CN110300763A

CN110300763A - For treating the anti-human CXCR3 antibody of leucoderma

Info

Publication number: CN110300763A
Application number: CN201780086634.5A
Authority: CN
Inventors: W·H·布龙迪克; R·楚; T·D·康纳斯; S·帕克; H·邱; M·约德; J·哈里斯; J·里奇满
Original assignee: Sanofi SA; University of Massachusetts UMass
Current assignee: Sanofi SA; University of Massachusetts UMass
Priority date: 2016-12-22
Filing date: 2017-12-21
Publication date: 2019-10-01
Also published as: EP3559036A1; JP2020514273A; KR20190095942A

Abstract

Provide the method and purposes of anti-CXCR3 Antybody therapy CXCR3 associated disorders such as leucoderma.In certain embodiments, the anti-CXCR3 antibody is Humanized anti-human CXCR3 antibody, has the effector function of enhancing for the cell for expressing CXCR3 on the surface.

Description

For treating the anti-human CXCR3 antibody of leucoderma

Cross reference to related applications

The U.S. Provisional Patent Application No. 62/437,889 and 2017 year 12 submitted this application claims on December 22nd, 2016 The equity for the European Patent Application No. 17306770.3 that the moon is submitted on the 14th, entire contents are herein incorporated by reference.

Background technique

The present invention relates to antibody and use the side of Antybody therapy obstacle (such as leucoderma) relevant to CXCR3 signal transduction Method.

Leucoderma is a kind of disfiguring autoimmune skin disease, wherein autoreactivity CD8⁺T cell is killed in epidermis Melanocyte, lead to patch shape pigmentation.Although it is one of most common autoimmune disease, the whole world 1% is influenced Population, but there is presently no the therapy of vitiligo methods of FDA approval.

C-X-C motif CC chemokine receptor 3 (CXCR3) be it is a kind of mainly the T cell (memory T cell) of experience antigen, The chemokine receptors expressed on effector T cell and activating T cell participates in raising these T cell subgroups to tissue inflammation portion Position is with response in its major ligand: CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I-TAC).CXCR3 and CXCL10 exist (Uno etc., Endocr J 57:991-96 (2010) is expressed in people T1D patient；Roep etc., Clin Exp Immunol 159: 338-43(2003)；Tanaka etc., Diabetes 58:2285-2291 (2009)).In these patients, CXCL10 is in pancreas islet In it is remaining generate insulin β cell in express.CXCR3 is expressed in peri-islet invasion T cell.Similar expression mould Formula reappears (Morimoto etc., J Immunol in non-obese diabetes (NOD) mouse (mouse models of type 1 diabetes) 173:7017-24(2004)；Li et al., (2005) World J Gastroenterol 11 (30): 4750-2；Sarkar etc., Diabetes 61(2):436-46(2012))。

CXCR3 is by skin CD3⁺Lymphocyte and plasmacytoid dendritic cells expression, and its chemokine ligand CXCL10 and CXCL9 raises ((2001) Rottman etc., Lab Invest 81 (3): 335-47 in psoriatic lesions；Chen Deng (2010) Arch Dermatol Res 302 (2): 113-23).CXCR3 is also present in the certain form of inflammatory tissue It is expressed on infiltrating T cells, and CXCL9, CXCL10 and CXCL11 are usually generated by the local cells in inflammatory lesion.

The up-regulation of CXCR3 is related to a series of autoimmune disorders.CXCR3 expression is substantially absent in Naive T cells, It is raised after with antigenic activation.These cells (including 1 type of t helper cell (Th1)) are raised the position for arriving tissue inflammation by CXCR3 With response in its major ligand.β cell in Langerhans pancreas islet expresses CXCL9 and CXCL10 (Frigerio etc., Nat Med 8:1414-20 (2002)), and infiltrated pancreas T cell expression CXCR3 (Christen etc., J Immunol 171: 6838-45(2003)；Van Halteren etc., Diabetologia 48:75-82 (2005)；Uno etc., Endocr J 57: 991-96(2010)；Roep etc., Clin Exp Immunol 159:338-43 (2003)；Tanaka etc., Diabetes 58: 2285-2291(2009)；(2012) Sarkar etc., Diabetes 61 (2): 436-46).The U.S. Patent number 8 of Youd etc., 865,870 describe anti-CXCR3 antibody.

In view of leucoderma and other be related to the generally existing of the obstacle of CXCR3, need to target CXCR3 signal transduction for example with To treat the obstacle (such as leucoderma) of patient or reduce the other methods of its progress.

Summary of the invention

There is provided herein by giving specific binding CXCR3 humanized antibody or its antigen-binding fragment treat The method of CXCR3 related disease and obstacle (such as leucoderma).In certain embodiments, it is provided for methods herein and purposes Anti- CXCR3 antibody has the ability for instructing CXCR3 expression cell depletion, or can be engineered and have the guidance of enhancing The ability of CXCR3 expression cell depletion is to treat CXCR3 related disease and obstacle (such as leucoderma).

In some embodiments, the humanized antibody or its antigen-binding fragment tool provided for methods herein and purposes There is at least 0.885 (when comparing all residues of the VH and VL chain in addition to the area the D residue of VH) or at least 0.950 (when only comparing When such as by the residue of the IMTG framework region determined) hair tonic ability score.In some embodiments, humanization provided herein Antibody or its antigen-binding fragment have at least 1x 10^-9The KD of M.In some embodiments, humanized antibody provided herein Or its antigen-binding fragment has less than 7x 10^-5The kd of 1/Ms.In some embodiments, humanized antibody provided herein Or its antigen-binding fragment at least 0.885 hair tonic ability score (when comparing VH the and VL chain in addition to the area the D residue of VH When all residues) or at least 0.950 (when only comparing the residue such as by the IMTG framework region determined) hair tonic ability score and At least 1x 10^-9The KD of M, and at least 0.885 (when comparing all residues of the VH and VL chain in addition to the area the D residue of VH) or The hair tonic ability score of at least 0.950 (when only comparing the residue such as by the IMTG framework region determined).In some embodiments In, humanized antibody provided herein or its antigen-binding fragment have at least 0.885 (when comparing in addition to the area the D residue of VH When all residues of VH and VL chain) or at least 0.950 (when only comparing the residue such as by the IMTG framework region determined) hair tonic Ability score, at least 1x 10^-9The KD of M, and it is less than 7x 10^-5The kd of 1/Ms.

In certain embodiments, the source of people of the specific light chain variable region comprising matching with specific heavy chain variable region is provided Change CXCR3 antibody.In certain embodiments, humanization CXCR3 antibody provided herein includes the area variant human IgG1 Fc, Assign the effector function of the enhancing for the cell for expressing people CXCR3 on the surface.

In the first aspect, there is provided herein used in the method for the CXCR3 expression cell in depletion subject Humanized anti-human CXCR3 antibody or its pharmaceutical composition, wherein the Humanized anti-human CXCR3 antibody includes to have weight chain variable The heavy chain (HC) in area (VH) and the light chain (LC) with light chain variable region (VL).In some embodiments, CD4+T cell is consumed Subtract.In some embodiments, CD8+T cell is by depletion.In some embodiments, CD4+T cell and CD8+T cell are consumed Subtract.In some embodiments, CD4+ memory T cell is by depletion.In some embodiments, CD8+ memory T cell is by depletion. In some embodiments, CD4+ memory T cell and CD8+ memory T cell are by depletion.In one embodiment, subject suffers from The autoimmune disease for thering is T cell to mediate.In another embodiment, subject suffers from leucoderma.

In an embodiment in the first aspect, VH the and VL packet of Humanized anti-human CXCR3 antibody provided herein Amino acid sequence containing sequence pair shown in table 1, and HC also includes the area human IgG1 Fc, and the area the human IgG1 Fc includes The amino acid sequence of any of SEQ ID NO:2,3,4,5,6,7,8,9,10 or 11.

Table 1

In another embodiment of first aspect, the VH of Humanized anti-human CXCR3 antibody provided herein includes SEQ The amino acid sequence of ID NO:20, and VL includes the amino acid sequence of SEQ ID NO:24.In some embodiments, described The humanization area human IgG1 Fc includes SEQ ID NO:2's or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 Amino acid sequence.

In another embodiment of first aspect, the VH of Humanized anti-human CXCR3 antibody provided herein includes SEQ The amino acid sequence of ID NO:18, and VL includes the amino acid sequence of SEQ ID NO:22.In some embodiments, described Antibody includes the area human IgG1 Fc.In some embodiments, the area the human IgG1 Fc includes SEQ ID NO:2 or SEQ ID The amino acid sequence of NO:9 or SEQ ID NO:10 or SEQ ID NO:11.

In other embodiments of first aspect, the HC and LC of Humanized anti-human CXCR3 antibody provided herein include The amino acid sequence of SEQ ID NO couple shown in table 2.

Table 2

In another embodiment in the first aspect, the HC of Humanized anti-human CXCR3 antibody provided herein includes The area human IgG1 Fc with reduced fucose content.In some embodiments, the Humanized anti-human CXCR3 antibody provided It is to be generated in following host cell, the host cell is cultivated in the culture medium containing glycosylation inhibitor.In a reality It applies in scheme, the glycosylation inhibitor is base husband alkali (kifunensine).

In the second aspect, there is provided herein for using in the method for the autoimmune disease that treatment T cell mediates Humanized anti-human CXCR3 antibody or its pharmaceutical composition, wherein the Humanized anti-human CXCR3 antibody include have heavy chain can Become the heavy chain (HC) of area (VH) and the light chain (LC) with light chain variable region (VL).In one embodiment, the T cell is situated between The disease led is leucoderma.

In the third aspect, the method for the autoimmune disease mediated there is provided herein treatment T cell comprising will include The Humanized anti-human CXCR3 of heavy chain (HC) with heavy chain variable region (VH) and the light chain (LC) with light chain variable region (VL) is anti- Body is given to subject in need thereof.In some embodiments, the autoimmune disease that the T cell mediates is leucoderma Wind.

In the fourth aspect, the method for the autoimmune disease mediated there is provided herein treatment T cell comprising providing will Humanized anti-human comprising the heavy chain (HC) with heavy chain variable region (VH) and the light chain (LC) with light chain variable region (VL) CXCR3 antibody is given to the specification of subject in need thereof.In some embodiments, the T cell mediate from Body immunological diseases are leucoderma.

The 5th aspect, there is provided herein for treat T cell mediation autoimmune disease kit, it includes Humanized anti-human containing the heavy chain (HC) with heavy chain variable region (VH) and the light chain (LC) with light chain variable region (VL) CXCR3 antibody, and the Humanized anti-human CXCR3 is given to the specification of human experimenter in need thereof.

In some embodiments of first, second, third, fourth and fifth aspect provided herein, humanization is anti- The VH and VL of people's CXCR3 antibody include the amino acid sequence of sequence pair shown in table 1, and HC includes the area human IgG1 Fc, institute State the amino acid sequence that the area human IgG1 Fc includes SEQ ID NO:2,3,4,5,6,7,8,9,10 or 11.In another embodiment party In case, VH includes the amino acid sequence of SEQ ID NO:20, and VL includes the amino acid sequence of SEQ ID NO:24.Another In a embodiment, VH includes the amino acid sequence of SEQ ID NO:20 and VL includes the amino acid sequence of SEQ ID NO:24 Column, and the area human IgG1 Fc includes SEQ ID NO:2 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 Amino acid sequence.

In other embodiments in terms of second, third, fourth, fifth provided herein, the 6th and the 7th, VH packet The amino acid sequence of the NO:18 of ID containing SEQ, and VL includes the amino acid sequence of SEQ ID NO:22.In some embodiments In, the area humanization human IgG1 Fc includes SEQ ID NO:2 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID The amino acid sequence of NO:11.

In other embodiments of first, second, third, fourth and fifth aspect provided herein, the antibody HC and LC include sequence pair shown in table 2 amino acid sequence.

In other embodiments of first, second, third, fourth and fifth aspect provided herein, provided herein is Humanized anti-human CXCR3 antibody HC include with reduced fucose content the area human IgG1 Fc.In some embodiments In, the Humanized anti-human CXCR3 antibody provided is generated in following host cell, and the host cell is containing glycosylation suppression It is cultivated in the culture medium of preparation.In some embodiments, the glycosylation inhibitor is base husband's alkali.

Detailed description of the invention

Fig. 1 is a series of six figures, it is shown that after following Antybody therapy in blood each T cell subgroup amount: " hamster CXCR3-173 " (Hamster anti-mouse CXCR3), " CXCR3 mIgG2a Dab " (are engineered to replace mouse IgG 2a with mutation Constant region removes the hamster CXCR3-173 of effector function), " CXCR3 mIgG2a WT " (be engineered to replace mouse wild The hamster CXCR3-173 of raw type IgG2a constant region), " CXCR3 mIgG1-agly " (be engineered to be mutated and be replaced with N297G The hamster CXCR3-173 of 1 constant region of mouse IgG) and " not treating " (representing untreated control).

Fig. 2A is a series of six figures, is depicted through the measurement of surface plasma body resonant vibration (Biacore) binding assay Combination of the recombined small-mouse Fc γ RI (mFcRI) to the various Fc engineered forms of CXCR3-173.BMP5, anti-BMP5 mIgG1 are same Kind type control；MIgG1 agly is mutated the CXCR3-173 for replacing 1 constant region of mouse IgG with N297G through being engineered；mIgG2a WT replaces the CXCR3-173 of mouse wild-type IgG2a constant region through being engineered；MIgG2a Dab takes through engineering mutation The CXCR3-173 of effector function is removed for mouse IgG 2a constant region；MIgG3 replaces mouse wild-type through being engineered The CXCR3-173 of IgG3 constant region；Hamster CXCR3, parent hamster mAb CXCR3-173.

Fig. 2 B is a series of six figures, depicts the recombined small-mouse Fc γ RIIb measured by Biacore binding assay (mFcRIIb) to the combination of the various Fc engineered forms of CXCR3-173.The Antibody Designation is identical as Fig. 2A.

