Chimeric Antigen Receptor Targeting CD22 and CD19 and Application thereof

Abstract

The present invention discloses a nucleic acid molecule for encoding a chimeric antigen receptor targeting CD22 and CD19. The chimeric antigen receptor of the present invention can be used for treatment of CD19.sup.+ and CD22.sup.+ B-cell hematological tumors, as well as combined treatment with CD19 CAR-T cells or CD22 CAR-T cells.

Claims

1. A nucleic acid molecule for encoding a chimeric antigen receptor targeting CD22 and CD19, wherein the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular signal transduction region, and the extracellular region encoded by the nucleic acid molecule comprises a CD22 and CD19 binding domain consisting of a CD22 single-chain fragment variable and a CD19 single-chain fragment variable; and the CD22 single-chain fragment variable and the CD19 single-chain fragment variable are arranged according to an amino acid sequence shown in SEQ ID No.9, an amino acid sequence shown in SEQ ID No.10, an amino acid sequence shown in SEQ ID No.11, an amino acid sequence shown in SEQ ID No. 12, an amino acid sequence shown in SEQ ID No. 23, or an amino acid sequence shown in SEQ ID No. 24 in order.

2. The nucleic acid molecule according to claim 1, wherein the extracellular region encoded by the nucleic acid molecule further comprises a signal peptide constructed at an amino terminal of the chimeric antigen receptor or an amino acid sequence having 90%-99% homology with the signal peptide, and the signal peptide is a signal peptide sequence in CD8α or GM-CSF, preferably signal peptide shown in SEQ ID NO.13.

3. The nucleic acid molecule according to claim 1, wherein the CD22 and CD19 binding domains encoded by the nucleic acid molecule are connected to the transmembrane region encoded by the nucleic acid molecule through a hinge region, and the hinge region is preferably a hinge region sequence in CD8α; and the transmembrane region is a transmembrane domain selected from the following proteins or an amino acid sequence having 90%-99% homology with the proteins: α, β or ζ chains of T cell receptors, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB or CD154.

4. The nucleic acid molecule according to claim 1, wherein the intracellular signal transduction region encoded by the nucleic acid molecule further comprises a costimulatory factor.

5. The nucleic acid molecule according to claim 4, wherein the costimulatory factor is one or more of functional signal domains obtained by selecting from the following proteins or the amino acid sequence having 90%-99% homology with the proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49α, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11α, ITGAM, CD11b, ITGAX, CD11c, CD29, ITGB1, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, CD226, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, CD162, LTBR, LAT, GADS or SLP-76.

6. The nucleic acid molecule according to claim 5, wherein the costimulatory factor is CD28 or 4-1BB or an amino acid sequence having 90%-99% homology therewith.

7. The nucleic acid molecule according to claim 1, wherein a sequence of the nucleic acid molecule is shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

8. A chimeric antigen receptor targeting CD22 and CD19, wherein the chimeric antigen receptor is encoded by the nucleic acid molecule according to claim 1.

9. The chimeric antigen receptor according to claim 8, wherein an amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO. NO.4, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18.

10. A vector, comprising the nucleic acid molecule according to claim 1.

11. A cell, comprising the nucleic acid molecule according to claim 1.

12. Application of the nucleic acid molecule according to claim 1 in preparation of anti-B-cell hematological tumor drugs, wherein B-cell hematological tumors are preferably B-cell lymphoma or acute B-lymphocytic leukemia.

13. Application of the chimeric antigen receptor according to claim 9 in preparation of anti-B-cell hematological tumor drugs, wherein B-cell hematological tumors are preferably B-cell lymphoma or acute B-lymphocytic leukemia.

14. Application of the vector according to claim 10 in preparation of anti-B-cell hematological tumor drugs, wherein B-cell hematological tumors are preferably B-cell lymphoma or acute B-lymphocytic leukemia.

15. Application of the cell according to claim 11 in preparation of anti-B-cell hematological tumor drugs, wherein B-cell hematological tumors are preferably B-cell lymphoma or acute B-lymphocytic leukemia.

16. The application according to claim 12, wherein the application is application in the preparation of the anti-B cell hematological tumor drugs for simultaneous expression of CD19 and CD22 or for recurrence or ineffectiveness of a patient after CD19 CAR-T treatment due to antigenic variation.

17. A pharmaceutical composition, comprising the nucleic acid molecule according to claim 1, and a pharmaceutically acceptable vector.

18. A pharmaceutical composition, comprising the chimeric antigen receptor according to claim 9, and a pharmaceutically acceptable vector.

19. Application of the pharmaceutical composition according to claim 17 in treatment of B-cell hematological tumors.

20. A method for treatment of B-cell hematological tumors, using the pharmaceutical composition according to claim 17.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1A and FIG. 1B are restriction endonuclease digestion segment electrophoresis identification diagrams of lentiviral expression vectors of CD19scFv-CD22scFv-CD8α-4-1 BB-CD3ζ, CD22scFv-CD19scFv-CD8α-4-1 BB-CD3ζ, CD19 V.sub.L-CD22 V.sub.L-CD22 V.sub.H-CD19 V.sub.H-CD8α- 4-1 BB-CD3ζ and CD22 V.sub.L-CD19 V.sub.L-CD19 V.sub.H-CD22 V.sub.H-CD8α-4-1 BB-CD3ζ in an embodiment of the present invention; in FIG. 1A, 1 is a 15kb nucleic acid molecular weight marker lane, 2 is a DNA segment (2314bp) and a vector segment (7228bp) encoding CD19 scFv-CD22 scfv-CD8α-4-1BB-CD34 obtained by double digestion of a lentiviral expression plasmid CD19 scFv-CD22 scFv-CD8α-4-1 BB-CD3ζ with endonucleases Nhel and Notl, and 3 is a DNA segment (2314bp) and a vector segment (7228bp) encoding CD22 scFv-CD19 scfv-CD8α-4-1 BB-CD3ζ obtained by double digestion of a lentiviral expression plasmid CD22 scFv-CD19 scFv-CD8α-4-1BB-CD3ζ with endonucleases Nhe I and Not I; and in FIG. 1B, 4 is a 15kb nucleic acid molecular weight marker lane, 5 is a DNA segment (2233bp) and a vector segment (7234bp) encoding CD19V.sub.L-CD22V.sub.L-CD22V.sub.H-CD19V.sub.H-CD8α-4-1BB-CD3ζ obtained by double digestion of a lentiviral expression plasmid CD19 V.sub.L-CD22V.sub.L-CD22V.sub.H-CD19V.sub.H-CD8α-4-1BB-CD3ζ with endonucleases Nhel and Notl, and 6 is a DNA segment (2233bp) and a vector segment (7234bp) encoding CD22 V.sub.L-CD19 V.sub.L-CD19V.sub.H-CD22V.sub.H-CD8α-4-1BB-CD3ζ obtained by double digestion of a lentiviral expression plasmid CD22 V.sub.L-CD19 V.sub.L-CD19V.sub.H-CD22V.sub.H-CD8α-4-1 BB-CD3ζ with endonucleases Nhel and Notl.

