed-type hypersensitivity(DTH) response is one of the main mechanisms causing autoimmune diseases such as ditis, rheumatoid arthritis and type 1. diabetes when this response is directed against ntigens (Actor, J.K. and Ampel, N.M. (December 2009) Hypersensitivity: T Lymphocyte— mediated (Type. IV). In: Encyclopedia of Life es (ELS) .

John Wiley & Sons, Ltd: Chichester). T cell—Dendritic Cell interaction is critical for DTH responses. Thus the inhibition of T cell-DC interaction is believed to be useful to treat those diseases. We investigated the effect of the anti—human XCRl, 567 Mab, on a model of DTH reaction, Mycobacterium (M.) butyricum~ induced DTH response, in human XCRl knocked—in mice (Mihara, M. et a1, Immunology Letters 2002, 84: 223-229; Mohan K et al, Eur.

J. Immunol. 2005, 35: 1702—1711) .

(Methods) The engineered knoked-in mice. in which human XCRl is expressed instead of amuse XCRl, were immunized subcutaneously with heat—killed M. cum (100 ug/head) with mineral oil on PCT/JPZOlZ/072667 day 0. A 567 Mab or a control mouse 196 (Jackson Laboratory), were intraperitoneally injected at the dose of 500 pg/head on day 1, day 3, day 7 and day 9. 10 days after the immunization with M. butyricum, the mice were challenged with M. cum ded in mineral oil on the right footpad (20 ug/foot, M. butyricum challenge), and mineral oil on alone left footpad (control nge). One day after the challenge injection, the DTH response was ted by measuring the d thickness of each footpad. The footpad ng was calculated according to the following a.

Footpad swelling = ([A]-[B])-.([C}-[D]) [A] = thickness of right footpad after M. butyricum challenge [B] = thickness of right footpad before M. butyricum challenge [C] = thickness of left footpad after control challenge [D] = ess of left d before control challenge (Results) The result showed 'that the M. butyricum—induced DTH response in mice treated with 5G? Mab showed significantly lower DTH response compared to the mice treated with the control IgG (Fig. 24) .

( Conclusion) The data showed the efficacy of anti-XCRl antibody treatment in the DTH response. It is suggested that the use of anti—XCRl antibodies may be beneficial in the treatment of DTH—driven autoimmune diseases such as thyroiditis, rheumatoidarthritis and type 1 diabetes .

Exgnple 13 Effect of 567 Mab on M06 37-50 Eptide mediated EAR Multiple sclerosis (MS) is a chronic demyelinating disease of the human central nervous system (CNS) which can be characterized clinically by a remitting—relapsing or a chronic progressive course. The most intensively studied animal model of MS, mental autoimmune encephalomyelitis (EAE) , classically leads to t in motor functions. Many s showed that T cells play l roles in the pathogenesis of ~ MS and EAE.

Therefore, we performed an EAE model experiment to investigate the inhibitory activity of 5G7 Mab on the pathogenesis of MS.

(Experimental Method) 1. Sample Mice Human XCRl in mice (7-12 weeks old), in which human XCRl is expressed instead of mouse XCRl on C'57BL/6 background, were used for the experiment. 2. Induction of EAE The induction of EAE was performed according to the method' reported in the journal Eur. J. Immunol. 2005, 35: 76-85, in which the probable role of CD8+ T cells was indicated in the EAE development. Briefly, the human XCRl knock—in mice were injected subcutaneously, with 200 - pg of myelin oligodendrocyte glycoprotein 37—50 e (MOG 37—50) emulsified in Freund’s complete adjuvant (CFA) containing 20 mg/ml of Mycobacterium tuberculosis H37Ra. 200 ng of pertussis toxin was stered intravenously on days 0 and 2, post-immunization. 3. Method for Administering Antibodies Anti—human XCRl mouse monoclonal antibody (5G7) and its control antibody, i.e. , mouse IgG (Jackson Laboratory), were prepared in PBS to a final concentration of 2 mg/mL. Each of the above antibodies was intravenously administered into the mice with the volume of 250 se (500 pg/mouse) on day 7, day 10, day 14 and day 17. 4. Scoring of the pathology of this model Clinical symptom of EAE was monitored from the day of the immunization, and was scored on a scale of 0—5, based on the following criteria: grade 0: no disease, grade 0.5: ndld—tail paralysis, grade 1: tail paralysis, grade 2: uneven gait, grade 2.5: one paralyzed rear leg, grade 3: rear limb paralysis, grade 4: paralyzed front and rear legs: and grade 5: nd or death.

(Experimental result and conclusion) The obtained clinical scores of the mice administered with 5G7 Mab showed lower levels than those in the mice administered with the control 196 (Fig. 25). The data indicated that the ent with anti—XCRl antibody showed a certain level cf suppression in the EAE development, and suggested that the treatment with anti- XCRl antibodies may be beneficial for MS in human.