Fig. 2 C is a series of six figures, depicts the recombined small-mouse Fc γ RIII measured by Biacore binding assay (mFcRIII) to the combination of the various Fc engineered forms of CXCR3-173.The Antibody Designation is identical as Fig. 2A.

Fig. 2 D is a series of six figures, depicts the recombined small-mouse Fc γ RIV measured by Biacore binding assay (mFcRIV) to the combination of the various Fc engineered forms of CXCR3-173.Various Antibody Designations are identical as Fig. 2A.

Fig. 3 A is a table, summarizes structure-effector of the anti-human CXCR3 antibody with engineering human IgG1's constant region Functional character.

Fig. 3 B is bar chart, depicts and compares target with various anti-human CXCR3 antibody with the effector for indicating concentration and 5:1 (E:T) cracking that the external antibody-dependent cytotoxicity (ADCC) of the CHO- people's CXCR3 target cell carried out mediates.Effect is thin Born of the same parents are natural kill (NK) cells from single donor.IgG, human IgG1's isotype controls.Test anti-human CXCR3 mAb be 4 (CXCR3 CL4) are cloned, 12 (CXCR3 CL12) are cloned, clone 82 (CXCR3 CL82), clone 135 (CXCR3 CL135), 53Hu37, and engineering Fc variant M1, M2 and M3 of the 53Hu37 as described in Fig. 3 A.Kif, base husband's alkali process.ALEM, A Lun Monoclonal antibody (alemtuzumab).

Fig. 3 C is bar chart, is depicted with various antibody to indicate what the effector of concentration and 3:1 was carried out than target (E:T) The cracking that the external ADCC of CHO- people's CXCR3 target cell is mediated.Effector cell comes from NK like cell system (NK92-CD16V). IgG, human IgG1's isotype controls.The anti-human CXCR3 mAb of test is the Fc variant of 53Hu37 and engineering as shown in Figure 3A M1.CXCR3 CL4, anti-human CXCR3 mAb clone 4.Kif, base husband's alkali process.ALEM is alemtuzumab.

Fig. 4 is a table, summarizes display to people Fc γ RIIa (rhFc γ RIIa), people Fc γ RIII-F158 (rhFc γ RIII-F158), people Fc γ RIII-V158 (rhFc γ RIII-V158) and mouse Fc γ RIV (rmFc γ RIV), which has, combines parent With the 53Hu37 of power KD and the Biacore data of instruction variant.M1-M3 as described in Fig. 3 A, and kif i.e. go it is fucosylated 53Hu37。

Fig. 5 A is a series of six figures, is depicted with the food crab for the instruction Antybody therapy given by the dosage of 2mg/kg weight The depletion of the T cell subgroup indicated in monkey body.The S239D/I332E variant of M1:53Hu37；Kif: fucosylated 53Hu37 is removed； Veh: vehicle control.CXCR3 antibody group N=8.Carrier group N=6.

Fig. 5 B is a figure, depicts the combination pharmacokinetics number that the concentration of antibody is indicated in assessment machin serum According to the machin is in advance with the time of the instruction Antybody therapy indicatrix for the single dose given by the dosage of 2mg/kg weight. Test anti-human CXCR3 mAb be 53Hu37, base husband's alkali process 53Hu37 (53Hu37 kif) and 53Hu37 M1 variant (53Hu37 M1)。

Fig. 6 is a table, it is shown that SEQ ID No and corresponding sequence.

Specific embodiment

Now with detailed reference to according to certain exemplary implementation schemes of the present disclosure, some embodiments are in the accompanying drawings It illustrates.

There is provided herein the purposes of the humanized antibody or its antigen-binding fragment of method and specific binding people CXCR3. In certain embodiments, anti-CXCR3 antibody provided herein has the ability for instructing CXCR3 expression cell depletion, Huo Zhejing It crosses engineering and has what is enhanced to instruct the ability of CXCR3 expression cell depletion to treat CXCR3 related disease and obstacle.One In a little embodiments, the therapy that leucoderma is treated for targeting CXCR3 is disclosed

Antibody

As used herein, term " antibody " refers to immunoglobulin molecules, and it includes pass through disulfide bond interconnected four Polypeptide chain, two weight (H) chains and two light (L) chain and its polymer (such as IgM).Each heavy chain includes weight chain variable (it is abbreviated as V in area_HOr VH) and heavy chain constant region (C_HOr CH).Heavy chain constant region includes three structural domains, C_H1、C_H2 and C_H3.Heavy chain Fc part include C_H2 and C_H3。

Every light chain includes that light chain variable region (is abbreviated as V_L) and constant region of light chain (C_LOr CL).Constant region of light chain includes one A structural domain (C_L1)。

V_HAnd V_LRegion can be further subdivided into denatured region, referred to as complementary determining region (CDR), be scattered with More conservative region, referred to as framework region (FR).Each V_HAnd V_LIt is made of three CDR and four FR, in the following order from amino End is arranged to carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

As used herein, " antigen-binding fragment " of term antibody include it is any it is naturally occurring, can enzymatic obtain, close At or genetically engineered polypeptide or glycoprotein, molecule of the antigen binding is to form compound.The antigen binding fragment of antibody Any suitable standard technique can be used in section, such as proteolytic digestion or is related to manipulation and expression encoding antibody variable domains It is such as derivative from complete antibody molecule with the recombination engineering technology of the DNA of optional constant domain.Antigen-binding portion thereof Non-limitative example includes: (i) Fab segment；(ii)F(ab')₂Segment；(iii) Fd segment；(iv) Fv segment；(v) scFv (scFv) molecule；(vi) dAb segment；(vii) atom (example of the amino acid residue comprising analog antibody hypervariable region Such as, isolated complementary determining region (CDR)).Other Engineering chemoattractant molecule, such as bispecific antibody, three-specific antibody, four specificity Antibody and miniantibody are also included in the statement of " antigen-binding fragment ".

In certain embodiments, CXCR3 antibody or antigen-binding fragment include at least one antigen-binding domains.? In some embodiments, antibody or segment have polyspecific, include two or more (for example, 2,3,4,5 or more) Antigen-binding domains enable antibody or segment to combine two or more CXCR3 molecules of identical or different epitope, Or it can be with high-affinity combination CXCR3 and other at least one antigens.Antigen-binding portion thereof may include one or more antibody Segment, the antibody fragment retain the ability of molecule of the antigen binding.These segments may include from parental antibody or come from The heavy chain variable region and/or light chain variable region of the variant of parental antibody.

As used herein, term " antigen " refers to the binding site or epitope of antibody or the identification of its antigen-binding fragment.

" epitope " or " antigenic determinant " is a part of antigen molecule, is responsible for and the antigen-binding domains of antibody Specificity interaction.

As used herein, " in conjunction with " about antibody or its antigen-binding fragment refers to that antibody or its antigen-binding fragment are logical The noncovalent interaction crossed between antibody and the antibody combining site of antigen forms one or more non-covalent with isogeneic The ability of key.Antigen can be the antigen of separation, or can associate and exist with another entity, more such as on cell surface Under the background of peptide.

As used herein, term " specific binding " refers to antibody or its antigen-binding fragment and Kd is at least about 1x 10^- ⁶M、1x 10^-7M、1x 10^-8M、1x 10^-9M、1x 10^-10M、1x 10^-11M、1x 10^-12M or the ability of higher antigen binding. In certain embodiments, the term refers to the ability of antibody or its antigen-binding fragment and antigen binding, affinity ratio Its at least 2 times of affinity height to heterogenetic antigen.It will be appreciated, however, that antibody or its antigen-binding fragment can be special Property combine two or more sequences on relevant antigen (such as people and machin CXCR3).Non-specific binding usually has low Affinity and medium to high capacity.If it is necessary, non-specific binding can be reduced substantially by changing conjugation condition It does not influence to specifically bind.These conditions are well known in the art, and can be selected using the technical staff of routine techniques Select suitable condition.The condition is generally according to antibody concentration, the ionic strength of solution, temperature, the binding time of permission and resistance The concentration of disconnected molecule (such as seralbumin and milk casein) defines.

Affinity costant can be determined by the Standard kinetic method of antibody response, such as immunoassays (such as ELISA) Or surface plasma body resonant vibration (SPR).Instrument and method for real-time detection and monitoring association rate are known and are It is commercially available (for example, Biacore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences)。

As used herein, " complementary determining region " or " CDR " refers in each variable region of antibody or antigen-binding fragment One of multiple portions, the multiple part are formed together the antigen binding site of antibody.There are three each Variable domain contains CDR, referred to as CDR1, CDR2 and CDR3.Correspondingly, variable heavy chain domain (VH) includes CDR-H1, CDR-H2 and CDR-H3, and And variable light chain domain (VL) includes CDR-L1, CDR-L2 and CDR-L3.Three CDR are along linear amino acid sequence It is discontinuous, but approached in the polypeptide of folding.CDR is located in the ring of the parallel chain of the β-pleated sheet of connection variable domains.

Term " frame (FR) amino acid residue " as used herein those of refers in Ig chain framework region amino acid.Such as this Term used in text " framework region " or " area FR " include as variable region a part but not be CDR a part amino acid it is residual Base.Therefore, the length of variable framework is between about 100-120 amino acid, but only includes amino acid those of except CDR.

As used herein, term " % is identical " or " percentage is identical " mean two sequences on relatively specific region When, the two sequences have the identical residue specified number in same position.Term " % is similar " or " percentage is similar " have Similar meaning, but other than the quantity of identical amino acid between two sequences, it is also contemplated that amino acid is not identical but to protect The case where keeping property replaces.Can be used known computerized algorithm (such as BLASTP, BLASTN and FASTA program (Altschul, SF etc., J Mol Biol 215:403 (1990)), use such as Pearson etc., Proc Natl Acad Sci USA 85: Default parameters in 2444 (1988) determines percentage identity.For example, National Biotechnology Information Center can be used (NCBI) the BLAST function of database determines identity.

In certain embodiments, antibody provided herein is humanized antibody." humanized antibody " is to combine required resist Former antibody molecule has one or more CDR (for example, mouse antibodies) from non-human species, and has and come from people The framework region of immunoglobulin molecules and/or at least some parts of constant domain.Known people Ig sequence is disclosed in for example ncbi.nlm.nih.gov/entrez-/query.fcgi；atcc.org/phage/hdb.html；sciquest.com； abcam.com；antibodyresource.com/onlinecomp.html；And Kabat etc., Sequences of Proteins of Immunological Interest,U.S.Dept.Health(1983).As known in the art, it imports Human sequence can be used for reducing immunogenicity or reduction, enhancing or modification binding affinity, association rate, dissociation rate, Affinity, specificity, half-life period or any other suitable feature.For antibody humanization art recognized methods with It is described in Publication about Document: Jones etc., Nature 321:522 (1986)；Verhoeyen etc., Science 239:1534 (1988)； Sims etc., J Immunol 151:2296 (1993)；Chothia and Lesk, J Mol Biol 196:901 (1987)；Carter Deng Proc Natl Acad Sci USA 89:4285 (1992)；Presta etc., J Immunol 151:2623 (1993)；Beauty State's patent No. 5,589,205,5,565,332,6,180,370,6,632,927,7,241,877,7,244,615,7,244, 832,7,262,050；And U.S. Patent Publication No. 2004/0236078 (submission on April 30th, 2004), entire contents are logical It crosses and is incorporated herein by reference.

In certain embodiments, certain Framework residues in humanized antibody provided herein are supplied from CDR The corresponding residue of body antibody replaces, such as is replaced by the Framework residues from mouse anti human CXCR3 antibody, with change (for example, changing It is kind) antigen binding.These framework substitutions are identified by the interaction modeling to CDR and Framework residues, with identification pair The important Framework residues of antigen binding, and carried out sequence and compared abnormal Framework residues to identify specific location.? In some embodiments, humanized antibody variants of the present disclosure are prepared using 4D humanization.Referring to WO2009/032661 (it is incorporated herein by reference in their entirety), for example, the method for 4D humanization of [0037]-[0044] section.In brief, 4D humanization may include: that (a) establishes the 3-D model for needing the variable domains of humanization；(b) using the 3- of the structural domain The molecular dynamics simulation of D model identifies the flexible residue in the variable domains；(c) by by the 3-D model Immediate human germline is identified compared with the molecular dynamics track of 49 human germlines in molecular dynamics track；And (d) flexible residue that will not belong to a part of CDR sports its human germline's counterpart (as identified in step (c)).

In some embodiments, provide the humanization CXCR3 antibody of VH and VL sequence comprising being listed in table 1 or its Antigen-binding fragment.

In some embodiments, the humanization of the heavy chain (HC) comprising listing in table 2 and light chain (LC) sequence is provided CXCR3 antibody or its antigen-binding fragment.