[0058] FIG. 2 is a schematic diagram with a lentiviral expression vector of CD22 scFv-CD19 scFv-CD8α-4-1 BB-CD3ζ as an example in an embodiment of the present invention, where a counterclockwise sequence is a forward gene segment, and a clockwise sequence is a reverse gene segment.

[0059] FIG. 3A shows results(represented as 19-22 CAR-T, 22-19 CAR-T, 19×22 CAR-T, 22×19 CAR-T, respectively) of testing expression of CAR molecules in dual CAR-target CD19 scFv-CD22 scFv-CD8α-4-1BB-CD3ζ, CD22 scFv-CD19 scFv-CD8α-4-1BB-CD3ζ, CD19 V.sub.L-CD22 V.sub.L-CD22 V.sub.H-CD19 V.sub.H-CD8α-4- 1BB-CD3ζ and CD22 V.sub.L-CD19 V.sub.L-CD19 V.sub.H-CD22 V.sub.H-CD8α-4-1BB-CD3ζ modified T cells constructed in an embodiment of the present invention using flow cytometry, and also shows results (represented as 19 CAR-T and 22 CAR-T, respectively) of expression of CAR molecules in single CAR-target CD19 scFv-CD8α-4-1 BB-CD3ζ and CD22 scFv-CD8α-4-1 BB-CD3ζ modified T cells, to compare functions of single-target and dual-target CAR-T. GFP is expression of marker proteins carried by a vector. F(ab′).sub.2 is rabbit anti-mouse IgG to detect (or label) CD22 scFv and CD19 scFv on the surface of T cells. FIG. 3B shows results (represented as1922-BB, 1922-28B, 1922-B28, and 1922-2828, respectively) of testing expression of CAR molecules in dual-target CD19 scFv-CD8α-4-1BB-CD3ζ-T2A-CD22 scFv-CD8α-4-1 BB-CD3ζ, CD19 scFv-CD8α-CD28-CD3ζ-T2A-CD22 scFv-CD8α-4-1 BB-CD3ζ, CD19 scFv-CD8α-4-1BB-CD3ζ-T2A-CD22 scFv-CD8α-CD28-CD3ζ and CD19 scFv-CD8α-28-CD3ζ-T2A-CD22 scFv-CD8α-28-CD3ζ-modified T cells constructed in an embodiment of the present invention using flow cytometry.

[0060] FIG. 4A and FIG. 4B show results of testing expression of target cells applied in an embodiment of the present invention: a Burkitt lymphoma cell line Namalwa, a chronic granulocytic leukemia cell line MV4-11-CD19 infected with CD19 antigen molecules, a chronic granulocytic leukemia cell line MV4-11-CD22 infected with CD22 antigen molecules, and CD19 and CD22 antigen molecules in wild-type MV4-11 cells by using flow cytometry, where FIG. 4A is expression rate of CD19 antigen molecules; and FIG. 4B is expression rate of CD22 antigen molecules.

[0061] FIG. 5A to FIG. 5L show results of residual tumor cells after co-culture of T cells with target tumor cells in an embodiment of the present invention using flow cytometry, where vector-T is a control group of transfected empty vector-T cells; 19CAR-T is an experimental group of CD19 scFv-CD8α-4-1BB-CD3ζ-modified T cells, 22CAR-T is an experimental group of CD22 scFv-CD8α-4-1 BB-CD3ζ-modified T cells, 19-22CAR-T is an experimental group of CD19 scFv-CD22 scFv-CD8α-4-1BB-CD3ζ-modifiedT cells, 22-19 CAR-T is an experimental group of CD22 scFv-CD19 scFv-CD8α-4-1BB-CD3ζ-modifiedT cells, 19×22 CAR-T is an experimental group of CD19 V.sub.L-CD22V.sub.L-CD22V.sub.HCD19V.sub.HCD8a-4-1BB-CD3ζ-modified T cells, and 22×19 CAR-T is an experimental group of CD22 V.sub.L-CD19V.sub.L-CD19V.sub.HCD22V.sub.H CD8α-4-1 BB-CD3ζ-modified T cells. FIG. 5A and FIG. 5B respectively show percentages of residual tumor cells after co-culture of vector-T, 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T with target tumor cells for 24 hours at different effector: target ratios (E:T ratio) of 1:8, 1:4, 1:2 and 1:1, respectively, and a schematic flow diagram of residual tumor cells percentages after co-culture for 48 hours at E:T ratio of 1:1 when the target cells were Namalwa cell lines. FIG. 5C and FIG. 5D respectively show percentages of residual tumor cells after co-culture of vector-T, 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T with target tumor cells for 24 hours at E:T ratio of 1:8, 1:4, 1:2 and 1:1, respectively, and a schematic flow diagram of percentages of residual tumor cells after co-culture for 48 hours at E:T ratio of 1:1 when the target cells were MV4-11-CD19 cell lines. FIG. 5E and FIG. 5F respectively show percentages of residual tumor cells after co-culture of vector-T, 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T with target cells for 24 hours at E:T ratio of 1:8, 1:4, 1:2 and 1:1, respectively, and a schematic flow diagram of percentages of residual tumor cells after co-culture for 48 hours at E:T ratio of 1:1 when the target cells were MV4-11-CD22 cell lines. FIG. 5G and FIG. 5H respectively show percentages of residual tumor cells after co-culture of Vec-T, 19-22 CAR-T and 22-19 CAR-T with target cells for 24 hours at E:T ratio of 1:8, 1:4, 1:2 and 1:1, respectively, and a schematic flow diagram of percentages of residual tumor cells after co-culture for 48 hours at E:T ratio of 1:1 when the target cells were wild-type MV4-11 cell lines. FIG. 5I shows percentages of residual tumor cells after co-culture of vector-T, 19×22 CAR-T and 22×19 CAR-T with target cells for 24 hours at E:T ratio of 1:2, 1:1 and 2:1 when the target cells were MV4-11-CD19 and MV4-11-CD22 mixed at a ratio of 1:1. FIG. 5J shows percentages of residual tumor cells after co-culture of vector-T, 19×22 CAR-T and 22×19 CAR-T with target cells four 24 hours at E:T ratio of 1:2, 1:1 and 2:1, respectively, when the target cells were MV4-11. FIG. 5K shows percentages of residual tumor cells after co-culture of vector-T,1922-BB CAR-T, 1922-28B CAR-T, 1922-B28 CAR-Tand 1922-2828 CAR-T with target cells for 48 hours at E:T ratio of 1:1, 1:3 and 1:9, respectively, when the target cells were MV4-11-CD19 and MV4-11-CD22 mixed at a ratio of 1:1. FIG. 5L shows percentages of residual tumor cells after co-culture of vector-T, 1922-BB CAR-T, 1922-28B CAR-T, 1922-B28 CAR-T and 1922-2828 CAR-T with target cells for 48 hours at E:T ratio of 1:1, 1:3 and 1:9, respectively, when the target cells were MV4-11.