‘Exgmple 14 Inhibition of Human xcnl Binding to Human XCRI-Egpressing Cells with Mbuse Anti-Human XCRI Antibodies (2H6, 567, and 11H2) To determine whether mouse uman XCRl antibodies (2H6, 5G7, and 11H2) inhibit human XCLl binding to human XCRl, a competitive ligand binding assay was performed.

First, the binding of human SS—His(10) to human XCRl-EGFP- expressing BaF3 cells were determined according to the following procedure cells . Parent BaF3 cells and human XCRl-EGFP-expressing BaF3 were mixed at a 1:1 ratio, and suspended in a FACS buffer (1% FBS- containing PBS‘ (Sigma)). The cells were incubated for 30 minutes on ice with an increasing concentration of human XCLlesSS—His(10) in the ce or absence of 2.5 uM soluble XCLl (R&D. #695-LT—025/CF) in the FACS buffer. Next, the cells were washed with the FACS buffer three times, and then incubated for 20 s on ice with x His tag antibody (BETHYL, #A190-114A, diluted at 1:100 in the FACS buffer). The cells were again washed with the FACS buffer three times, and then incubated for 20 minutes on ice with PE-labeled anti-rabbit IgG antibody (Jackson, #711—166~152, diluted at 1:50 in FACS buffer). Next, the cells were once again washed three times with the FACS buffer, and then suspended in the FACS buffer. The scence intensity was measured using a FACSCanto II cell analyzer (BD Bioscience). Specific binding was determined by subtracting the non-specific g (in the presence of 2.5 uM soluble XCLl) from the total g (in the absence of 2.5 uM soluble XCLl).

The competitive ligand binding assay waS'performed according to the following procedure. Parent BaF3 cells and human XERl—EGFP— expressing BaF3 cells were mixed at a 1:1 ratio, and suspended in a FACS buffer (1% FBS~containing PBS" (Sigma)). The cells were blocked for 10 minutes on ice with a FACS buffer ning 10% rat serum. The cells were then incubated for 20 ndnutes on ice with House anti-human XCRI antibodies (2H6, 567, or 11H2), mouse e control antibodies, IgGZa (eBioscience, #16—4724485), or IgGZb (eBioscience, #1685) at various concentrations from 0 to 150 ug/mL. Next the cells were incubated for 30 minutes on ice with human XCLl-SSS-His(10) at a saturating concentration of 0.12 ug/mL. The cells were washed with the FACS buffer-three times, and then incubated for 20 minutes on ice with x His tag antibody (BETHYL, #A190—114A, diluted at 1:100 in FACS buffer). The cells were again_washed with the FACS buffer three times, and then incubated for 20 minutes on ice with PE-labeled anti—rabbit IgG antibody on, #71l152, diluted at 1:50 in FACS buffer). Next the cells were once again washed three times with the FACS buffer, and then suspended in FACS . The fluorescence intensity was measured using a FACSCanto II cell analyzer (BD ence).

Human XCLl binding to human XCRl-EGFP—expressing BaF3 cells was' inhibited with mouse anti-human XCRl antibodies (2H6, 567, and 11H2), and the ICE of the antibodies was ; 37.0, 6.9, and 23.8 nM, respectively.

On the other hand, control antibodies did not inhibit human XCLl binding to human XCR-EGFP-expressing BaF3 cells.