Antibody mediated effect subfunction/depletion activity

In certain embodiments, anti-CXCR3 antibody disclosed herein has the energy for instructing CXCR3 expression cell depletion Power, or can have what is enhanced to instruct the ability of CXCR3 expression cell depletion to treat CXCR3 related disease by engineering And obstacle.It can may include CD4 by the CXCR3 expression cell of antibody depletion disclosed herein⁺T cell and/or CD8⁺T cell.It can May include CD4 by the CXCR3 expression cell of antibody depletion disclosed herein⁺Memory T cell and/or CD8⁺Memory T cell.Such as It is used herein, about CXCR3⁺" depletion " of cell (that is, cell that CXCR3 is expressed on cell surface) refers to from cell mass Remove these cells.To referring to including complete or partial depletion for depletion.In addition, depletion can be it is permanent or temporary, And it can have in amplitude and/or position in various degree.Depletion may be cell death as a result, such as Apoptosis or bad Extremely.It can be by before and after being exposed to antibody or antigen-binding fragment provided herein, or the absence and presence of this In the case where antibody or antigen-binding fragment that text provides, using any method known in the art (for example, flow cytometry, Immunohistochemical method etc.) measure CXCR3 in cell mass⁺The quantity of cell, to assess depletion situation.Be exposed to provided herein is Antibody or antigen-binding fragment after, CXCR3⁺Cell can with depletion at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

In certain embodiments, anti-to the corresponding humanization with the area wild type Fc (such as wild type human IgG1 Fc) People's CXCR3 antibody is compared, and Humanized anti-human CXCR3 antibody goes out the effect enhanced to the cells show for expressing people CXCR3 on the surface Answer subfunction.As used herein, " effector function of enhancing " refer under the same conditions, with same antigen specificity and The reference antibody in the area wild type human IgG1 Fc is compared, and antibody is directed to the antibody-dependent cytotoxicity of suitable target cell (ADCC), any one in the phagocytosis (ADCP) of the cytotoxicity (CDC) or antibody dependent cellular mediation of complement-mediated Item or multinomial measurable ability increase.In certain embodiments, the reference antibody includes the area variant people Fc.Certain In embodiment, the effector function is ADCC, ADCP or CDC, or any combination thereof.In certain embodiments, described Effector function is ADCC or CDC or ADCC and CDC.In certain embodiments, the effector function is ADCC.At certain In a little embodiments, the effector function is CDC.In certain embodiments, the effector function is ADCC and CDC. In certain embodiments, the effector function is ADCP.

As used herein, " area variant human IgG1 Fc " refers to compared with wild type human IgG1 Fc, by being engineered or repairing It adorns and the area human IgG1 Fc including one or more amino acid mutations or amino acid modification.It is described wild in certain embodiments The type area human IgG1 Fc includes amino acid sequence SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (EU number 192-446) (SEQ ID NO:2)。

In certain embodiments, the area variant human IgG1 Fc includes at least one of following amino acid substitution: G236A, S239D, S267E, H268F, S324T, I332E (Eu number) or any combination thereof.In certain embodiments, described The variant area human IgG1 Fc includes at least one of following amino acid substitution group: S239D/I332E, G236A/S267E/ H268F/S324T/I332E and S239D/H268F/S324T/I332E (Eu number).In certain embodiments, the variant The area human IgG1 Fc includes amino acid substitution S239D/I332E.In other embodiments, the area variant human IgG1 Fc includes Amino acid substitution G236A/S267E/H268F/S324T/I332E.In still other embodiments, the variant human IgG1 Fc Area includes amino acid substitution S239D/H268F/S324T/I332E.

For example, in certain embodiments, the area variant human IgG1 Fc includes SEQ ID NO:3,4,5,6,7 or 8 Amino acid sequence.

In certain embodiments, the area variant human IgG1 Fc include with any one of SEQ ID NO:3-8 or Multiple at least 90% identical sequences, condition are to maintain specific amino acid substitution in each case.In each embodiment In, the area variant human IgG1 Fc includes identical as any one or more of SEQ ID NO:3-8 at least 90%, at least 91% is identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, extremely Few 97% is identical, at least 98% identical or at least 99% identical sequence, condition are to maintain specific amino in each case Acid replaces.

In certain embodiments, the area variant human IgG1 Fc includes at least one of following amino acid substitution group: S239D/I332E, G236A/S267E/H268F/S324T/I332E and S239D/H268F/S324T/I332E (Eu number).Example Such as, in certain embodiments, the area variant human IgG1 Fc includes the amino acid sequence of SEQ ID NO:9,10 or 11.

In certain embodiments, at least one amino acid substitution is S239D/I332E (Eu number).In other embodiment party In case, at least one amino acid substitution is G236A/S267E/H268F/S324T/I332E (Eu number).In certain embodiments In, at least one amino acid substitution is S239D/H268F/S324T/I332E (Eu number).

In certain embodiments, the variant area human IgG1 Fc of the anti-CXCR3 antibody of humanization provided herein include with The identical sequence of any of SEQ ID NO:9-11 at least 90%, condition are to maintain specific amino in each case Acid replaces and the effector function of enhancing.In each embodiment, the area variant human IgG1 Fc includes and SEQ ID NO: Any one or more of 9-11 at least 90% is identical, at least 91% identical, at least 92% identical, at least 93% identical, extremely Few 94% is identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% is identical Sequence, condition is to maintain specific amino acid substitution in each case.

CDR variant:

Other than foregoing embodiments, there is provided herein the anti-CXCR3 antibody comprising 6 CDR, wherein the VH includes CDR with amino acid sequence below:

(i) SEQ ID NO:12, SEQ ID NO:35 and SEQ ID NO:14；

(ii) SEQ ID NO:12, SEQ ID NO:35 and SEQ ID NO:45；

(iii) SEQ ID NO:12, SEQ ID NO:36 and SEQ ID NO:45；

(iv) SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:14；

(v) SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:45；

(vi) SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:46

(vii) SEQ ID NO:12, SEQ ID NO:38 and SEQ ID NO:14；

(viii) SEQ ID NO:12, SEQ ID NO:38 and SEQ ID NO:45；

(ix) SEQ ID NO:12, SEQ ID NO:39 and SEQ ID NO:14；

(x) SEQ ID NO:12, SEQ ID NO:39 and SEQ ID NO:47；

(xi) SEQ ID NO:12, SEQ ID NO:40 and SEQ ID NO:14；

(xii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:45

(xiii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:46；

(xiv) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:48；

(xv) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:49；

(xvi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:50；

(xvii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:51；

(xviii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:52；

(xix) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:53；

(xx) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:54；

(xxi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:55；

(xxii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:56；

(xxiii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:57；

(xxiv) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:58；

(xxv) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:59；

(xxvi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:60；

(xxvii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:61；

(xxviii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:62；

(xxix) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:63；

(xxx) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:64；

(xxxi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:65；

(xxxii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:66；

(xxviii) SEQ ID NO:34, SEQ ID NO:13 and SEQ ID NO:14；

(xxxiv) SEQ ID NO:12, SEQ ID NO:41 and SEQ ID NO:14；

(xxxv) SEQ ID NO:12, SEQ ID NO:41 and SEQ ID NO:46；

(xxxvi) SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:14；

(xxxvi) SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:45；

(xxxviii) SEQ ID NO:12, SEQ ID NO:43 and SEQ ID NO:14；

(xxxix) SEQ ID NO:12, SEQ ID NO:44 and SEQ ID NO:14；

(xl) SEQ ID NO:12, SEQ ID NO:41 and SEQ ID NO:14；Or

(xli) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:68；With

Wherein the VL includes the CDR with amino acid sequence below:

(i) SEQ ID NO:69, SEQ ID NO:16 and SEQ ID NO:17；

(ii) SEQ ID NO:70, SEQ ID NO:16 and SEQ ID NO:17；

(iii) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:17；

(iv) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:75；

(v) SEQ ID NO:72, SEQ ID NO:16 and SEQ ID NO:17；

(vi) SEQ ID NO:73, SEQ ID NO:16 and SEQ ID NO:17；

(vii) SEQ ID NO:74, SEQ ID NO:16 and SEQ ID NO:17；

(viii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:75；

(ix) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:76；

(x) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:77；

(xi) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:78；Or

(vii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:79.

(i) SEQ ID NO:12, SEQ ID NO:35 and SEQ ID NO:14；

(ii) SEQ ID NO:12, SEQ ID NO:35 and SEQ ID NO:45；

(iii) SEQ ID NO:12, SEQ ID NO:36 and SEQ ID NO:45；

(iv) SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:14；

(v) SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:45；

(vi) SEQ ID NO:12, SEQ ID NO:37 and SEQ ID NO:46；

(vii) SEQ ID NO:12, SEQ ID NO:38 and SEQ ID NO:14；

(viii) SEQ ID NO:12, SEQ ID NO:38 and SEQ ID NO:45；

(ix) SEQ ID NO:12, SEQ ID NO:39 and SEQ ID NO:14；

(x) SEQ ID NO:12, SEQ ID NO:39 and SEQ ID NO:47；Or

(xi) SEQ ID NO:12, SEQ ID NO:40 and SEQ ID NO:14；And

Wherein the VL includes the CDR with amino acid sequence below:

(i) SEQ ID NO:69, SEQ ID NO:16 and SEQ ID NO:17；

(ii) SEQ ID NO:70, SEQ ID NO:16 and SEQ ID NO:17；

(iii) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:17；

(iv) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:100；

(v) SEQ ID NO:72, SEQ ID NO:16 and SEQ ID NO:17；

(vi) SEQ ID NO:73, SEQ ID NO:16 and SEQ ID NO:17；

(vii) SEQ ID NO:74, SEQ ID NO:16 and SEQ ID NO:17；

(viii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:75；

(ix) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:76；

(x) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:77；

(xi) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:78；Or

(vii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:79.

(i) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:45；

(ii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:46；

(iii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:48；

(iv) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:49；

(v) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:50；

(vi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:51；

(vii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:52；

(viii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:53；

(ix) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:54；

(x) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:55；Or

(xi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:56；With

Wherein the VL includes the CDR with amino acid sequence below:

(i) SEQ ID NO:69, SEQ ID NO:16 and SEQ ID NO:17；

(ii) SEQ ID NO:70, SEQ ID NO:16 and SEQ ID NO:17；

(iii) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:17；

(iv) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:75；

(v) SEQ ID NO:72, SEQ ID NO:16 and SEQ ID NO:17；

(vi) SEQ ID NO:73, SEQ ID NO:16 and SEQ ID NO:17；

(vii) SEQ ID NO:74, SEQ ID NO:16 and SEQ ID NO:17；

(viii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:75；

(ix) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:76；

(x) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:77；

(xi) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:78；Or

(vii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:79.

(i) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:57；

(ii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:58；

(iii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:59；

(iv) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:60；

(v) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:61；

(vi) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:62；

(vii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:63；

(viii) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:64；

(ix) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:65；Or

(x) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:66；With

Wherein the VL includes the CDR with amino acid sequence below:

(i) SEQ ID NO:69, SEQ ID NO:16 and SEQ ID NO:17；

(ii) SEQ ID NO:70, SEQ ID NO:16 and SEQ ID NO:17；

(iii) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:17；

(iv) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:75；

(v) SEQ ID NO:72, SEQ ID NO:16 and SEQ ID NO:17；

(vi) SEQ ID NO:73, SEQ ID NO:16 and SEQ ID NO:17；

(vii) SEQ ID NO:74, SEQ ID NO:16 and SEQ ID NO:17；

(viii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:75；

(ix) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:76；

(x) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:77；

(xi) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:78；Or

(vii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:79.

(i) SEQ ID NO:34, SEQ ID NO:13 and SEQ ID NO:14；

(ii) SEQ ID NO:12, SEQ ID NO:41 and SEQ ID NO:14；

(iii) SEQ ID NO:12, SEQ ID NO:41 and SEQ ID NO:71；

(iv) SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:14；

(v) SEQ ID NO:12, SEQ ID NO:42 and SEQ ID NO:70；

(vi) SEQ ID NO:12, SEQ ID NO:43 and SEQ ID NO:14；

(vii) SEQ ID NO:12, SEQ ID NO:44 and SEQ ID NO:14；

(viii) SEQ ID NO:12, SEQ ID NO:41 and SEQ ID NO:14；Or

(ix) SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:68；With

Wherein the VL includes the CDR with amino acid sequence below:

(i) SEQ ID NO:69, SEQ ID NO:16 and SEQ ID NO:17；

(ii) SEQ ID NO:70, SEQ ID NO:16 and SEQ ID NO:17；

(iii) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:17；

(iv) SEQ ID NO:71, SEQ ID NO:16 and SEQ ID NO:75；

(v) SEQ ID NO:72, SEQ ID NO:16 and SEQ ID NO:17；

(vi) SEQ ID NO:73, SEQ ID NO:16 and SEQ ID NO:17；

(vii) SEQ ID NO:74, SEQ ID NO:16 and SEQ ID NO:17；

(viii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:75；

(ix) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:76；

(x) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:77；

(xi) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:78；Or

(vii) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:79.

Other than foregoing embodiments, there is provided herein the anti-CXCR3 antibody comprising following VH, the VH includes following Amino acid sequence:

SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89 or SEQ ID NO:90.

SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101.

Other than foregoing embodiments, there is provided herein the anti-CXCR3 antibody comprising following VH, the VH includes to be selected from Amino acid sequence below:

SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、

SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、

SEQ ID NO:110 or SEQ ID NO:111.

SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、

SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:119、

SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122.

Other than foregoing embodiments, there is provided herein the anti-CXCR3 antibody comprising following VL, the VL includes following Amino acid sequence:

SEQ ID NO:123、SEQ ID NO:124、SEQ ID NO:125、SEQ ID NO:126、

SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、

SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133 or SEQ ID NO:134.

In certain embodiments, anti-human CXCR3 antibody includes the VH/VL respectively containing amino acid sequence shown in table 3 To (indicated variation is respectively relative to corresponding 53Hu37 VH or VL sequence (SEQ ID NO:20 and 24)):

Table 3

Neutralizing antibody

In certain embodiments, Humanized anti-human CXCR3 antibody provided herein is CXCR3 neutralizing antibody.Certain In exemplary implementation scheme, in addition to the effector function of enhancing, CXCR3 antibody also has neutralization activity.Whenever need reduce or When eliminating effect (such as recruitment of T cell) that CXCR3 is mediated, CXCR3 is neutralized and CXCR3⁺The combined effect of cell depletion can It can be advantageous.

" CXCR3 neutralizing antibody " is in conjunction with CXCR3 and blocks the activity of receptor, is such as produced in conjunction with CXCR3 by CXCR3 ligand Raw characteristic physiological and hereditary response.Neutralization activity can be (100% neutralize) completely or part (for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% (or any percentage therebetween) or more) it neutralizes, And various factors known to technical staff will be depended on, will be lived such as antibody concentration, affinity and epitope and for evaluating to neutralize The particular assay of property.The neutralization activity of CXCR3 neutralizing antibody can be by measure to the combination of such as ligand binding, GTP, calcium It mobilizes, the measurement of the inhibition of cell chemotaxis and/or receptor internalisation is shown.For measuring neutralizing antibody (in especially CXCR3 And antibody) active many measurements be that and verifying specific antibodies can be easily adaptable whether known to technical staff Neutrality.