[0062] FIG. 6A and FIG. 6B show results of degranulation assays for a killing effect of vector-T and CAR-T on Namalwa, MV4-11-CD19, MV4-11-CD22 and MV4-11 (E:T ratio of 1:1) in an embodiment of the present invention, where CAR-T in FIG. 6A are 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T, respectively, and CAR-T in FIG. 6B are 19×22 CAR-T and 22×19 CAR-T, respectively.

[0063] FIG. 7A to FIG. 7C show results of levels of cytokines TNF-α (FIG. 7A) IL-2 (FIG. 7B) and IFN-γ(FIG. 7C) released from T cells after co-culture of vector-T, 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T with four target cells Namalwa, MV4-11-CD19, MV4-11-CD22 and MV4-11, respectively, for 48 hours at E:T ratio of 1:1 in an embodiment of the present invention.

[0064] FIG. 8 shows expression results of CD22 and CD19 target antigen molecules in bone marrow mononuclear cells (BMMCs) of B-ALL patients using flow cytometry, where P1 to P4 represent patient numbers.

[0065] FIG. 9 shows results of testing percentages of residual tumor cells by flow cytometry after co-culture of 22-19 CAR-T and 19-22 CAR-T with BMMCs from B-ALL patients at E:T ratio of 1:4 for 48 hours in an embodiment of the present invention.

[0066] FIG. 10 shows results of degranulation assays for a killing effect of 22-19 CAR-T and 19-22 CAR-T on BMMCs from B-ALL patients in an embodiment of the present invention.

[0067] FIG. 11A to FIG. 11D show evaluation results of an in vivo effect of 22-19 CAR-T and 19-22 CAR-T in xenograft mice in an embodiment of the present invention. Female NOD/SCID mice at 6-8 weeks were selected and randomly divided into two groups, and 5×10.sup.6 Namalwa cells were inoculated via tail vein on day 0, and 1×10.sup.7 vec-T cells or CAR-T cells (as shown in FIG. 11A) were administered intravenously on day 5, 9 and 12, respectively, and the mice were monitored weekly for weight change (as shown in FIG. 11B) after T cell injection. Tumor load plots are shown in FIG. 11C. Survival curves of the mice are shown in FIG. 11D. Survival time was calculated using SPSS software.

[0068] FIG. 12A and FIG. 12B show evaluation results of an in vivo effect of 22×19 CAR-T in mice in an embodiment of the present invention. Female NOD/SCID mice at 6-8 weeks were selected and randomly divided into two groups, 2.5×10.sup.6 Namalwa cells were inoculated via tail vein on day 0, and 1×10.sup.7 vec-T cells or CAR-T cells were administered intravenously on day 5, 9 and 12 (as shown in FIG. 12A), respectively. Survival curves of the mice are shown in FIG. 12B. Survival time was calculated using SPSS software.

SEQUENCE DESCRIPTION

[0069] SEQ ID NO.1 is an amino acid sequence of CD19 scFv-CD22 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0070] SEQ ID NO.2 is an amino acid sequence of CD22 scFv-CD19 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0071] SEQ ID NO.3 is an amino acid sequence of CD19 VL-CD22 VL-CD22 VH-CD19 V.sub.H-CD8α-4-1 BB-CD3ζ of the present invention;

[0072] SEQ ID NO.4 is an amino acid sequence of CD22 VL-CD19 VL-CD19 VH-CD22 VH-CD8α-4-1BB-CD3ζ of the present invention;

[0073] SEQ ID NO.5 is a nucleic acid sequence of CD19 scFv-CD22 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0074] SEQ ID NO.6 is a nucleic acid sequence of CD22 scFv-CD19 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0075] SEQ ID NO.7 is a nucleic acid sequence of CD19 VL-CD22 VL-CD22 VH-CD19 VH-CD8α-4-1BB-CD3ζ of the present invention;

[0076] SEQ ID NO.8 is a nucleic acid sequence of CD22 VL-CD19 VL-CD19 VH-CD22 VH-CD8α-4-1BB-CD3ζ of the present invention;

[0077] SEQ ID NO.9 is an amino acid sequence of a CD19 scFv-CD22 scFv-CD8α-4-1 BB-CD3ζ antigen recognition region of the present invention;

[0078] SEQ ID NO.10 is an amino acid sequence of a CD22 scFv-CD19 scFv-CD8α-4-1 BB-CD3ζ antigen recognition region of the present invention;

[0079] SEQ ID NO.11 is an amino acid sequence of a CD19 VL-CD22 VL-CD22 VH-CD19 VH-CD8α-4-1 BB-CD3ζ antigen recognition region of the present invention;

[0080] SEQ ID NO.12 is an amino acid sequence of a CD22 VL-CD19 VL-CD19 VH-CD22 VH-CD8α-4-1 BB-CD3ζ antigen recognition region of the present invention;

[0081] SEQ ID NO.13 is an amino acid sequence of a signal peptide in a chimeric antigen receptor targeting CD19 and CD22 of the present invention;

[0082] SEQ ID NO.14 is an amino acid sequence of CD8α-4-1BB-CD3ζ in a chimeric antigen receptor targeting CD19 and CD22 of the present invention;

[0083] SEQ ID NO.15 is an amino acid sequence of CD19 scFv-CD8α-4-1BB-CD3ζ-T2A-CD22 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0084] SEQ ID NO.16 is an amino acid sequence of CD19 scFv-CD8α-CD28-CD3ζ-T2A-CD22 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0085] SEQ ID NO.17 is an amino acid sequence of CD19 scFv-CD8α-4-1BB-CD3ζ-T2A-CD22 scFv-CD8α-CD28-CD3ζ of the present invention;