Claims

[Claim 1] An antibody binding to human XCRl, wherein the antibody the antibody comprising a heavy chain variable region comprising heavy chain CDRs l to 3 described in (g) to (i) below and a light chain variable region comprising light chain CDRs l to 3 bed in (j) to (1) below; the antibody comprising a heavy chain le region comprising heavy chain CDRs l to 3 described in (m) to (0) below and a light chain variable region comprising light chain CDRs l to 3 described in (p) to (r) below; or the antibody comprising a heavy chain variable region comprising heavy chain CDRs l to 3 described in (a) to (c) below and a light chain variable region comprising light chain CDRs l to 3 described in (d) to (f) below: (a) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 41, (b) a heavy chain CDR 2 ting of the amino acid sequence of SEQ ID NO: 42, (c) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 43; (d) a light chain CDR 1 consisting of the amino acid ce of SEQ ID NO: 44, (e) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 45, and (f) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 46; (g) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 17, (h) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 18, (i) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 19; (j) a light chain CDR 1 ting of the amino acid ce of SEQ ID NO: 20, (k) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 21, (l) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 22; (m) a heavy chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 29, (n) a heavy chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 30, (o) a heavy chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 31; (p) a light chain CDR 1 consisting of the amino acid sequence of SEQ ID NO: 32, (q) a light chain CDR 2 consisting of the amino acid sequence of SEQ ID NO: 33, and (r) a light chain CDR 3 consisting of the amino acid sequence of SEQ ID NO: 34.
[Claim 2] The antibody according to Claim 1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60 or 64, and a light chain variable region comprising an amino acid ce of SEQ ID NO: 68 or 72.
[Claim 3] The antibody according to Claim 1 or 2, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 60, and a light chain variable region sing an amino acid sequence of SEQ ID NO: 68.
[Claim 4] The antibody according to Claim 1 or 2, wherein the antibody comprises a heavy chain variable region sing an amino acid ce of SEQ ID NO: 64, and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 72.
[Claim 5] The antibody according to any one of Claims 1 to 4, n the antibody comprises a human constant region.
[Claim 6] The antibody according to any one of Claims 1 to 5, wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 59, and a light Chain comprising an amino acid sequence of SEQ ID NO: 67.
[Claim 7] The antibody according to any one of Claims 1 to 5, wherein the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 63, and a light chain comprising an amino acid sequence of SEQ ID NO: 71.
[Claim 8] The antibody according to any one of Claims 1 to 7 comprising an Fc region, wherein the Fc region is mutated to induce a change in ADCC activity.
[Claim 9] The antibody ing to Claim 8, wherein the Fc region is mutated to lower ADCC activity.
[Claim 10] The antibody according to any one of Claims 1 to 9, wherein the antibody is ated to a cytotoxic molecule.
[Claim 11] The antibody according to any one of Claims 1 to 10, wherein the antibody inhibits interaction between human XCRl and human XCLl.
[Claim 12] The antibody according to any one of Claims 1 to 11, wherein the antibody inhibits cell ion of dendritic cells.
[Claim 13] The antibody according to any one of Claims 1 to 12, wherein the antibody sses the activity of cytotoxic T lymphocytes.
[Claim 14] A pharmaceutical composition comprising the antibody according to any one of Claims 1 to 13 and a pharmaceutically acceptable r or additive.
[Claim 15] The pharmaceutical composition according to Claim 14, wherein the pharmaceutical composition is a therapeutic agent for an immune disease.
[Claim 16] The pharmaceutical composition according to Claim 15, wherein the immune disease is an immune disease of the skin.
[Claim 17] The pharmaceutical ition according to Claim 16, wherein the immune disease of the skin is psoriasis, parapsoriasis, atopic dermatitis, contact itis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease (pemphigus, pemphigoid, or acquired epidermolysis bullosa), pustulosis, herpes gestationis, linear IgA bullous dermatosis, alopecia areata, go vulgaris, skin disease associated with collagenosis (systemic lupus erythematosus, Sjogren me, or mixed connective tissue disease), skin disease associated with Addison's disease, skin disease associated with graft—versus—host disease (GVHD), eczema, or urticaria.
[Claim 18] The pharmaceutical composition according to Claim 16, wherein the immune disease of the skin is psoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, or ion body myositis.
[Claim 19] The pharmaceutical composition ing to Claim 16, wherein the immune disease of the skin is atopic dermatitis or contact dermatitis.
[Claim 20] The pharmaceutical composition according to Claim 15, wherein the immune disease is thyroiditis, rheumatoid arthritis, type 1 diabetes, or multiple sclerosis.
[Claim 21] A nucleic acid comprising a nucleotide sequence encoding the dy according to any one of Claims 1 to 13.
[Claim 22] Use of an antibody according to any one of Claims 1 to 14 or a pharmaceutical composition according to Claim 15, in the manufacture of a ment for treating an immune disease.
[Claim 23] The use according to Claim 22, wherein the immune disease is an immune disease of the skin.
[Claim 24] The use according to Claim 23, wherein the immune disease of the skin is psoriasis, parapsoriasis, atopic dermatitis, contact dermatitis, dermatomyositis, polymyositis, inclusion body myositis, autoimmune blistering disease (pemphigus, pemphigoid, or acquired epidermolysis bullosa), pustulosis, herpes gestationis, linear IgA bullous dermatosis, ia areata, vitiligo vulgaris, skin e associated with collagenosis (systemic lupus erythematosus, n syndrome, or mixed connective tissue disease), skin disease associated with Addison's disease, skin disease associated with versus—host disease (GVHD), eczema, or urticaria.
[Claim 25] The use ing to Claim 23, wherein the immune disease of the skin is psoriasis, atopic dermatitis, contact itis, dermatomyositis, polymyositis, or inclusion body myositis.
[Claim 26] The use ing to Claim 22, wherein the immune disease is thyroiditis, toid arthritis, type 1 diabetes, or multiple sclerosis.
[Claim 27] The antibody according to claim 1, substantially as herein described with reference to any one of the Examples and/or