For example, in some embodiments, the neutralization activity of anti-CXCR3 antibody can be assessed by chemotactic assay, Substantially such as by 49801 production of clone and by the package insert of the R&D Systems antibody (catalog number (Cat.No.): MAB160) sold It is described.Neutralize (the ND of dosage -50₅₀) be defined as under specific rhCXCL11 concentration generating cell surface in response cell line Antibody concentration needed for the half maximum suppression of recombined human CXCL11 (rhCXCL11) response that CXCR3 is mediated.It is anti-in order to measure Body blocks the ability of the chemotaxis of the rhCXCL11 induction of hCXCR3 transfection BaF/3 cell, and the rhCXCL11 of 7ng/mL is added Into the bottom compartment of 96 hole chemotactic rooms (NeuroProbe, Cabin John, Md.).Then using the polycarbonate for being free of PVP Filter (5 μm of apertures) assembles chemotactic room.By the serial dilution (for example, 0.001 to 10000 μ g/mL) and 0.25x of antibody 10⁶Cells/well is added in the top-portion apertures of chemotactic room.At 37 DEG C in 5%CO₂After being incubated 3 hours in the incubator of humidification, dismantle The cell migrated to bottom compartment is transferred to working plate, and is carried out using such as Resazurin Fluorescence by chemotactic room It is quantitative.

Colvin etc., Mol Cell Biol 26:5838-49 (2006) are described and can be used in certain embodiments Other measurements, to determine the neutralization activity for neutralizing anti-CXCR3 antibody.In brief, 300-19 cell, i.e. function can be used Property expression CXCR4 mouse pre B cell Leukemia Cell Lines.After transfection, this cell line can other chemotactic factor (CF)s of functional expression by Body, such as people CXCR3 (see, for example, 201-209 sections of U.S. Patent Application Publication No. 2010/0061983, pass through reference It is incorporated herein).The 300-19 cell for expressing people CXCR3 can be in the complete RPMI culture medium for containing 10% fetal calf serum (FBS) Middle growth.In order to assess the combination of CXCR3 ligand and CXCR3 in candidate and in the presence of R3 antibody, by 400,000 CXCR3/ 300-19 cell is placed in combination buffer (0.5%BSA, the 5mM MgCl that total volume in tissue culturing plates with 96 hole is 150 μ L₂, 1mM CaCl₂, 50mM HEPES, pH 7.4) in.It can be by 0.04nM's in total¹²⁵CXCL10 (the New England of I label Nuclear, Boston, Mass.) or CXCL11 (Amersham Biosciences Piscataway, N.J.) and 5x 10⁶nM Unmarked CXCL10 or CXCL11 (Peprotech, Rocky Hill, N.J.) to 500nM are added in cell and in room temperature Lower incubated under agitation 90 minutes.Cell is transferred to 96 hole filter plates being immersed in 0.3% polyethyleneimine in advance On (Millipore, Billerica, Mass.), and washed three times with the 200 μ L combination buffers for being supplemented with 0.5M NaCl.It will Plate is dry, and Wallac Microbeta scintillation counter (Perkin-Elmer Life Sciences, Boston, Mass. radioactivity is measured after addition scintillation solution in).The combination of CXCL10 and CXCL11 assessment CXCL9 can be similar to.

In certain embodiments, antibody disclosed herein can be prevented or reduced into the calcium in CXCR3 expression cell Flux.In some embodiments, calcium flux can be detected in cell such as CXCR3/300-19 cell.By about 5x 10⁶It is a Cell is suspended in 2mL and contains in the RPMI culture medium of 1% bovine serum albumin(BSA) (BSA).Add the Fura-2 of 15 micrograms (Molecular Probes, Eugene, OR) incubates cell 20 minutes at 37 DEG C.Cell is washed twice in PBS, and It is resuspended in 2mL calcium flux buffer (145mM NaCl, 4mM KCl, 1mM NaHPO₄, 1.8mM CaCl₂, 25mM HEPES, 0.8mM MgCl₂With 22mM glucose) in.In 37 DEG C of measurement DeltaRAM fluorimeter (Photon Technology International, Lawrenceville, N.J.) fluorescence reading.In addition chemotactic factor (CF) (for example, CXCL9, CXCL10 Or CXCL11) before and after, intracellular calcium concentration is recorded as exciting in response to the sequence at 340nm and 380nm in 510nm Locate the excitation fluorescence intensity of transmitting, and is expressed as the relative scale at 340nm with fluorescence at 380nm.

In certain embodiments, it can be neutralized by measuring the reduction of receptor internalisation to evaluate CXCR3.In some implementations It, can be by will about 2.5x 10 in scheme⁵A cell (such as CXCR3/300-19 cell) is placed in the RPMI culture medium containing 1%BSA In incubated 30 minutes together with CXCL10, CXCL11 or CXCL9 of various concentration at 37 DEG C and carry out receptor internalisation measurement.Then Cell can be washed with ice-cold flow cytometry dye solution, then using the table of the CXCR3 antibody analysis CXCR3 of PE conjugation Face expression.

As assessed by any of above measurement, in certain embodiments, neutralizing anti-CXCR3 antibody may have greatly The ND of the μ g/mL of about 0.01,0.02,0.05,0.1,0.2,0.5,1,2,3,4,5,6,7,8,9,10,15,20,40,50 or 100₅₀。 In special embodiment, the ND₅₀It can be 0.5-12 μ g/mL, and In a more specific embodiment, be 1-6 μ g/ mL。

Antibody or antigen-binding fragment pair provided herein can be assessed by any method known to those skilled in the art The inhibition of cell migration, recruitment or accumulation.These methods may include, for example, by immunohistochemical method to biopsy Look into analyzed, flow cytometry, RT-PCR etc., to assess one or more cell masses or in vivo or one or more in organ Cell (such as CXCR3 of a position⁺Cell) quantity.In the case where antibody provided herein or antigen-binding fragment is not present Cell migration, recruitment or accumulation compare, cell migration, recruitment or accumulation can be suppressed at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or More.

Nucleotide sequence

The nucleotide sequence for encoding amino acid sequence disclosed herein is also provided herein.In some embodiments, It is nucleotide sequence coded being capable of in vitro and/or internal depletion CXCR3⁺The antibody or segment of cell.In certain embodiments, Nucleotide sequence can be used for preparing expression vector, the expression (example for CXCR3 antibody anti-in cell or its antigen-binding fragment Such as, the expression in mammalian cell).

In certain embodiments, there is disclosed herein with encode the basic phase of those of amino acid sequence disclosed herein Same polynucleotides.Essentially identical sequence can be polymorphic sequence, i.e., alternative sequence or allele in group.Basic phase Same sequence also may include mutagenized sequences, including the sequence comprising silent mutation.Mutation may include one or more nucleosides The insertion of sour residue variation, the missing of one or more nucleotide residues or one or more additional nucleotides residues.Due to core The degeneracy of acid encoding, essentially identical sequence also may be embodied in any given ammonia in amino acid sequence disclosed herein The various nucleotide sequences of same amino acid are encoded at base acid position.It further include one of encoding antibody in essentially identical sequence Or the sequence of multiple chains, the antibody retain depletion CXCR3 in vitro and/or in vivo⁺The ability of cell.

In certain embodiments, one or more of nucleic acid encode provided herein antibody provided herein or segment The amino acid sequence of chain, the antibody or segment can depletion CXCR3 expression cell or the nucleic acid can be in stringent condition It is lower to hybridize with the nucleic acid of the amino acid sequence of one or more chains in encoding said antibody or its antigen-binding fragment.

In certain embodiments, disclosed herein is polynucleotide sequences, and it includes encode anti-CXCR3 antibody or its antigen The nucleotide sequence of the amino acid sequence of the VH structural domain of binding fragment, and the nucleotides sequence with the heavy chain of encoding said antibody Arrange at least about 80%-100% (for example, about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween)) it is identical.In certain embodiments, the polynucleotide sequence can To include that the nucleotide sequence of heavy chain relative to encoding said antibody has 0,1,2,3,4,5,6,7,8,9 or 10 mutation The nucleotide sequence of (including addition, missing and substitution, such as conservative replaces).

In certain embodiments, disclosed herein is polynucleotide sequence, it includes encode anti-CXCR3 antibody or segment The nucleotide sequence of the amino acid sequence of VL structural domain, and at least about with the nucleotide sequence of the light chain of encoding said antibody 80%-100% is (for example, about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or 100% (or any percentage therebetween)) identical.In certain embodiments, the polynucleotide sequence may include phase For encoding said antibody light chain nucleotide sequence have 0,1,2,3,4,5,6,7,8,9 or 10 mutation (including addition, Missing and replace, such as conservative replaces) nucleotide sequence.

In special embodiment, polynucleotide sequence disclosed herein include with VH amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (appointing between or What percentage) identical nucleotide sequence, or with VL amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, the identical nucleotides sequence of 94%, 95%, 96%, 97%, 98%, 99% or 100% (any percentage between or) Column, wherein the heavy chain and light-chain amino acid sequence of nucleotide sequence coded any antibody disclosed herein.

Disclosed polynucleotides can be obtained by any method known in the art.For example, if the nucleotide of antibody Sequence be it is known, then the polynucleotides of encoding said antibody can be assembled by chemically synthesized oligonucleotides.This may relate to, For example, the overlapping oligonucleotide of the part of sequence of the synthesis containing encoding said antibody, anneals and connects those oligonucleotides, so The oligonucleotides connected afterwards by PCR amplification.Disclosed polynucleotides can also be raw from any other suitable nucleic acid source At such as antibody cDNA library, or from any tissue or cell for expressing the antibody (for example, from selection to express antibody Hybridoma) separation cDNA library.

The expression of anti-CXCR3 antibody or its antigen-binding fragment

After the nucleic acid that operation encodes the anti-CXCR3 antibody of humanization provided herein or its antigen-binding fragment, typical case will Code nucleic acid is inserted into expression vector to be introduced into host cell, and the host cell can be used for generating the desired amount of encoding antibody Or its antigen-binding fragment.Suitable carrier for expression is known in the art.Suitable host cell includes, such as CHO, COS, Sf9 and/or other mankind or nonhuman cells system.In some embodiments, when cultivating in the medium, Su Zhuxi Born of the same parents are of short duration on carrier or steadily express nucleic acid, to provide the method for generating antibody disclosed herein or segment.

Term " carrier " used herein or " expression vector " description are for gene needed for introducing and expressing in cell Carrier.As it is known by the man skilled in the art, examples of such carriers includes, such as plasmid, bacteriophage, virus and retrovirus. In general, suitable carrier may include selected marker, restriction site appropriate to promote the clone of required gene, and Into eukaryon or prokaryotic cell and/or in the ability wherein replicated.

It can be using many kinds of expression vector systems for expressing anti-CXCR3 antibody provided herein.For example, a kind of carrier Using from animal virus such as bovine papilloma virus, polyomavirus, adenovirus, vaccinia virus, baculoviral, retrovirus The DNA element of (RSV, MMTV or MOMLV) or SV40 virus.Other carrier classes are related to using with Internal Ribosome Binding Site Polycistronic system.Furthermore it is possible to by being introduced into one or more labels select that DNA is integrated into its chromosome Cell, one or more labels allow to select the host cell through transfecting.The label can be provided to auxotroph Former nutrition, biocide resistance (such as antibiotic) or the resistance to heavy metal such as copper of host.Selected marker can be with It directly connect with DNA sequence dna to be expressed, or is introduced into same cell by cotransformation.It it may also be desirable to additional element to come most MRNA is synthesized goodly.These elements may include signal sequence, splicing signal and transcripting promoter, enhancer and termination signal. In some embodiments, the variable region gene of clone is inserted into expression together with heavy chain as described above and light chain constant region gene In carrier.In some embodiments, the heavy chain and constant region of light chain are the mankind.

In other embodiments, can be used polycistronic con-struct express anti-CXCR3 antibody provided herein or its Antigen-binding fragment.In such expression system, a variety of interested genes can be generated from single polycistronic con-struct and are produced Object, such as the heavy chain and light chain of antibody.Internal ribosome entry site (IRES) is advantageously used in eucaryon host in these systems The polypeptide provided herein of relative high levels is provided in cell.Compatible IRES sequence is public in U.S. Patent number 6,193,980 It opens, is incorporated herein by reference.It will be understood by those skilled in the art that such expression system can be used for effectively generating in the application Disclosed complete series polypeptide.

Once being prepared for encoding antibody or the carrier or DNA sequence dna of its antigen-binding fragment, so that it may draw expression vector Enter in suitable host cell.That is, the host cell can be converted.It can be by known to those skilled in the art Various technologies plasmid is introduced into host cell to realize.The including but not limited to transfection of these technologies (including electrophoresis and electricity are worn Hole), protoplast fusion, calcium phosphate precipitation, infect with the cell fusion of coating DNA, microinjection and intact virus.Referring to Ridgway, A.A.G. " Mammalian Expression Vectors " the 24.2nd chapter, the 470-472 pages Vectors, Rodriguez and Denhardt edits (Butterworths, Boston, Mass.1988).In some embodiments, plasmid Host is introduced by electroporation.The cell of conversion is in the item for being suitable for generating encoding amino acid sequence (such as antibody light chain and heavy chain) It is grown under part, and the generation of the encoding amino acid sequence described in these raji cell assay Rajis.Exemplary assay techniques include enzyme linked immunological Determining adsorption (ELISA), radiommunoassay (RIA), flow cytometry, immunohistochemistry etc..

" host cell ", which refers to have had been incorporated into, constructs using recombinant nucleic acid technology and encodes at least one exogenous proteins Carrier cell.When description is for process from recombinant host isolated polypeptide, term " cell " and " cell culture " can It is used interchangeably with the source of the protein of presentation code (such as antibody or its antigen-binding fragment), unless expressly stated otherwise,. In other words, recycling polypeptide can refer to the full cell from centrifugation from " cell ", or from containing culture medium and suspension cell two It is recycled in the cell culture of person.