[0086] SEQ ID NO.18 is an amino acid sequence of CD19 scFv-CD8α-28-CD3ζ-T2A-CD22 scFv-CD8α-28-CD3ζ of the present invention;

[0087] SEQ ID NO.19 is a nucleic acid sequence of CD19 scFv-CD8α-4-1BB-CD3ζ-T2A-CD22 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0088] SEQ ID NO.20 is a nucleic acid sequence of CD19 scFv-CD8α-CD28-CD3ζ-T2A-CD22 scFv-CD8α-4-1 BB-CD3ζ of the present invention;

[0089] SEQ ID NO.21 is a nucleic acid sequence of CD19 scFv-CD8α-4-1 BB-CD3ζ-T2A-CD22 scFv-CD8α-CD28-CD3ζ of the present invention;

[0090] SEQ ID NO.22 is a nucleic acid sequence of CD19 scFv-CD8α-CD28-CD3ζ-T2A-CD22 scFv-CD8α-CD28-CD3ζ of the present invention;

[0091] SEQ ID NO.23 is an amino acid sequence of a CD19 scFv-CD8α-4-1 BB-CD3ζ antigen recognition region of the present invention;

[0092] SEQ ID NO.24 is an amino acid sequence of a CD22 scFv-CD8α-CD28-CD3ζ antigen recognition region of the present invention;

[0093] SEQ ID NO.25 is an amino acid sequence of CD8α-CD28-CD3ζ in a chimeric antigen receptor targeting CD19 and CD22 of the present invention; and

[0094] SEQ ID NO. 26 is an amino acid sequence of T2A in a dual-target chimeric antigen receptor targeting CD19 and CD22 in the present invention.

