CN110300763A - For treating the anti-human CXCR3 antibody of leucoderma - Google Patents
For treating the anti-human CXCR3 antibody of leucoderma Download PDFInfo
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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
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 areaHOr VH) and heavy chain constant region (CHOr CH).Heavy chain constant region includes three structural domains, CH1、CH2 and CH3.Heavy chain
Fc part include CH2 and CH3。
Every light chain includes that light chain variable region (is abbreviated as VL) and constant region of light chain (CLOr CL).Constant region of light chain includes one
A structural domain (CL1)。
VHAnd VLRegion 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 VHAnd VLIt 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.
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;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.
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: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.
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: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.
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: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.
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: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.
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: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%Man5-9(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 1a | Hair tonic Capability index 2b |
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
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)
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 SWXLThe TSK SW of guard columnXLSize 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 Thy1a/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
<210> 1
<211> 368
<212> PRT
<213>homo sapiens
<400> 1
Met Val Leu Glu Val Ser Asp His Gln Val Leu Asn Asp Ala Glu Val
1 5 10 15
Ala Ala Leu Leu Glu Asn Phe Ser Ser Ser Tyr Asp Tyr Gly Glu Asn
20 25 30
Glu Ser Asp Ser Cys Cys Thr Ser Pro Pro Cys Pro Gln Asp Phe Ser
35 40 45
Leu Asn Phe Asp Arg Ala Phe Leu Pro Ala Leu Tyr Ser Leu Leu Phe
50 55 60
Leu Leu Gly Leu Leu Gly Asn Gly Ala Val Ala Ala Val Leu Leu Ser
65 70 75 80
Arg Arg Thr Ala Leu Ser Ser Thr Asp Thr Phe Leu Leu His Leu Ala
85 90 95
Val Ala Asp Thr Leu Leu Val Leu Thr Leu Pro Leu Trp Ala Val Asp
100 105 110
Ala Ala Val Gln Trp Val Phe Gly Ser Gly Leu Cys Lys Val Ala Gly
115 120 125
Ala Leu Phe Asn Ile Asn Phe Tyr Ala Gly Ala Leu Leu Leu Ala Cys
130 135 140
Ile Ser Phe Asp Arg Tyr Leu Asn Ile Val His Ala Thr Gln Leu Tyr
145 150 155 160
Arg Arg Gly Pro Pro Ala Arg Val Thr Leu Thr Cys Leu Ala Val Trp
165 170 175
Gly Leu Cys Leu Leu Phe Ala Leu Pro Asp Phe Ile Phe Leu Ser Ala
180 185 190
His His Asp Glu Arg Leu Asn Ala Thr His Cys Gln Tyr Asn Phe Pro
195 200 205
Gln Val Gly Arg Thr Ala Leu Arg Val Leu Gln Leu Val Ala Gly Phe
210 215 220
Leu Leu Pro Leu Leu Val Met Ala Tyr Cys Tyr Ala His Ile Leu Ala
225 230 235 240
Val Leu Leu Val Ser Arg Gly Gln Arg Arg Leu Arg Ala Met Arg Leu
245 250 255
Val Val Val Val Val Val Ala Phe Ala Leu Cys Trp Thr Pro Tyr His
260 265 270
Leu Val Val Leu Val Asp Ile Leu Met Asp Leu Gly Ala Leu Ala Arg
275 280 285
Asn Cys Gly Arg Glu Ser Arg Val Asp Val Ala Lys Ser Val Thr Ser
290 295 300
Gly Leu Gly Tyr Met His Cys Cys Leu Asn Pro Leu Leu Tyr Ala Phe
305 310 315 320
Val Gly Val Lys Phe Arg Glu Arg Met Trp Met Leu Leu Leu Arg Leu
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Gly Cys Pro Asn Gln Arg Gly Leu Gln Arg Gln Pro Ser Ser Ser Arg
340 345 350
Arg Asp Ser Ser Trp Ser Glu Thr Ser Glu Ala Ser Tyr Ser Gly Leu
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<213>homo sapiens
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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 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 Ile 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
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Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
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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 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 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 Ile 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> 4
<211> 255
<212> PRT
<213>synthetic peptide
<400> 4
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 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 Ile 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> 5
<211> 255
<212> PRT
<213>synthetic peptide
<400> 5
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 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 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 Ile 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> 6
<211> 255
<212> PRT
<213>synthetic peptide
<400> 6
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 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 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 Ile 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> 7
<211> 255
<212> PRT
<213>artificial sequence
<220>
<223>synthetic peptide
<400> 7
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 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
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> 8
<211> 255
<212> PRT
<213>artificial sequence
<220>
<223>synthetic peptide
<400> 8
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 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
225 230 235 240
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
245 250 255
<210> 9
<211> 255
<212> PRT
<213>artificial sequence
<220>
<223>synthetic peptide
<400> 9
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 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>
<223>synthetic peptide
<400> 10
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 (32)
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
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 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
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.
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
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.
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.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US201662437889P | 2016-12-22 | 2016-12-22 | |
US62/437,889 | 2016-12-22 | ||
EP17306770 | 2017-12-14 | ||
EP17306770.3 | 2017-12-14 | ||
PCT/US2017/067988 WO2018119288A1 (en) | 2016-12-22 | 2017-12-21 | Anti-human cxcr3 antibodies for treatment of vitiligo |
Publications (1)
Publication Number | Publication Date |
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CN110300763A true CN110300763A (en) | 2019-10-01 |
Family
ID=61003387
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Application Number | Title | Priority Date | Filing Date |
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CN201780086634.5A Pending CN110300763A (en) | 2016-12-22 | 2017-12-21 | For treating the anti-human CXCR3 antibody of leucoderma |
Country Status (4)
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EP (1) | EP3559036A1 (en) |
JP (1) | JP2020514273A (en) |
KR (1) | KR20190095942A (en) |
CN (1) | CN110300763A (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070092521A1 (en) * | 2005-10-21 | 2007-04-26 | Mcpherson John M | Antibody-based therapeutics with enhanced adcc activity |
WO2013109974A2 (en) * | 2012-01-20 | 2013-07-25 | Genzyme Corporation | Anti-cxcr3 antibodies |
WO2014186842A1 (en) * | 2013-05-22 | 2014-11-27 | Monash University | Antibodies and uses thereof |
-
2017
- 2017-12-21 JP JP2019534287A patent/JP2020514273A/en active Pending
- 2017-12-21 CN CN201780086634.5A patent/CN110300763A/en active Pending
- 2017-12-21 KR KR1020197020838A patent/KR20190095942A/en unknown
- 2017-12-21 EP EP17832143.6A patent/EP3559036A1/en not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070092521A1 (en) * | 2005-10-21 | 2007-04-26 | Mcpherson John M | Antibody-based therapeutics with enhanced adcc activity |
WO2013109974A2 (en) * | 2012-01-20 | 2013-07-25 | Genzyme Corporation | Anti-cxcr3 antibodies |
CN104507967A (en) * | 2012-01-20 | 2015-04-08 | 建新公司 | Anti-cxcr3 antibodies |
WO2014186842A1 (en) * | 2013-05-22 | 2014-11-27 | Monash University | Antibodies and uses thereof |
Non-Patent Citations (1)
Title |
---|
JILLIAN RICHMOND等: ""Vitiligo International Symposium Rome 2-3 December 2016"", 《IFPCS》 * |
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KR20190095942A (en) | 2019-08-16 |
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