It in one embodiment, is mammalian origin for the host cell line of antibody expression；Art technology Personnel can determine the particular host cell system for being best suited for the gene product needed for wherein expressing.Exemplary host cells system packet Include but be not limited to DG44 and DUXB11 (Chinese hamster ovary system, DHFR-), HELA (human cervical carcinoma), CVI (monkey kidney system), COS (derivative of the CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast), (mouse is at fiber by BALBC/3T3 Cell), SP2/O (mouse myeloma), BFA-1c1BPT (bovine aortic endothelial cells), RAJI (human lymphocyte), 293 (people's kidneys).? In one embodiment, NS0 cell can be used.In some embodiments, using Chinese hamster ovary celI.Host cell line is typically It can be from commerce services, American tissue Culture collection (American Tissue Culture Collection) or openly Document in obtain.

In one embodiment, the cell line provides for example non-rock algae of glycosylation of the change of the antibody by its expression Glycosylation is (for example, PER.C6.RTM (Crucell) or FUT8 knocks out CHO cell line (Potelligent.RTM.Cells) (Biowa, Princeton, N.J.)).Alternatively, one or more sugar needed for cell may lack N- glycan early stage processing Glycosides enzyme and/or condition of culture may be such that the activity of these one or more glycosidases is suppressed.For example, cell may lack one Kind or a variety of glycosidases, such as alpha-Glucosidase I, alpha-Glucosidase II and alpha-Mannosidase I.In addition, alternatively, work Journey cell can be with the suppression of one or more glycosidases (such as alpha-Glucosidase I, alpha-Glucosidase II and alpha-Mannosidase I) Preparation contact.In certain embodiments, the inhibitor is the inhibitor of alpha-Mannosidase I, such as alpha-Mannosidase I Specific inhibitor, base husband's alkali.With the illustrative methods of base husband alkali and other inhibitor culture host cells in U.S. Patent number It discloses, is incorporated herein by reference in their entirety in 8,071,336.In certain embodiments, base husband alkali process, which generates, has extremely Few 50%Man_5-9(GlcNAc)₂The antibody of N- glycan, wherein the N- glycan containing Man8 and Man9 is main species together.

Produced in vitro allows to scale up to provide a large amount of required polypeptide.It is dynamic for lactation under conditions of tissue culture The technology of object cell culture is known in the art and (such as in airlift reactor or continuously stirs including homogeneous suspension culture Mix in reactor), or on agarose microballon or immobilization or embedding on ceramic box cell culture (such as in hollow fibre In dimension, microcapsules).If necessary and/or if needs, conventional chromatographic methods, such as gel filtration, ion exchange can be passed through The solution of chromatography, the chromatography on DEAE- cellulose and/or (immune) affinity chromatography purified polypeptide.

The nucleic acid for encoding anti-CXCR3 antibody provided herein or its segment can also be in nonmammalian cells (such as bacterium Or yeast or plant cell) in expression.In this respect, it should be understood that various unicellular non mammali microorganisms (such as bacterium) Those of it can also be used for expressing antibody and its antigen-binding fragment provided herein, can be grown in culture or fermentation.It closes Suitable bacterium includes enterobacteriaceae member, such as Escherichia coli or the bacterial strain of Salmonella；Bacillaceae, such as bacillus subtilis Bacterium；Pneumococcus；Streptococcus and haemophilus influenzae.It should also be understood that polypeptide, which can become, to be included when expressing in bacterium A part of body.It must separate, purified polypeptide, then assemble them into functional molecular.

In addition to prokaryotes, eukaryotic microorganisms can also be used.Saccharomyces cerevisiae or common Saccharomyces cerevisiae are eukaryotic microorganisms In it is most common, although many other bacterial strains are typically available.For the expression in saccharomyces, usually using such as matter Grain YRp7 (Stinchcomb etc., Nature, 282:39 (1979)；Kingsman etc., Gene, 7:141 (1979)；Tschemper Deng Gene, 10:157 (1980)).This plasmid has contained TRP1 gene, and the gene provides what shortage was grown in tryptophan The selected marker of the mutant yeast strains of ability, such as No. ATCC 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).Then, the presence of Trpl damage provides in the case where no tryptophan as yeast host cell genome signature The effective environment converted by growth detection.

Medicament preparation and medication

There is provided herein the drugs comprising Humanized anti-human CXCR3 antibody and pharmaceutically acceptable carrier disclosed herein Composition.

Preparation and to the method that subject gives antibody or its antigen-binding fragment provided herein be those skilled in the art Member is known or is easy determination.Antibody or its segment give approach be take orally, parenteral it is (such as intravenous, intramuscular, intraperitoneal Or it is subcutaneous), suck or administer locally to.In some embodiments, antibody provided herein is formulated for intravenous administration.One In a little embodiments, the suitable pharmaceutical composition for injection includes buffer (such as acetate, phosphate or citrate Buffer), surfactant (such as polysorbate), optional stabilizer (such as human albumin) etc..

Being suitable for the pharmaceutical composition that injection uses includes aseptic aqueous solution (water-soluble) or dispersion liquid, and for facing When preparation sterile injectable solution or dispersion liquid aseptic powdery.Carrier can be solvent or decentralized medium, the solvent or point Dispersion media contains such as water, ethyl alcohol, polyalcohol (for example, glycerol, propylene glycol and liquid macrogol) and its suitable mixing Object.For example, by using coating such as lecithin, by maintaining required granularity in the case where dispersion and by using surface Activating agent can maintain mobility appropriate.Prevent the effect of microorganism can be by a variety of antibacteriums and antifungal agent (example Such as, p-hydroxybenzoate, anesin, phenol, ascorbic acid, thimerosal etc.) Lai Shixian.In many cases it is preferred to It in the composition include isotonic agent, such as sugar, polyalcohol (such as mannitol, D-sorbite) or sodium chloride.By at described group The medicament such as aluminum monostearate and gelatin absorbed in object comprising delay is closed, the extension that Injectable composition may be implemented absorbs.

It is in any case possible to by by reactive compound (for example, antibody itself or with other activating agents combine) with Required amount mixes in solvent appropriate, and subsequent filtration sterilization prepares sterile injectable solution, and the solvent has as needed There is the combination of a kind of ingredient or Multiple components enumerated herein.In general, by the way that the reactive compound is mixed sterile carrier In prepare dispersion liquid, the sterile carrier contain basic dispersion medium and needed for those listed above other at Point.In the case where being used to prepare the aseptic powdery of sterile injectable solution, preferably preparation method is that vacuum drying and freezing are dry It is dry, the vacuum drying and freeze-drying by the solution that had previously been sterile filtered generate active constituent and it is any it is additional needed for ingredient Powder.The preparation processing that will be used to inject is filled into container such as ampoule, sack, bottle, syringe or bottle, and according to this Method known to field aseptically seals.This product preferably has label or package insert, indicates relevant group Closing object can be used for treating the subject for suffering from or being susceptible to suffer from autoimmunity or tumour obstacle.

The dosage of antibody provided herein for treating above-mentioned illness or its antigen-binding fragment according to it is many it is different because Element and change, the factor includes that mode of giving, target site, the physiological status of patient, patient are people or animal, give Other drugs and treatment are preventative or therapeutic.In general, patient is people, but it also can treat and fed including transgenosis Non-human mammal including newborn animal.

In some embodiments, the dosage may range from, for example, about 0.0001 to 100mg/kg or 0.01 to 5mg/kg host's weight.

Can it is daily, the next day, given to subject weekly or by any other determining period planning is rule of thumb analyzed Such dosage.Antibody provided herein or its antigen-binding fragment can repeatedly be given.Interval between single-dose can be with It is, such as daily, weekly, monthly or every year.As indicated by horizontal by blood polypeptide in measurement patient or target molecules, It is irregular every being also possible to.

Antibody provided herein or its antigen-binding fragment are optionally and for treating obstruction and illness in need for the treatment of Other medicaments of (for example, preventative or therapeutic treatment) combine together to be given.Other preferred medicaments are art-recognized And medicament those of is given for specific obstacle standard.

The method for treating CXCR3 related disease or obstacle

CXCR3 antibody or its antigen-binding fragment provided herein can be used for antagonism CXCR3 activity.In some embodiments In, antibody and antigen-binding fragment are in following methods, the method to be to inhibit CXCR3 and one or more ligands (such as CXCL9, CXCL10 and/or CXCL11) it combines；Inhibit CXCR3⁺Migration, accumulation, recruitment or the infiltration of cell are (such as to inflammation portion Position)；And/or depletion CXCR3⁺Cell.In some embodiments, the antibody and antigen-binding fragment are used for internal depletion CXCR3⁺In the method for cell.CXCR3⁺Cell includes but is not limited to CXCR3⁺/CD4⁺T cell, CXCR3⁺/CD8⁺T cell and CXCR3⁺/CD19⁺B cell subgroup.

In certain embodiments, it provides anti-comprising one or more CXCR3 by being given to subject in need The pharmaceutical composition of body or its antigen-binding fragment is come the method for the treatment of CXCR3 related disease or obstacle.In an embodiment In, provide the autoimmune disease that treatment T cell mediates or the method for reducing its progress.The method includes being disclosed herein Humanized anti-human CXCR3 antibody or its antigen-binding fragment give to subject in need thereof, thus treat T cell Jie The autoimmune disease led or the step of reduce its progress.In certain embodiments, the autoimmunity disease that the T cell mediates Disease is leucoderma.In some embodiments, subject in need thereof includes being diagnosed with CXCR3 related disease Or the autoimmune disease that mediates of T cell or tend to develop what CXCR3 related disease or T cell as described herein mediated The subject of autoimmune disease.

Subject to be treated by method provided herein may include people or other mammals.In an embodiment In, the subject is people.In each embodiment, can to subject carry out prophylactic treatment, or with exception Destroying for the CXCR3 relevant any illness of activity or in which CXCR3 signal transduction can beneficial any illness hair in the treatment It is treated after work.

In some embodiments, prophylactic treatment can be carried out to subject, or treated after leucoderma breaking-out.

" subject with leucoderma " be be usually itself immunogene because or unknown cause patch shape skin-color The plain calm any subject for reducing (it is consistent with the medical diagnosis of leucoderma).Involving region may include face, hand, foot Portion, arm, leg, lip or any combination thereof, but other positions can be related to, such as neck or trunk.In some cases, Hair pigmentation is reduced.

Embodiment

It will be further clarified by the following the antibody, composition of matter and method, these embodiments should not be explained Further to limit.Through sequence table, attached drawing and all bibliography of the application reference, patent and disclosed patent application Content is expressly incorporated into herein.

Embodiment 1: the generation of the anti-CXCR3 monoclonal antibody of humanization

The anti-CXCR3 monoclonal antibody of generation as described in WO2013/109974.It is as described below to generate 53 list of humanization clone Clonal antibody.

It is prepared for the humanization form of clone 53, the depletion of CXCR3 expression cell can be instructed.Generating has such as table 4 Shown in VH and VK sequence humanization clone 53 monoclonal antibodies.

The hair tonic Capability index score of the every kind of humanization variants shown in table 4.By heavy chain and IGHV3-23*03/ IGHJ4*03 Germline sequences are compared；Light chain is compared with IGKV1-9*01/IGKJ2*01 Germline sequences.

Table 4

Hu antibody	VH SEQ ID NO	VL SEQ ID NO	Hair tonic Capability index 1^a	Hair tonic Capability index 2^b
					23	18	21	0.885	0.950
24	19	21	0.872	0.933
					25	20	21	0.877	0.939
26	18	22	0.890	0.955
					27	19	22	0.877	0.939
29	20	22	0.881	0.944
					30	18	23	0.890	0.955
31	19	23	0.877	0.939
					33	20	23	0.881	0.944
34	18	24	0.895	0.961
					35	19	24	0.881	0.944
37	20	24	0.886	0.950

^aHair tonic Capability index (1) weight: the pairs of comparison of all residues in addition to the area D those of occupies

It is light: the pairs of comparison of all residues

^bHair tonic Capability index (2) weight: only the pairs of of all Framework residues compares (such as defined and defined by IMTG)

Light: only the pairs of of all Framework residues compares (such as defined and defined by IMTG)

The binding characteristic of every kind of Humanized monoclonal antibodies provided herein is shown in Table 5.

5 binding kinetics of table

Hu antibody	ka(1/Ms)	kd(1/s)	KD(M)
				23	7.56E+04	2.45E-05	3.22E-10
24	1.01E+05	6.16E-06	6.14E-11
				25	9.95E+04	6.21E-05	6.23E-10
26	1.01E+05	5.46E-05	5.37E-10
				27	1.13E+05	8.34E-06	7.37E-11
29	1.15E+05	3.50E-05	3.03E-10
				30	9.37E+04	1.26E-04	1.34E-09
31	1.11E+05	5.00E-05	4.49E-10
				33	9.66E+04	6.35E-05	6.56E-10
34	8.89E+04	9.19E-05	1.03E-09
				35	1.02E+05	4.73E-06	4.56E-11
37	6.74E+04	6.48E-05	9.57E-10

Table 6 shows clone 53 (53), chimerical clone 53 (Ch53) and clone 53 humanization forms (53Hu1-53Hu20) Binding characteristic and hair tonic ability

Table 6

It is light: the pairs of comparison of all residues

As indicated by above-mentioned data, humanized antibody provided herein has the binding characteristic significantly improved, has simultaneously Advantageous hair tonic Capability index.

Embodiment 2:CDR optimization

It is prepared for many VH CDR and/or VL CDR variant.Use Biacore measurement and the combination of recombined human CXCR3 parent With joint efforts.The mutant with combination strong at least and as 53Hu37 is shown in table 3.