TABLE-US-00001 SEQUENCE LIST Number Sequence SEQ ID NO.1 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSSEAAAKEAAAKEAAAKEAAAKE AAAKDIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAW YQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSVKA EDLAVYYCQQSYSYPFTFGSGTKLEIKRGGGGSGGGGSGGGGS QVKLQQSGPELVKPGASVKISCKASGYDFSISWMNWVRQRPGQG LEWIGRIYPGDGDSNYNGKFEGKATLTADKSSSTAYMQLSGLTSV DSAVYFCARTTTMIALYAMDYWGQGTTVTVSSEFTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO.2 DIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQK PGQSPKLLlYWASTRESGVPDRFTGSGSGTDFTLTVSSVKAEDLA VYYCQQSYSYPFTFGSGTKLEIKRGGGGSGGGGSGGGGSQVKL QQSGPELVKPGASVKISCKASGYDFSISWMNWVRQRPGQGLEWI GRIYPGDGDSNYNGKFEGKATLTADKSSSTAYMQLSGLTSVDSAV YFCARTTTMIALYAMDYWGQGTTVTVSSEAAAKEAAAKEAAAKEA AAKEAAAKDIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWY QQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSK DLADYFCQQYNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQ VQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQG LEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSE DSAVYFCARKTISSVVDFYFDYWGQGTTLTVSSEFTTTPAPRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO.3 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSDIELTQSPSSLAVSVGEKVTMS CKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPD RFTGSGSGTDFTLTVSSVKAEDLAVYYCQQSYSYPFTFGSGTKLEI KRGSTSGSGKPGSGEGSTKGQVKLQQSGPELVKPGASVKISCKA SGYDFSISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKFEGK ATLTADKSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMDYWG QGTTVTVSSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTAD KSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTL TVSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR SEQ ID NO.4 DIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQK PGQSPKLLlYWASTRESGVPDRFTGSGSGTDFTLTVSSVKAEDLA VYYCQQSYSYPFTFGSGTKLEIKRGGGGSDIVLTQSPKFMSTSVG DRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPD RFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEI KRGSTSGSGKPGSGEGSTKGQVQLQQSGAELVRPGSSVKISCKA SGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQ ATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYW GQGTTLTVSSGGGGSQVKLQQSGPELVKPGASVKISCKASGYDF SISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKFEGKATLTAD KSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMDYWGQGTTVT VSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITLYKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR SEQ ID NO.5 gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtgagcgt gacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaacccg gccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccgacag atttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagagcaaa gacctggccgactacttctgccagcagtacaacagatacccctacaccagcggaggagga acaaagctggagatcaagagaggtggtggtggttctggcggcggcggctccggtggtggtg gttctcaagtgcaactgcaacagagcggagccgaactggtgagacccggaagcagcgtga agatcagctgcaaggcttccggctacgcctttagcagctactggatgaactgggtgaagcag agacctggacagggactggaatggatcggccagatttaccctggagacggcgacacaaac tacaacggcaagttcaagggccaagctacactgaccgccgacaaaagcagcagcaccgc ctatatgcagctgagcggactgaccagcgaagatagcgctgtgtacttctgcgccagaaaga ccatcagcagcgtggtggacttctacttcgactactggggacaaggcaccaccctgacagtg agcagcgaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgct gctaaggaagccgctgctaaggacattgagctcacccagtctccatcctccctagctgtgtca gttggagagaaggttactatgagctgcaagtccagtcagagccttttatatagtagcaatcaaa agaactatttggcctggtaccagcagaaaccagggcagtctcctaaactgctgatttactggg catccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagatttcac tctcaccgtcagcagtgtgaaggctgaagacctggcagtttattactgtcagcaatcttatagtta tccattcacattcggctcgggcaccaagctggaaatcaaacggggtggtggtggttctggcgg cggcggctccggtggtggtggttctcaggtcaaactgcagcagtcaggacctgaactggtga agcctggggcctcagtgaagatttcctgcaaagcttctggctacgatttcagtatttcttggatga actgggtgaggcagaggcctggacagggtcttgagtggattggacggatttatcctggagatg gagatagtaactacaatgggaagttcgagggcaaggccacactgactgcagacaaatcctc cagcacagcctacatgcagctcagcggcctgacctctgtggactctgcggtctatttttgtgcaa gaaccaccactatgattgccctctatgctatggactactggggccaagggaccacggtcacc gtctcctcagaattcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatc gcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg cacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtg gggtccttctcctgtcactggttatcaccctttacaaacggggcagaaagaaactcctgtatatat tcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccga tttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagac gcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaaga gaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccg agaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcgga ggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcc tttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctg ccccctcgc SEQ ID gacattgagctcacccagtctccatcctccctagctgtgtcagttggagagaaggttactatgag NO.6 ctgcaagtccagtcagagccttttatatagtagcaatcaaaagaactatttggcctggtaccag cagaaaccagggcagtctcctaaactgctgatttactgggcatccactagggaatctggggtc cctgatcgcttcacaggcagtggatctgggacagatttcactctcaccgtcagcagtgtgaagg ctgaagacctggcagtttattactgtcagcaatcttatagttatccattcacattcggctcgggcac caagctggaaatcaaacggggtggtggtggttctggcggcggcggctccggtggtggtggttc tcaggtcaaactgcagcagtcaggacctgaactggtgaagcctggggcctcagtgaagattt cctgcaaagcttctggctacgatttcagtatttcttggatgaactgggtgaggcagaggcctgga cagggtcttgagtggattggacggatttatcctggagatggagatagtaactacaatgggaagt tcgagggcaaggccacactgactgcagacaaatcctccagcacagcctacatgcagctca gcggcctgacctctgtggactctgcggtctatttttgtgcaagaaccaccactatgattgccctct atgctatggactactggggccaagggaccacggtcaccgtctcctcagaagccgctgctaag gaagccgctgctaaggaagccgctgctaaggaagccgctgctaaggaagccgctgctaag gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtgagcgt gacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaacccg gccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccgacag atttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagagcaaa gacctggccgactacttctgccagcagtacaacagatacccctacaccagcggaggagga acaaagctggagatcaagagaggtggtggtggttctggcggcggcggctccggtggtggtg gttctcaagtgcaactgcaacagagcggagccgaactggtgagacccggaagcagcgtga agatcagctgcaaggcttccggctacgcctttagcagctactggatgaactgggtgaagcag agacctggacagggactggaatggatcggccagatttaccctggagacggcgacacaaac tacaacggcaagttcaagggccaagctacactgaccgccgacaaaagcagcagcaccgc ctatatgcagctgagcggactgaccagcgaagatagcgctgtgtacttctgcgccagaaaga ccatcagcagcgtggtggacttctacttcgactactggggacaaggcaccaccctgacagtg agcagcgaattcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgc gtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgca cacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggg gtccttctcctgtcactggttatcaccctttacaaacggggcagaaagaaactcctgtatatattc aaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgattt ccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacg cccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagag aggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccga gaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggag gcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctt taccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgc cccctcgc SEQ ID NO.7 gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtgagcgt gacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaacccg gccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccgacag atttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagagcaaa gacctggccgactacttctgccagcagtacaacagatacccctacaccagcggaggagga acaaagctggagatcaagagaggcggcggaggttctgacattgagctcacccagtctccat cctccctagctgtgtcagttggagagaaggttactatgagctgcaagtccagtcagagcctttta tatagtagcaatcaaaagaactatttggcctggtaccagcagaaaccagggcagtctcctaa actgctgatttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtgga tctgggacagatttcactctcaccgtcagcagtgtgaaggctgaagacctggcagtttattactg tcagcaatcttatagttatccattcacattcggctcgggcaccaagctggaaatcaaacggggc agcacaagcggctctggcaagcctggatctggcgagggctctaccaagggccaggtcaaa ctgcagcagtcaggacctgaactggtgaagcctggggcctcagtgaagatttcctgcaaagc ttctggctacgatttcagtatttcttggatgaactgggtgaggcagaggcctggacagggtcttg agtggattggacggatttatcctggagatggagatagtaactacaatgggaagttcgagggca aggccacactgactgcagacaaatcctccagcacagcctacatgcagctcagcggcctgac ctctgtggactctgcggtctatttttgtgcaagaaccaccactatgattgccctctatgctatggact actggggccaagggaccacggtcaccgtctcctcaggcggcggaggttctcaagtgcaact gcaacagagcggagccgaactggtgagacccggaagcagcgtgaagatcagctgcaag gcttccggctacgcctttagcagctactggatgaactgggtgaagcagagacctggacaggg actggaatggatcggccagatttaccctggagacggcgacacaaactacaacggcaagttc aagggccaagctacactgaccgccgacaaaagcagcagcaccgcctatatgcagctgag cggactgaccagcgaagatagcgctgtgtacttctgcgccagaaagaccatcagcagcgtg gtggacttctacttcgactactggggacaaggcaccaccctgacagtgagcagcgaattcac cacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgt ccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggct ggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtc actggttatcaccctttacaaacggggcagaaagaaactcctgtatatattcaaacaaccattt atgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaag aagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtac cagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgat gttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaa ccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtga gattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtct cagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO.8 gacattgagctcacccagtctccatcctccctagctgtgtcagttggagagaaggttactatgag ctgcaagtccagtcagagccttttatatagtagcaatcaaaagaactatttggcctggtaccag cagaaaccagggcagtctcctaaactgctgatttactgggcatccactagggaatctggggtc cctgatcgcttcacaggcagtggatctgggacagatttcactctcaccgtcagcagtgtgaagg ctgaagacctggcagtttattactgtcagcaatcttatagttatccattcacattcggctcgggcac caagctggaaatcaaacggggcggcggaggttctgatattgtgctgacccagagccccaag ttcatgagcaccagcgtgggcgatagagtgagcgtgacctgcaaggcaagccagaacgtg ggaacaaacgtggcctggtaccaacagaaacccggccaaagccctaagcccctgatttac agcgccacctacagaaatagcggcgtgcccgacagatttacaggaagcggcagcggaac cgatttcacactgaccatcaccaacgtgcagagcaaagacctggccgactacttctgccagc agtacaacagatacccctacaccagcggaggaggaacaaagctggagatcaagagagg cagcacaagcggctctggcaagcctggatctggcgagggctctaccaagggccaagtgca actgcaacagagcggagccgaactggtgagacccggaagcagcgtgaagatcagctgca aggcttccggctacgcctttagcagctactggatgaactgggtgaagcagagacctggacag ggactggaatggatcggccagatttaccctggagacggcgacacaaactacaacggcaag ttcaagggccaagctacactgaccgccgacaaaagcagcagcaccgcctatatgcagctg agcggactgaccagcgaagatagcgctgtgtacttctgcgccagaaagaccatcagcagcg tggtggacttctacttcgactactggggacaaggcaccaccctgacagtgagcagcggcggc ggaggttctcaggtcaaactgcagcagtcaggacctgaactggtgaagcctggggcctcagt gaagatttcctgcaaagcttctggctacgatttcagtatttcttggatgaactgggtgaggcaga ggcctggacagggtcttgagtggattggacggatttatcctggagatggagatagtaactaca atgggaagttcgagggcaaggccacactgactgcagacaaatcctccagcacagcctacat gcagctcagcggcctgacctctgtggactctgcggtctatttttgtgcaagaaccaccactatga ttgccctctatgctatggactactggggccaagggaccacggtcaccgtctcctcagaattcac cacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgt ccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggct ggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtc actggttatcaccctttacaaacggggcagaaagaaactcctgtatatattcaaacaaccattt atgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaag aagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtac cagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgat gttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaa ccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtga gattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtct cagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc SEQ ID NO.9 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSSEAAAKEAAAKEAAAKEAAAKE AAAKDIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAW YQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSVKA EDLAVYYCQQSYSYPFTFGSGTKLEIKRGGGGSGGGGSGGGGS QVKLQQSGPELVKPGASVKISCKASGYDFSISWMNWVRQRPGQG LEWIGRIYPGDGDSNYNGKFEGKATLTADKSSSTAYMQLSGLTSV DSAVYFCARTTTMIALYAMDYWGQGTTVTVSS SEQ ID NO.10 DIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQK PGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSVKAEDLA VYYCQQSYSYPFTFGSGTKLEIKRGGGGSGGGGSGGGGSQVKL QQSGPELVKPGASVKISCKASGYDFSISWMNWVRQRPGQGLEWI GRIYPGDGDSNYNGKFEGKATLTADKSSSTAYMQLSGLTSVDSAV YFCARTTTMIALYAMDYWGQGTTVTVSSEAAAKEAAAKEAAAKEA AAKEAAAKDIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWY QQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSK DLADYFCQQYNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQ VQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQG LEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSE DSAVYFCARKTISSVVDFYFDYWGQGTTLTVSS SEQ ID NO.11 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSDIELTQSPSSLAVSVGEKVTMS CKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPD RFTGSGSGTDFTLTVSSVKAEDLAVYYCQQSYSYPFTFGSGTKLEI KRGSTSGSGKPGSGEGSTKGQVKLQQSGPELVKPGASVKISCKA SGYDFSISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKFEGK ATLTADKSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMDYWG QGTTVTVSSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTAD KSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTL TVSS SEQ ID NO.12 DIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQK PGQSPKLLlYWASTRESGVPDRFTGSGSGTDFTLTVSSVKAEDLA VYYCQQSYSYPFTFGSGTKLEIKRGGGGSDIVLTQSPKFMSTSVG DRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPD RFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEl KRGSTSGSGKPGSGEGSTKGQVQLQQSGAELVRPGSSVKISCKA SGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQ ATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYW GQGTTLTVSSGGGGSQVKLQQSGPELVKPGASVKISCKASGYDF SISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKFEGKATLTAD KSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMDYWGQGTTVT VSS SEQ ID NO.13 MALPVTALLLPLALLLHAARP SEQ ID NO.14 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO.15 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQlYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSSFETTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYlFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPRAAAEGRGSLLTCGDVEENP GPSGATMALPVTALLLPLALLLHAARPGSDIELTQSPSSLAVSVGE KVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTVSSVKAEDLAVYYCQQSYSYPFTFGS GTKLEIKRGGGGSGGGGSGGGGSQVKLQQSGPELVKPGASVKIS CKASGYDFSISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKF EGKATLTADKSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMD YWGQGTTVTVSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR SEQ ID NO.16 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSSFETTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPRAAAEGRGSLLTCGDVEENP GPSGATMALPVTALLLPLALLLHAARPGSDIELTQSPSSLAVSVGE KVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTVSSVKAEDLAVYYCQQSYSYPFTFGS GTKLEIKRGGGGSGGGGSGGGGSQVKLQQSGPELVKPGASVKIS CKASGYDFSISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKF EGKATLTADKSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMD YWGQGTTVTVSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY IFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR SEQ ID NO.17 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSSFETTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPRAAAEGRGSLLTCGDVEENP GPSGATMALPVTALLLPLALLLHAARPGSDIELTQSPSSLAVSVGE KVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTVSSVKAEDLAVYYCQQSYSYPFTFGS GTKLEIKRGGGGSGGGGSGGGGSQVKLQQSGPELVKPGASVKIS CKASGYDFSISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKF EGKATLTADKSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMD YWGQGTTVTVSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR SEQ ID DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP NO.18 KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSSFETTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPRAAAEGRGSLLTCGDVEENP GPSGATMALPVTALLLPLALLLHAARPGSDIELTQSPSSLAVSVGE KVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRE SGVPDRFTGSGSGTDFTLTVSSVKAEDLAVYYCQQSYSYPFTFGS GTKLEIKRGGGGSGGGGSGGGGSQVKLQQSGPELVKPGASVKIS CKASGYDFSISWMNWVRQRPGQGLEWIGRIYPGDGDSNYNGKF EGKATLTADKSSSTAYMQLSGLTSVDSAVYFCARTTTMIALYAMD YWGQGTTVTVSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR SEQ ID NO.19 gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtgagcgt gacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaacccg gccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccgacag atttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagagcaaa gacctggccgactacttctgccagcagtacaacagatacccctacaccagcggaggagga acaaagctggagatcaagagaggtggtggtggttctggcggcggcggctccggtggtggtg gttctcaagtgcaactgcaacagagcggagccgaactggtgagacccggaagcagcgtga agatcagctgcaaggcttccggctacgcctttagcagctactggatgaactgggtgaagcag agacctggacagggactggaatggatcggccagatttaccctggagacggcgacacaaac tacaacggcaagttcaagggccaagctacactgaccgccgacaaaagcagcagcaccgc ctatatgcagctgagcggactgaccagcgaagatagcgctgtgtacttctgcgccagaaaga ccatcagcagcgtggtggacttctacttcgactactggggacaaggcaccaccctgacagtg agcagcttcgaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgc gtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgca cacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggg gtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatata ttcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccg atttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcaga cgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaag agaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagcc gagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcgg aggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatgg cctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccc tgccccctcgcgcggccgctgagggcagaggaagtcttctaacatgcggtgacgtggagga gaatcccggcccttccggagccaccatggccttaccagtgaccgccttgctcctgccgctggc cttgctgctccacgccgccaggccgggatccgacattgagctcacccagtctccatcctcccta gctgtgtcagttggagagaaggttactatgagctgcaagtccagtcagagccttttatatagtag caatcaaaagaactatttggcctggtaccagcagaaaccagggcagtctcctaaactgctga tttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggac agatttcactctcaccgtcagcagtgtgaaggctgaagacctggcagtttattactgtcagcaat cttatagttatccattcacattcggctcgggcaccaagctggaaatcaaacggggtggtggtgg ttctggcggcggcggctccggtggtggtggttctcaggtcaaactgcagcagtcaggacctga actggtgaagcctggggcctcagtgaagatttcctgcaaagcttctggctacgatttcagtatttc ttggatgaactgggtgaggcagaggcctggacagggtcttgagtggattggacggatttatcct ggagatggagatagtaactacaatgggaagttcgagggcaaggccacactgactgcagac aaatcctccagcacagcctacatgcagctcagcggcctgacctctgtggactctgcggtctattt ttgtgcaagaaccaccactatgattgccctctatgctatggactactggggccaagggaccac ggtcaccgtctcctcagaattcaccacgacgccagcgccgcgaccaccaacaccggcgcc caccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcgggggg cgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgg gacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaac tcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctg tagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcagg agcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatcta ggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggg ggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagata agatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggg gcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcaca tgcaggccctgccccctcgctaa SEQ ID NO.20 gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtgagcgt gacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaacccg gccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccgacag atttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagagcaaa gacctggccgactacttctgccagcagtacaacagatacccctacaccagcggaggagga acaaagctggagatcaagagaggtggtggtggttctggcggcggcggctccggtggtggtg gttctcaagtgcaactgcaacagagcggagccgaactggtgagacccggaagcagcgtga agatcagctgcaaggcttccggctacgcctttagcagctactggatgaactgggtgaagcag agacctggacagggactggaatggatcggccagatttaccctggagacggcgacacaaac tacaacggcaagttcaagggccaagctacactgaccgccgacaaaagcagcagcaccgc ctatatgcagctgagcggactgaccagcgaagatagcgctgtgtacttctgcgccagaaaga ccatcagcagcgtggtggacttctacttcgactactggggacaaggcaccaccctgacagtg agcagcttcgaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgc gtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgca cacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggg gtccttctcctgtcactggttatcaccctttactgcaggagtaagaggagcaggctcctgcacag tgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatg ccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgc ccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagaga ggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgag aaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggagg cctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttt accagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgcc ccctcgcgcggccgctgagggcagaggaagtcttctaacatgcggtgacgtggaggagaat cccggcccttccggagccaccatggccttaccagtgaccgccttgctcctgccgctggccttgc tgctccacgccgccaggccgggatccgacattgagctcacccagtctccatcctccctagctgt gtcagttggagagaaggttactatgagctgcaagtccagtcagagccttttatatagtagcaat caaaagaactatttggcctggtaccagcagaaaccagggcagtctcctaaactgctgatttac tgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggacagat ttcactctcaccgtcagcagtgtgaaggctgaagacctggcagtttattactgtcagcaatcttat agttatccattcacattcggctcgggcaccaagctggaaatcaaacggggtggtggtggttctg gcggcggcggctccggtggtggtggttctcaggtcaaactgcagcagtcaggacctgaactg gtgaagcctggggcctcagtgaagatttcctgcaaagcttctggctacgatttcagtatttcttgg atgaactgggtgaggcagaggcctggacagggtcttgagtggattggacggatttatcctgga gatggagatagtaactacaatgggaagttcgagggcaaggccacactgactgcagacaaa tcctccagcacagcctacatgcagctcagcggcctgacctctgtggactctgcggtctattt.sup.ttgt gcaagaaccaccactatgattgccctctatgctatggactactggggccaagggaccacggt caccgtctcctcagaattcaccacgacgccagcgccgcgaccaccaacaccggcgcccac catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgc agtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggac ttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcct gtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtag ctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggag cgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctagg acgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggggg aaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataag atggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggc acgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatg caggccctgccccctcgc SEQ ID NO.21 gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtgagcgt gacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaacccg gccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccgacag atttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagagcaaa gacctggccgactacttctgccagcagtacaacagatacccctacaccagcggaggagga acaaagctggagatcaagagaggtggtggtggttctggcggcggcggctccggtggtggtg gttctcaagtgcaactgcaacagagcggagccgaactggtgagacccggaagcagcgtga agatcagctgcaaggcttccggctacgcctttagcagctactggatgaactgggtgaagcag agacctggacagggactggaatggatcggccagatttaccctggagacggcgacacaaac tacaacggcaagttcaagggccaagctacactgaccgccgacaaaagcagcagcaccgc ctatatgcagctgagcggactgaccagcgaagatagcgctgtgtacttctgcgccagaaaga ccatcagcagcgtggtggacttctacttcgactactggggacaaggcaccaccctgacagtg agcagcttcgaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgc gtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgca cacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggg gtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatata ttcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccg atttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcaga cgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaag agaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagcc gagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcgg aggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatgg cctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccc tgccccctcgcgcggccgctgagggcagaggaagtcttctaacatgcggtgacgtggagga gaatcccggcccttccggagccaccatggccttaccagtgaccgccttgctcctgccgctggc cttgctgctccacgccgccaggccgggatccgacattgagctcacccagtctccatcctcccta gctgtgtcagttggagagaaggttactatgagctgcaagtccagtcagagccttttatatagtag caatcaaaagaactatttggcctggtaccagcagaaaccagggcagtctcctaaactgctga tttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgggac agatttcactctcaccgtcagcagtgtgaaggctgaagacctggcagtttattactgtcagcaat cttatagttatccattcacattcggctcgggcaccaagctggaaatcaaacggggtggtggtgg ttctggcggcggcggctccggtggtggtggttctcaggtcaaactgcagcagtcaggacctga actggtgaagcctggggcctcagtgaagatttcctgcaaagcttctggctacgatttcagtatttc ttggatgaactgggtgaggcagaggcctggacagggtcttgagtggattggacggatttatcct ggagatggagatagtaactacaatgggaagttcgagggcaaggccacactgactgcagac aaatcctccagcacagcctacatgcagctcagcggcctgacctctgtggactctgcggtctattt ttgtgcaagaaccaccactatgattgccctctatgctatggactactggggccaagggaccac ggtcaccgtctcctcagaattcaccacgacgccagcgccgcgaccaccaacaccggcgcc caccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcgggggg cgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgg gacttgtggggtccttctcctgtcactggttatcaccctttactgcaggagtaagaggagcaggc tcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattac cagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggag cgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctagg acgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggggg aaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataag atggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggc acgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatg caggccctgccccctcgc SEQ ID NO.22 gatattgtgctgacccagagccccaagttcatgagcaccagcgtgggcgatagagtga gcgtgacctgcaaggcaagccagaacgtgggaacaaacgtggcctggtaccaacagaaa cccggccaaagccctaagcccctgatttacagcgccacctacagaaatagcggcgtgcccg acagatttacaggaagcggcagcggaaccgatttcacactgaccatcaccaacgtgcagag caaagacctggccgactacttctgccagcagtacaacagatacccctacaccagcggagg aggaacaaagctggagatcaagagaggtggtggtggttctggcggcggcggctccggtggt ggtggttctcaagtgcaactgcaacagagcggagccgaactggtgagacccggaagcagc gtgaagatcagctgcaaggcttccggctacgcctttagcagctactggatgaactgggtgaag cagagacctggacagggactggaatggatcggccagatttaccctggagacggcgacaca aactacaacggcaagttcaagggccaagctacactgaccgccgacaaaagcagcagcac cgcctatatgcagctgagcggactgaccagcgaagatagcgctgtgtacttctgcgccagaa agaccatcagcagcgtggtggacttctacttcgactactggggacaaggcaccaccctgaca gtgagcagcttcgaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccat cgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagt gcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt ggggtccttctcctgtcactggttatcaccctttactgcaggagtaagaggagcaggctcctgca cagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccc tatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcag acgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaa gagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagc cgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcg gaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatg gcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggcc ctgccccctcgcgcggccgctgagggcagaggaagtcttctaacatgcggtgacgtggagg agaatcccggcccttccggagccaccatggccttaccagtgaccgccttgctcctgccgctgg ccttgctgctccacgccgccaggccgggatccgacattgagctcacccagtctccatcctccct agctgtgtcagttggagagaaggttactatgagctgcaagtccagtcagagccttttatatagta gcaatcaaaagaactatttggcctggtaccagcagaaaccagggcagtctcctaaactgctg atttactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctggga cagatttcactctcaccgtcagcagtgtgaaggctgaagacctggcagtttattactgtcagca atcttatagttatccattcacattcggctcgggcaccaagctggaaatcaaacggggtggtggt ggttctggcggcggcggctccggtggtggtggttctcaggtcaaactgcagcagtcaggacct gaactggtgaagcctggggcctcagtgaagatttcctgcaaagcttctggctacgatttcagtat ttcttggatgaactgggtgaggcagaggcctggacagggtcttgagtggattggacggatttat cctggagatggagatagtaactacaatgggaagttcgagggcaaggccacactgactgcag acaaatcctccagcacagcctacatgcagctcagcggcctgacctctgtggactctgcggtct atttttgtgcaagaaccaccactatgattgccctctatgctatggactactggggccaagggacc acggtcaccgtctcctcagaattcaccacgacgccagcgccgcgaccaccaacaccggcg cccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcgggg ggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggcc gggacttgtggggtccttctcctgtcactggttatcaccctttactgcaggagtaagaggagcag gctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcatt accagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcagg agcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatcta ggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggg ggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagata agatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggg gcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcaca tgcaggccctgccccctcgc SEQ ID NO.23 DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSP KPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQ YNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAE LVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG DGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCAR KTISSVVDFYFDYWGQGTTLTVSS SEQ ID NO.24 DIELTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSVKAE DLAVYYCQQSYSYPFTFGSGTKLEIKRGGGGSGGGGSGGGGSQ VKLQQSGPELVKPGASVKISCKASGYDFSISWMNWVRQRPGQGL EWIGRIYPGDGDSNYNGKFEGKATLTADKSSSTAYMQLSGLTSVD SAVYFCARTTTMIALYAMDYWGQGTTVTVSS SEQ ID NO.25 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO.26 EGRGSLLTCGDVEENPGP