Embodiment 3:CXCR3-173 is depletion antibody

Hamster anti-mouse CXCR3 monoclonal (clone CXCR3-173) is used as substitution antibody in preclinical laboratory.Previously CXCR3-173 has been described as to the blocking antibody of not depletion CD4+T cell in vivo.(referring to Uppaluri etc., Transplantation 86:137-47(2008))。

Prepare hamster CXCR3-173 mAb following Fc variant with the effector function of test antibody: mouse IgG 1 it is non- It glycosylates N297G variant (CXCR3 mIgG1 agly)；Wild-type mice IgG2a chimera (CXCR3 mIgG2a WT)；Through repairing It is decorated with the mouse IgG 2a chimera (CXCR3 mIgG2a Dab) for eliminating consumption ability；And wild-type mice IgG3 (CXCR3 mIgG3)。

Single 5mg/kg is given to the C57BL/6 mouse (Jackson Laboratories, every group of n=5) of 8 to 10 week old The antibody of intravenous dosages, then harvest blood is used to carry out flow cytometry using following marker after 24 hours: CD4 and CD8 T cell is analyzed by TCRab, CD4 and CD8；Memory CD4 is analyzed by TCRab, CD4, CD8, CD62L and CD44 With CD8 T cell.Cell is quantified using Count Bright pearl (Invitrogen) according to the scheme of manufacturer, with true Determine total cell number/microlitre blood.

The gate result from total T lymphocyte and T lymphocyte subgroup is shown in Fig. 1.As shown, astonishing Ground demonstrates hamster CXCR3-173 depletion CD4+ and the CD8+ memory T cell when giving mouse for the first time.

The effector function of the Fc variant of embodiment 4:CXCR3-173

The mouse of the Fc- engineered forms of CXCR3-173 is assessed with antibody capture method using 3000 instrument of Biacore Fc γ receptor combines.Recombinant protein A/G (Pierce) is covalently fixed to CM5 sensor core on piece using amination.It will CXCR3-173 antibody is diluted to 5 μ g/mL in HBS-EP buffer, and is infused with 10 L/ minutes flow velocitys of μ to albumin A/G chip It penetrates 30 seconds.By recombined small-mouse Fc γ RI (CD64), Fc γ RIIb (CD32), Fc γ RIII (CD16) from R&D Systems Dilute 3 times from 300 to 3.7nM in HBS-EP buffer with Fc γ IV (CD16-2), and in duplicate with 30 L/ minutes streams of μ Speed is injected into the antibody of capture.Make surface regeneration with glycine 2.0 (GE Healthcare).It will be normalized in conjunction with response For a-protein/G capture RU amount.As the result is shown in fig. 2 a-2d.

As shown, being modified has the hamster CXCR3-173 of wild-type mice IgG2a isotype and all four recombinations small Mouse (rm) Fc γ receptor combines, although dAB mutation significantly reduces this combination.There is wild-type mice IgG3 isotype through modification CXCR3-173 also in conjunction with all four recombined small-mouse Fc γ receptors.Original unmodified hamster CXCR3-173 ratio IgG2a isotype variant preferably combines rmFc γ RIIb and rmFc γ RIII, and nonglycosylated mIgG1 isotype variant is not In conjunction with any rmFc γ R.

Embodiment 5: vitro effect subfunction

The anti-CXCR3 mAb of humanization and its Fc- engineered forms are had studied in a series of ADCC measurement.Use standard side Method prepares the Fc- engineered forms of the anti-CXCR3 mAb of humanization.By cultivating expression humanization mAb in the presence of base husband's alkali Cell come prepare seaweed saccharification form.

Using primary NK cells of human beings or it is overexpressed the NK9.2 cell line (Conkwest) with the CD16 of valine polymorphism As effector cell, and the CHO transfection cell for being overexpressed people CXCR3 (A isotype) is used to carry out ADCC survey as target cell It is fixed.

For using measurement of the primary NK cells as effector, from the leucopak purified NK cells of Normal donor, and Cultivate 24 hours in IL-2, then with the E:T ratio of 5:1 with marked with chromium together with overnight CHO- people's CXCR3 target cell Tiling.Culture is incubated 3 hours in incubator for tissue culture, then wash and is cracked with 1%Tritron-X, is counted later in γ Supernatant is read on number device.

For using NK9.2 cell as the measurement of effector, according to the recommendation of manufacturer, by NK9.2 cell in IL-2 Middle amplification 2 weeks.On the day of measurement, simultaneously with Calcein-Safranine T (Invitrogen) label NK9.2 cell (70,000 cells) It incubates 30 minutes together with appropriate diluted antibody so that antibody is in conjunction with the CXCR3 on target cell.By NK cell with 3:1's Effector tiles than target cell ratio, and culture is incubated 1 hour in incubator for tissue culture.It is used at the end of culture period Triton X-100 cracks these cells, and reads plate using M5 microplate reader (492nm excitation and 515nm emit).

Human IgG1 (Sigma) is used as negative control, crosses table with the CD52 that alemtuzumab (monoclonal anti-CD 52 antibody) is handled The positive control of cracking is served as up to the cracking of Chinese hamster ovary celI.Signal is indicated with any flat fluorescent (AFU).Percentage of cytotoxicity It is indicated by (experiment cracking-Spontaneous lysis)/(maximum cracking-Spontaneous lysis) x 100%.

In ADCC measurement, the anti-CXCR3 mAb of humanization with human IgG1's Fc and Fc- engineered forms is tested 53Hu37.Fc- engineered forms M1 (S239D/D332E (EU representation), M2 (G236A/S267E/H268F/S324T/I332E (EU representation), " AEFTE ") and M3 (S239D/H268F/S324T/I332E (EU representation), " DFTE ") containing allow ADCC Or the amino acid variation of CDC increased activity.The cell line of wild-type antibodies is prepared by base husband's alkali process to generate the 4th kind of Fc- Engineered forms.

It is also tested for anti-human CXCR3 clone and clones 4 (CXCR3 CL4), 12 (CXCR3 CL12) of clone, clone 82 (CXCR3 CL82) and 135 (CXCR3 CL135) of clone.In independent experiment, with different effector cells, effector: target (E:T) than being measured with antibody concentration.It is representative as the result is shown in Fig. 3 B and Fig. 3 C.

Fig. 3 A summarizes the effector function for removing fucosylated form of M1, M2, M3 and 53Hu37.Fig. 3 B display uses former Measurement result for NK cell as effector.Fig. 3 C shows the measurement result for using NK9.2 cell as effector.

Embodiment 6:Fc γ receptor combines

Shape is engineered using the Fc- of the anti-CXCR3 mAb 53Hu37 and 53Hu37 of Biacore T200 Instrument Evaluation humanization The people of formula and mouse Fc γ receptor binding affinity.The albumin A from Sigma is fixed on CM5 series S chip using amination On.Antibody is injected into albumin A chip, and the recombined human of a variety of concentration and mouse Fc γ receptor (R&D Systems) are infused It is mapped in the antibody of capture.Low-affinity conjugate and high-affinity conjugate (1.2nM are crossed over using the receptor of wide concentration range To 5 μM).Two parts of each sample injections.1:1 dynamics binding model will be fitted in conjunction with sensing figure.Quantitative result summarizes In Fig. 4.

As shown in figure 4, M1 and M3 Fc- engineered forms all have the affine of improvement to hFc γ RIII and mFc γ RIV Power, M2 increases the combination of hFc γ RIIa, and compared with Fc- engineered forms, the 53Hu37 of base husband's alkali process is shown The moderate of hFc γ RIII and mFc γ RIV increases.

Embodiment 7:

Machin receive single intravenous infusion 2mg/kg weight 53Hu37, M1 variant or base husband's alkali process form it is (every A Antybody therapy group n=8) or vehicle control (n=6).Before infusion and then be transfused after 1,3,7 and 14 day collection blood Liquid sample, and pass through the total T cell of flow cytometry and T cell subgroup.For flow cytometry, erythrocyte splitting is used in combination Antibody dyes cell for following label to identify total CD4 and CD8 T cell and memory CD4 and CD8 T cell: CD3 (clone SP34-2), CD4 (clone OKT4), CD8a (clone RPA-T8), CD45RA (clone 5H9), CCR7 (clone G043H7) and CXCR3 (clone 1C6).Cell number/microlitre (cell is determined to cell quantification using Count Bright pearl (Invitrogen) Number/μ L).Data take the average percent of the value before blood for being expressed as described group of each cell subsets at any time.By making 4 antibody and suitable secondary antibody dyed through fixed sample sections come after studying infusion to CXCR3 are cloned with anti-human CXCR3 The histology of the 14th day spleen sample obtained from the subgroup of each treatment group.As the result is shown in Fig. 5 A-5C.Using peptide ELISA, The pharmacokinetics for also measuring the serum in blood sample, measures the cyclical level of given antibody.

As shown, the 53Hu37 that the M1 form and Kif of 53Hu37,53Hu37 are handled reduce effect memory CD4 T it is thin Born of the same parents, and the 53Hu37 of M1 form and the Kif processing of 53Hu37, Hu37 reduces effect memory CD8 T cell.In addition, The 53Hu37 of M1 form and the Kif processing of 53Hu37 is significantly reduced in spleen for 14 days after single intravenous infusion antibody CXCR3 dyeing.

Embodiment 8: biochemical analysis

Thermal stability, shearing resistance shearing stress and the inactivation of virus of the 53Hu37 of 53Hu37, M1 form of measurement and Ji Fu alkali process Stability, the stability of freeze thawing resistance stress, it is anti-agitation stress stability and pH stability.

Differential scanning calorimetry (DSC)

Sample is analyzed with high-throughput VP-DSC (Microcal).Sample is diluted to about 0.5mg/ with corresponding buffer ML, and be loaded on 96 orifice plates.Sweep parameter is formed by 25 DEG C and 100 DEG C of final temperature of initial temperature.Use sweeping for 200 DEG C/h Retouch rate.

Turbidity

With the sample at 5nm increment measurement 340-360nm on Spectramax Plus (Molecular Devices) Absorbance, and these values are average to generate final turbidity measurement.Use 96 hole UV flat undersides and 150-200 μ L material. It reads plate in advance before adding sample, and route inspection is applied to sample value." A based on Brigitte Eckhardt Turbidimetric Method to Determine Visual Appearance of Protein Solutions”, Technology Applications, volume 48, the 2nd phase, April in March, 1994 -), by sample and by the UV of turbidity standard object The value that absorbance obtains is compared to be classified to turbidity.

Size exclusion chromatography (SEC)

HP1100 or 1200 serial systems are equipped with and couple SW_XLThe TSK SW of guard column_XLSize exclusion column.Use 20mM The mobile phase of sodium phosphate, 500mM NaCl, pH 6.0 runs sample 35 minutes.Use 0.5mL/ minutes flow velocitys.Carry out 50 μ G injection simultaneously monitors UV signal at 280nm.

The opposite aggregation tendency (TI-RAP) of thermal induction

By in PBS buffer solution and 10mM histidine and 9% Sucrose buffer in 5 DEG C (controls), 64 DEG C, 67 DEG C, 70 DEG C or 73 DEG C by the anti-human CXCR3 antibody of 0.2mg/mL (single antibody or antibody mixture) incubate 10 minutes it is temperature-induced to generate Aggregation.After heat incubates, sample is centrifuged 2 minutes with 7000xg at 5 DEG C to remove insoluble protein sediment, and pass through sun Ion-exchange chromatography (CEX) analyzes supernatant.By using the peak area of control (5 DEG C) sample to the chromatographic peak of thermal stress sample Area is normalized to calculate the percentage of soluble and monomeric (and opposite aggregation tendency).

The opposite aggregation tendency (AI-RAP) of agitation induction

For the opposite aggregation tendency of agitation induction, make slow in PBS buffer solution and 10mM histidine and 9% sucrose at 5 DEG C The solution of anti-human CXCR3 antibody containing the final protein concentration of 0.2mg/mL in fliud flushing (containing 0 and 0.01% polysorbate80) Vigorous agitation stress is subjected in 5 DEG C.At full throttle with VX-2500 multitube turbula shaker (VWR, West Chester, PA) The solution for stirring anti-human CXCR3 antibody continues 24 hours in total.

It is analyzed by taking out small sample aliquot for CEX, analyzes the various time points in entire research.By sample Aliquot is centrifuged 2 minutes with 7000x g (at 5 DEG C) to remove insoluble protein sediment, and analyzes supernatant by CEX Liquid.The chromatographic peak area of agitation sample is normalized by using the peak area of control (0 hour) sample solvable to calculate Property monomer percentage (and opposite aggregation tendency).

Cation-exchange chromatography (CEX)

Using ProPac WCX-10 analytical column (weak cation exchange, 4x 250mm, Thermo Scientific) 25 CEX analysis is carried out DEG C on Agilent 1290infinity HPLC system.20 micrograms of protein quality samples are loaded on column, And it is analyzed with 0.8mL/ minutes flow velocitys.It is flat with buffer solution A (20mM sodium acetate, 0.0025% sodium azide, pH 5.2) Weigh the column, with from 0 to 100% linear gradient buffer solution B (20mM sodium acetate, 1M sodium chloride, 0.0025% sodium azide, PH 5.2) elution protein 40 minutes.It measures the absorbance at 280nm and integrates 280nm absorbance peak to determine protein peak Area.

Experimental design

PH/ temperature stress

Using Slide-A-Lyzers (Thermo Scientific PN 66810) by a part from each clone Material carries out dialysis in the 20mM sodium phosphate of pH 5.0 and the sodium phosphate of pH 7.0.This material is diluted using corresponding buffer, and Use Millex GV filter (Millipore PN SLGV033RB) filtering to about 2mg/mL.Before test, by pH 5 It is stored 3 weeks and 5 weeks with the phosphoric acid sodium sample of pH 7 at 37 DEG C.

Freeze thawing stress

It is using Slide-A-Lyzers (Thermo Scientific PN 66810) that the anti-human CXCR3 antibody of instruction is saturating It analyses in the 20mM sodium phosphate of pH 6.0.This material is diluted using corresponding buffer, and uses Millex GV filter (Millipore PN SLGV033RB) is filtered to about 1mg/mL.By freeze thawing stress sample -80 DEG C freezing and at room temperature Melt, carries out 5 times in total.After 1 time and 3 Frozen-thawed cycleds, takes out material and be used to be tested by selection.Final Complete one group of test analysis is carried out after freeze thawing.

Shear stress

Antibody samples are carried out dialysis into pH 6.0 using Slide-A-Lyzers (Thermo Scientific PN 66810) 20mM sodium phosphate in.This material is diluted using corresponding buffer, and uses Millex GV filter (Millipore PN SLGV033RB it) filters to about 1mg/mL.It repeats to pipette shear stress sample totally 50 times with 200 μ L pipettes.

Simulated virus inactivation

Antibody samples are carried out dialysis into pH 6.0 using Slide-A-Lyzers (Thermo Scientific PN 66810) 20mM sodium phosphate in.This material is diluted using corresponding buffer, and uses Millex GV filter (Millipore PN SLGV033RB it) filters to about 1mg/mL.Inactivation of virus sample is adjusted to pH 3.5 with 1N HCl, and keeps this at room temperature PH value 100 minutes.After retention period, sample is restored to pH 7.2 and kept for 100 minutes using 1N NaOH.Then 1N is used Sample is adjusted to pH 6.0 and tested by HCl.

As a result

Differential scanning calorimetry:

It was found that the stability of M1 form (D/E mutant) is slightly below Kif form, the stability of Kif form is slightly below again 53Hu37。

Shear stress:

It was found that the stability of M1 form (D/E mutant) is slightly below 53Hu37 and Kif form, both rear stability is substantially It is equal.

Inactivation of virus stress:

It was found that the stability of M1 form (D/E mutant) is higher than Kif form, the stability of the two is below 53Hu37.

Freeze thawing stress:

It was found that M1 form (D/E mutant), Kif form and 53Hu37 are roughly the same.

The opposite aggregation tendency of agitation induction:

About 80% precipitates in 2 hours of agitation in the antibody of form of ownership, although in pH 5.5, M1 form (D/E Mutant) it is relatively more more stable than other two kinds of forms.

Aggregation is formed:

Under the conditions of control (not the accelerating) of pH 5.0, M1 form (D/E mutant) and 53Hu37 are bases within 5 weeks It is stable in sheet, but Kif form becomes milky；Same mode is had found under acceleration conditions.PH 7.0 control (not Accelerate) under the conditions of, three kinds of forms of antibody are roughly the same within 5 weeks.Under acceleration conditions, the stability of DE mutant is omited Higher than 53Hu37, the stability of 53Hu37 is higher than Kif form again.

Embodiment 9: in the mouse model of leucoderma, depletion but be not non-depletion anti-CXCR3 be with protectiveness and It is therapeutic

Mouse.KRT14-Kitl*4XTG2Bjl (Krt14-Kitl*) mouse is given by University of Wisconsin BJ Longley It send.PMEL TCR transgenic mice is Thy1.1⁺, it is obtained from The Jackson Laboratory, stock number 005023, B6.Cg Thy1^a/CyTg(TcraTcrb)8Rest/J.All mouse in C57BL/6J background, maintain without pathogen Facility in, and according to NIH experimental animal nursing and guide for use carry out program.

Antibody.Δ AB CXCR3 is to neutralize the active antibody of CXCR3 (referred to herein as IgG2a-dAB, dAB or Δ AB).WT Mouse CXCR3 is the antibody with the active wild-type mice IgG2a Fc of depletion.

Leucoderma induction and Antybody therapy.As previously mentioned, leucoderma passes through premelanosome specificity (PMEL) CD8⁺T cell Adoptive transfer induction.Harris etc., J.Invest.Dermatol.132:1869-76 (2012).In brief, according to manufacture The explanation of quotient, by the Solid phase on microballon (Miltenyi Biotech), from PMEL TCR transgenic mice (the Jackson Laboratory, stock number 005023) spleen in separate PMEL CD8⁺T cell.By the CD8 of purifying⁺T cell (1x 10⁶It is a) it is injected intravenously into the Krt14-Kitl* host (the through sub- lethal exposure (preceding 1 day 500 ladd of transfer) Jackson Laboratory stock number 009687,8-12 week old).On the day of transfer, Recipient mice is also received in peritonaeum Inject 1x 10⁶Pfu rVV-hPMEL (N Restifo, NCI, NIH).

By the way that 100 μ g antibody of intraperitoneal injection or isotype controls are treated three times weekly during observation.

For Prevention Research, the 2-6 weeks treatment mouse after leucoderma induction.This period is critically important, because it sends out It gives birth to after the virus sweep for inducing disease, but before autoimmunity starts.Using based on the position for being easy to see at four The score value scale of the bleaching level at the place of setting quantifies leucoderma score, including foregoing ear, nose, rear foot pad and tail Bar.Harris etc., J.Invest.Dermatol.132:1869-76 (2012).It checks each position, and bleaching level is estimated For the percentage of the region of anatomy；Foot pad is estimated together after left and right ear and left and right, therefore is evaluated as single position.Score is sentenced Fixed as follows: the evidence (0%) not decolourized is scored at 0, and > 0-10%=1 points, > 10%-25%=2 points, > 25%-75%=3 Point,>75%-<100%=4 points, 100%=5 points." leucoderma score " is the score summation at all four positions, top score It is 20 points.

Pigment regeneration is studied, when disease normal table, the 12-20 weeks after leucoderma induction, with WT mouse CXCR3 depletion Ab or isotype controls treatment tail decoloration are more than 75% mouse.As previously mentioned, carrying out pigment again with ImageJ Analysis estranged.Agarwal etc., J.Invest.Dermatol.135:1080-8 (2015).

The influence that CXCR3 antibody counts blood and spleen cell.Every kind of antibody of 100 μ g is transfused to mouse, and 24 Hour assessment comes autoblood (the every microlitre of cell number measured using Countbright pearl) and spleen (using from Becton The total cell count of the Via-Cell XR instrument of Coulter) memory CD8⁺T cell counts.Because the antibody of administration is essence It goes up antibody (identical CDR) identical with the antibody for CXCR3 dyeing and staining antibodies and the knot of CXCR3 can be blocked It closes, passes through evaluation memory CD8 using the measurement independent of CXCR3⁺The quantity of T cell come assess depletion (such as by CD44 and CD62L dyeing definition) because being more than 50% these cells expression CXCR3.

Flow cytometry.Blood, tail, spleen and skin-draining lymph nodes are collected, and are divided by flow cytometry Analysis.Spleen is destroyed, and uses RBC lysis buffer (Sigma Aldrich) splitting erythrocyte.According to the scheme of manufacturer, Blood sample is dyed and cracks/fix buffer processing with Biolegend.In order to separate corium and epidermis, by tail skin sample With 5U mL^-1Dispase II (Roche) is incubated 1 hour at 37 DEG C.It removes epidermis and uses 70 μm of cell filter Mechanical Crushings, And by corium sample and 1mg mL^-1Clostridiopetidase A IV and 1-2mg mL^-1DNAse I (Sigma-Aldrich) is small in 37 DEG C of incubations 1 When.Before analysis, all cells are passed through into 70 μm of the screen to filtrates.In order to identify PMEL and its phenotype, it is used in FACS buffer solution Following antibody in (1%BSA in PBS) dyes sample by 1:200: CD45 AF700, CD8b PerCP-Cy5.5, Thy1.1 Pacific Blue, (Biolegend).Use the Live/Dead Blue of the 1:1000 in FACS buffer solution (Invitrogen) living cells is distinguished.Use BD LSR II flow cytometer (BD Biosciences) and FlowJo software Version 10 (Tree Star, Inc.) collects and analyzes data.

As a result: the influence that CXCR3 antibody counts blood and spleen cell.Compared with untreated mouse, in blood and In spleen, CD8 is remembered in WT mouse or the mouse of hamster CXCR3 Antybody therapy⁺The sum of T cell substantially reduces, and uses Δ The cell number of the mouse of AB CXCR3 treatment does not change.

Prevention.It (is neutralized with Hamster anti-mouse CXCR3 (depletion), WT Mouse CXCR3 (superior depletion), Δ AB CXCR3；It is non- Depletion) or Isotype control antibodies treatment mouse.Wherein depletion antibody WT Mouse CXCR3 shows most in terms of preventing clinical disease Good, 5 weeks significantly reduced scores can prove (one-way analysis of variance p=0.0066, Dunnett post-hoc tests pair after treatment Than isotype ns；Tukey post-hoc tests, which compare WT and Δ AB comparison hamster for Δ AB, has conspicuousness).This observation result with Instruction WT Mouse CXCR3 Ab is more consistent with active data are preferably consumed than hamster CXCR3 Ab.It is all to prevent in mouse The Ab of leucoderma test causes the PMEL in skin less.After any CXCR3 Antybody therapy, PMEL quantity is significant in epidermis It reduces (two-way analysis of variance p < 0.0001, Bonferroni post-hoc tests, compared with isotype controls), tends in the dermis In reduction.However, the effect of neutralizing antibody is not so good as depletion antibody although it has the ability for reducing PMEL quantity in skin, This may be since the PMEL of Low threshold quantity is enough the fact that cope with complete clinical disease.By measurement lymph node (LN), spleen, Host CD8 in blood, epidermis and corium⁺T cell and total CD45⁺The quantity of cell is thin to host T to assess CXCR3 depletion antibody The influence of born of the same parents.It is treated by hamster or WT Mouse CXCR3 Ab, the onlooker host CD8 in spleen and lymph node (LN)⁺T cell Quantity substantially reduces, but do not have then in skin (two-way analysis of variance p < 0.0001, Bonferroni post-hoc tests, and it is of the same race Type control is compared).However, after with any CXCR3 Ab treatment, CD45⁺The sum of cell does not change.

Pigment regeneration.Mouse WT CXCR3 Ab treats significant reversing clinical disease and reduces the PMEL quantity in epidermis.To the greatest extent Manage total CD45⁺Cell number does not change, but the host CD8 of the mouse through treating⁺T cell number is slightly reduced.In short, these data Instruction CXCR3 depletion Ab can reduce autoreactive T cell quantity and reverse disease, while to other compartments of immune system With minimum influence.

The embodiment of front is intended to illustrate and is never limited in present disclosure.In view of compound disclosed herein and side The explanation and practice of method, other embodiments of disclosed Compounds and methods for be to those skilled in the art it is aobvious and It is clear to.

Sequence table

<110>Sino is luxuriant and rich with fragrance (SANOFI)

<120>it is used for the anti-human CXCR3 antibody of therapy of vitiligo

<130> 597513: SA9-197PC

<150> 62/437,889

<151> 2016-12-22

<150> EP 17306770.3

<151> 2017-12-14

<160> 141

<170>PatentIn version 3 .5

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Ala Leu Phe Asn Ile Asn Phe Tyr Ala Gly Ala Leu Leu Leu Ala Cys

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Gly Leu Cys Leu Leu Phe Ala Leu Pro Asp Phe Ile Phe Leu Ser Ala

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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

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Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

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Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

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Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

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Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

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Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

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Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

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Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu His Glu Asp Pro

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Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

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Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

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Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

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Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

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Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

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Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

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Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro

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Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

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Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

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Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

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Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

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Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

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Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

35 40 45

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

50 55 60

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

65 70 75 80

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

85 90 95

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

100 105 110

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

115 120 125

Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

130 135 140

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

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Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

180 185 190

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

195 200 205

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

210 215 220

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

245 250 255

<210> 8

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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

35 40 45

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

50 55 60

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

65 70 75 80

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

85 90 95

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

100 105 110

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

115 120 125

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr

130 135 140

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

145 150 155 160

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

180 185 190

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

195 200 205

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

210 215 220

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

245 250 255

<210> 9

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Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

1 5 10 15

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

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His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp

35 40 45

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

50 55 60

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

65 70 75 80

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

85 90 95

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

100 105 110

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

115 120 125

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr

130 135 140

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

145 150 155 160

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

180 185 190

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

195 200 205

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

210 215 220

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

225 230 235 240

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

245 250 255

<210> 10

<211> 255

<212> PRT

<213>artificial sequence

<220>

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Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

1 5 10 15

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

20 25 30

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Ala Gly Pro Ser

35 40 45

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

50 55 60

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Glu Phe Glu Asp Pro

65 70 75 80

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

85 90 95

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

100 105 110

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

115 120 125

Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr

130 135 140

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

145 150 155 160

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

180 185 190

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

195 200 205

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

210 215 220

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

225 230 235 240

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

245 250 255

<210> 11

<211> 255

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 11

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

1 5 10 15

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

20 25 30

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp

35 40 45

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

50 55 60

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Phe Glu Asp Pro

65 70 75 80

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

85 90 95

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

100 105 110

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

115 120 125

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr

130 135 140

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

145 150 155 160

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

165 170 175

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

180 185 190

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

195 200 205

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

210 215 220

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

225 230 235 240

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

245 250 255

<210> 12

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 12

Gly Phe Thr Phe Thr Ser Tyr Ala

1 5

<210> 13

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 13

Ile Ser His Gly Gly Thr Tyr Thr

1 5

<210> 14

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 14

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 15

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 15

Ser Gly Val Asn Tyr

1 5

<210> 16

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 16

Phe Thr Ser

1

<210> 17

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 17

Gln Gln Phe Thr Ser Ser Pro Tyr Thr

1 5

<210> 18

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 18

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 19

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 20

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 21

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Pro Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 22

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 22

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Pro Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 23

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 24

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 25

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 26

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 26

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 27

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 27

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 28

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 28

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 29

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 29

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 30

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 30

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 31

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 31

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 32

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 32

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Glu Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 33

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 33

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser Phe Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Thr Asn Lys Ala Leu Pro Ala

325 330 335

Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 34

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 34

Gly Phe Thr Phe Arg Ser Tyr Ala

1 5

<210> 35

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 35

Ile Ser His Lys Gly Thr Tyr Thr

1 5

<210> 36

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 36

Ile Ser His Lys Gly Lys Tyr Thr

1 5

<210> 37

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 37

Ile Ser His Arg Gly Thr Tyr Thr

1 5

<210> 38

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 38

Ile Ser His Arg Gly Arg Tyr Thr

1 5

<210> 39

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 39

Ile Ser Arg Gly Gly Thr Tyr Thr

1 5

<210> 40

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 40

Ile Ser Ser Gly Gly Thr Tyr Thr

1 5

<210> 41

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 41

Ile Ser His Gly Gly Lys Tyr Thr

1 5

<210> 42

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 42

Ile Ser His Gly Gly Arg Tyr Thr

1 5

<210> 43

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 43

Ile Ser His Gly Gly Trp Tyr Thr

1 5

<210> 44

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 44

Ile Ser His Gly Gly Tyr Tyr Thr

1 5

<210> 45

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 45

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 46

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 46

Ala Arg His Pro Ile Tyr Ser Gly Trp Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 47

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 47

Ala Arg His Pro Ile Tyr Ser Gly Gln Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 48

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 48

Ala Arg His Pro Ile Tyr Ala Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 49

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 49

Ala Arg His Pro Ile Tyr Cys Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 50

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 50

Ala Arg His Pro Ile Tyr Asp Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 51

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 51

Ala Arg His Pro Ile Tyr Glu Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 52

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 52

Ala Arg His Pro Ile Tyr Phe Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 53

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 53

Ala Arg His Pro Ile Tyr Gly Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 54

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 54

Ala Arg His Pro Ile Tyr His Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 55

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 55

Ala Arg His Pro Ile Tyr Ile Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 56

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 56

Ala Arg His Pro Ile Tyr Lys Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 57

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 57

Ala Arg His Pro Ile Tyr Leu Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 58

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 58

Ala Arg His Pro Ile Tyr Met Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 59

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 59

Ala Arg His Pro Ile Tyr Asn Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 60

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 60

Ala Arg His Pro Ile Tyr Pro Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 61

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 61

Ala Arg His Pro Ile Tyr Gln Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 62

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 62

Ala Arg His Pro Ile Tyr Arg Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 63

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 63

Ala Arg His Pro Ile Tyr Thr Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 64

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 64

Ala Arg His Pro Ile Tyr Val Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 65

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 65

Ala Arg His Pro Ile Tyr Trp Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 66

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 66

Ala Arg His Pro Ile Tyr Tyr Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 67

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 67

Ile Ser His Gly Gly Arg Tyr Thr

1 5

<210> 68

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 68

Ala Arg His Pro Ile His Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

1 5 10 15

<210> 69

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 69

Ser Gly Val Ile Tyr

1 5

<210> 70

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 70

Ser Gly Val Lys Tyr

1 5

<210> 71

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 71

Ser Gly Val Arg Tyr

1 5

<210> 72

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 72

Ser Gly Val Ser Tyr

1 5

<210> 73

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 73

Ser Gly Val Trp Tyr

1 5

<210> 74

<211> 5

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 74

Ser Gly Val Tyr Tyr

1 5

<210> 75

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 75

Gln Gln Phe Thr Arg Ser Pro Tyr Thr

1 5

<210> 76

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 76

Gln Gln Phe Thr Ser Lys Pro Tyr Thr

1 5

<210> 77

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 77

Gln Gln Phe Lys Ser Ser Pro Tyr Thr

1 5

<210> 78

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 78

Gln Gln Phe Arg Ser Ser Pro Tyr Thr

1 5

<210> 79

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 79

Gln Gln Phe Tyr Ser Ser Pro Tyr Thr

1 5

<210> 80

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 80

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Lys Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 81

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 81

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Lys Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 82

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 82

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Lys Gly Lys Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 83

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 83

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Arg Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 84

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 84

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Arg Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 85

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 85

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Arg Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Trp Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 86

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 86

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Arg Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 87

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 87

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Arg Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 88

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 88

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 89

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 89

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Arg Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Gln Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 90

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 90

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 91

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 91

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 92

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 92

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Trp Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 93

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 93

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ala Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 94

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 94

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Cys Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 95

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 95

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Asp Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 96

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 96

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Glu Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 97

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 97

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Phe Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 98

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 98

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Gly Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 99

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 99

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr His Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 100

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 100

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ile Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 101

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 101

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Lys Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 102

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 102

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Leu Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 103

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 103

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Met Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 104

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 104

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Asn Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 105

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 105

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Pro Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 106

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 106

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Gln Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 107

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 107

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Arg Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 108

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 108

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Thr Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 109

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 109

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Val Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 110

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 110

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Trp Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 111

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 111

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Tyr Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 112

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 112

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 113

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 113

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Lys Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 114

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 114

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Lys Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Trp Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 115

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 115

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 116

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 116

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Phe Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 117

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 117

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Trp Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 118

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 118

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Tyr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 119

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 119

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Lys Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 120

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 120

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile His Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 121

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 121

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 122

<211> 123

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 122

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Trp Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 123

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 123

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Ile Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 124

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 124

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Lys Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 125

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 125

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Arg Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 126

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 126

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Arg Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Arg Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 127

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 127

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Ser Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 128

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 128

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Trp Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 129

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 129

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Tyr Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 130

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 130

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Arg Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 131

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 131

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Lys Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 132

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 132

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Lys Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 133

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 133

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Arg Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 134

<211> 106

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 134

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Tyr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 135

<211> 213

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 135

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 136

<211> 213

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 136

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 137

<211> 213

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 137

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Pro Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 138

<211> 213

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 138

Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Gly Val Asn Tyr Leu

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Phe Thr Ser Thr Leu Ala Pro Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 139

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 139

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 140

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 140

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

<210> 141

<211> 452

<212> PRT

<213>artificial sequence

<220>

<223>synthetic peptide

<400> 141

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser His Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg His Pro Ile Tyr Ser Gly Asn Tyr Gln Gly Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly

450

Claims

1. Humanized anti-human C-X-C motif CC chemokine receptor 3 used in a kind of method in treatment leucoderma (CXCR3) antibody or its pharmaceutical composition, wherein the Humanized anti-human CXCR3 antibody includes with heavy chain variable region (VH) Heavy chain (HC) and the light chain (LC) with light chain variable region (VL), wherein

A) VH and VL include be respectively selected from SEQ ID NO:24/20,22/18,80/130,81/24,82/130,82/24, 83/126、83/130、83/24、84/126、84/24、85/24、86/126、87/126、87/133、87/24、88/24、89/ 24、90/126、91/126、91/130、92/130、92/24、93/126、94/126、95/126、96/126、97/126、98/ 126、99/126、100/126、101/126、102/126、103/126、104/126、105/126、106/126、107/126、 108/126、109/126、110/126、111/126、121/24、113/130、113/24、114/130、115/126、115/ 130、115/24、116/126、116/130、116/24、117/126、118/126、20/123、20/124、20/125、20/ 126、20/127、20/128、20/129、20/130、20/131、20/132、20/133、20/134、119/126、122/126、 With the amino acid sequence of 120/130 sequence pair, and wherein

B) heavy chain include the area human IgG1 Fc, the area the human IgG1 Fc have selected from SEQ ID NO:2,3,4,5,6,7,8, 9,10 and 11 amino acid sequence.

2. the Humanized anti-human CXCR3 antibody of claim 1, wherein the VH includes the amino acid sequence of SEQ ID NO:20, And the VL includes the amino acid sequence of SEQ ID NO:24.

3. the Humanized anti-human CXCR3 antibody of claim 2, wherein the area the human IgG1 Fc includes the amino of SEQ ID NO:2 Acid sequence.

4. the Humanized anti-human CXCR3 antibody of claim 2, wherein the area the human IgG1 Fc includes the amino of SEQ ID NO:9 Acid sequence.

5. the Humanized anti-human CXCR3 antibody of claim 2, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:10 Base acid sequence.

6. the Humanized anti-human CXCR3 antibody of claim 2, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:11 Base acid sequence.

7. the Humanized anti-human CXCR3 antibody of claim 1, wherein the VH includes the amino acid sequence of SEQ ID NO:18, And the VL includes the amino acid sequence of SEQ ID NO:22.

8. the Humanized anti-human CXCR3 antibody of claim 7, wherein the area the human IgG1 Fc includes the amino of SEQ ID NO:2 Acid sequence.

9. the Humanized anti-human CXCR3 antibody of claim 8, wherein the area the human IgG1 Fc includes the amino of SEQ ID NO:9 Acid sequence.

10. the Humanized anti-human CXCR3 antibody of claim 8, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:10 Base acid sequence.

11. the Humanized anti-human CXCR3 antibody of claim 8, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:11 Base acid sequence.

12. the Humanized anti-human CXCR3 antibody or its medicament preparation of claim 35, wherein

A) HC includes the amino acid sequence of SEQ ID NO:25, and the LC includes the amino acid of SEQ ID NO:135 Sequence,

B) HC includes the amino acid sequence of SEQ ID NO:26, and the LC includes the amino acid of SEQ ID NO:135 Sequence,

C) HC includes the amino acid sequence of SEQ ID NO:27, and the LC includes the amino acid of SEQ ID NO:135 Sequence,

D) HC includes the amino acid sequence of SEQ ID NO:139, and the LC includes the amino acid of SEQ ID NO:135 Sequence,

E) HC includes the amino acid sequence of SEQ ID NO:31, and the LC includes the amino acid of SEQ ID NO:137 Sequence,

F) HC includes the amino acid sequence of SEQ ID NO:32, and the LC includes the amino acid of SEQ ID NO:137 Sequence,

G) HC includes the amino acid sequence of SEQ ID NO:32, and the LC includes the amino acid of SEQ ID NO:137 Sequence,

H) HC includes the amino acid sequence of SEQ ID NO:140, and the HC includes the amino acid of SEQ ID NO:137 Sequence.

13. the Humanized anti-human CXCR3 antibody or its medicament preparation of any one of claim 1-12, wherein the IgG1 The area Fc has reduced fucose content.

14. the Humanized anti-human CXCR3 antibody or its medicament preparation of any one of claim 1-12, wherein the antibody is It is generated in following host cell, the host cell is cultivated in the presence of glycosylation inhibitor.

15. the Humanized anti-human CXCR3 antibody or its medicament preparation of claim 14, wherein the glycosylation inhibitor is base Husband's alkali.

16. a kind of method for treating leucoderma comprising comprising the heavy chain (HC) with heavy chain variable region (VH) and will have light The Humanized anti-human CXCR3 antibody of the light chain (LC) of chain variable region (VL) is given to subject in need thereof, wherein

B) heavy chain includes the area human IgG1 Fc selected from SEQ ID NO:2,3,4,5,6,7,8,9,10 and 11.

17. the method for claim 16, wherein the VH includes the amino acid sequence of SEQ ID NO:20, and the VL includes The amino acid sequence of SEQ ID NO:24.

18. the Humanized anti-human CXCR3 antibody of claim 17, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:2 Base acid sequence.

19. the Humanized anti-human CXCR3 antibody of claim 17, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:9 Base acid sequence.

20. the Humanized anti-human CXCR3 antibody of claim 17, wherein the area the human IgG1 Fc includes SEQ ID NO:10's Amino acid sequence.

21. the Humanized anti-human CXCR3 antibody of claim 17, wherein the area the human IgG1 Fc includes SEQ ID NO:11's Amino acid sequence.

22. the method for claim 16, wherein the VH includes the amino acid sequence of SEQ ID NO:18, and the VL includes The amino acid sequence of SEQ ID NO:22.

23. the Humanized anti-human CXCR3 antibody of claim 22, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:2 Base acid sequence.

24. the Humanized anti-human CXCR3 antibody of claim 22, wherein the area the human IgG1 Fc includes the ammonia of SEQ ID NO:9 Base acid sequence.

25. the Humanized anti-human CXCR3 antibody of claim 22, wherein the area the human IgG1 Fc includes SEQ ID NO:10's Amino acid sequence.

26. the Humanized anti-human CXCR3 antibody of claim 22, wherein the area the human IgG1 Fc includes SEQ ID NO:11's Amino acid sequence.

27. the method for claim 16, wherein

28. the method for any one of claim 16-27, wherein the HC includes the human IgG1 with reduced fucose content The area Fc.

29. the method for any one of claim 16-27, wherein the antibody is generated in following host cell, the place Chief cell is cultivated in the presence of glycosylation inhibitor.

30. the method for claim 29, wherein the glycosylation inhibitor is base husband's alkali.

31. a kind of method for treating leucoderma comprising providing will be comprising heavy chain (HC) and tool with heavy chain variable region (VH) There is the Humanized anti-human CXCR3 antibody of the light chain (LC) of light chain variable region (VL) to give to the explanation of subject in need thereof Book, wherein

32. it is a kind of for treating the kit of leucoderma, it includes

A) Humanized anti-human CXCR3 antibody, the Humanized anti-human CXCR3 antibody include the heavy chain with heavy chain variable region (VH) (HC) and with light chain variable region (VL) light chain (LC), wherein

I) VH and VL include be respectively selected from SEQID NO:24/20,22/18,80/130,81/24,82/130,82/24, 83/126、83/130、83/24、84/126、84/24、85/24、86/126、87/126、87/133、87/24、88/24、89/ 24、90/126、91/126、91/130、92/130、92/24、93/126、94/126、95/126、96/126、97/126、98/ 126、99/126、100/126、101/126、102/126、103/126、104/126、105/126、106/126、107/126、 108/126、109/126、110/126、111/126、121/24、113/130、113/24、114/130、115/126、115/ 130、115/24、116/126、116/130、116/24、117/126、118/126、20/123、20/124、20/125、20/ 126、20/127、20/128、20/129、20/130、20/131、20/132、20/133、20/134、119/126、122/126、 With the amino acid sequence of 120/130 sequence pair, and wherein

Ii) HC also includes the area human IgG1 Fc, the area the human IgG1 Fc include selected from SEQ ID NO:2,3,4,5,6,7, 8,9,10 or 11 amino acid sequence；And

B) the Humanized anti-human CXCR3 is given to the specification of human experimenter in need thereof.