DETAILED DESCRIPTION OF THE INVENTION

[0095] The present invention discloses a CAR targeting CD22 and CD19 and application thereof, and those skilled in the art can learn from the content herein to appropriately modify the process parameters to achieve the same. It should be specifically noted that all such alternatives and modifications as would be obvious to those skilled in the art are deemed to be included in the present invention and that it is obvious to those skilled in the relevant art can implement and apply the technology of the present invention by making modifications or appropriate changes and combinations on the content described herein without departing from the content, spirit and scope of the present invention.

[0096] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0097] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

Example 1: Construction of a Dual-Target Chimeric Antigen Receptor Vector

[0098] 1. BamH I and EcoR I endonucleases were adopted to digest a plasmid containing CD8α-4-1BB-CD3ζ segment constructed by the inventor previously to obtain a CD8a-4-1BB-CD3ζ segment, an amino acid sequence of which is shown in SEQ ID NO.5, where the plasmid containing the CD8α-4-1BB-CD3ζ segment may be prepared by any suitable method in the prior art, for example, the patent No. ZL201510233748.0.

[0099] 2. CD22scFv-CD19scFv, CD19scFv-CD22scFv, CD19V.sub.L-CD22V.sub.L-CD22V.sub.H-CD19V.sub.H and CD22V.sub.L-CD19V.sub.L-CD19V.sub.H-CD22V.sub.H segments obtained by synthesis were incorporated to a target vector, respectively, and constructed CD22scFv-CD19 scFv-CD8α-4-1BB-CD3ζ CAR (22-19 CAR), CD19scFv-CD22 scFv-CD8α-4-1BB-CD3ζ CAR (19-22 CAR), CD19 V.sub.L-CD22V.sub.L-CD22V.sub.H-CD19V.sub.H-CD8α-4-1BB -CD3ζ (19×22 CAR) and CD22 V.sub.L-CD19 V.sub.L-CD19V.sub.H-CD22V.sub.H-CD8α-4-1BB-CD3ζ (22×19 CAR) target vectors were digested with endonucleases Nhe I and Not I for identification. The results are shown in FIG. 1A and FIG. 1B. The results of digestion showed that positive clones contained a target band and were correct after sequencing and identification. A schematic diagram of a vector of CD22 scFv-CD19 scFv-CD8α-4-1 BB-CD3ζ CAR is taken as an example, as shown in FIG. 2.

Example 2: Preparation of a Lentivirus-Modified T Cells of a Dual-Target Chimeric Antigen Receptor

[0100] 1. An EndoFree Plasmid Maxi plasmid extraction kit (QIAGEN) was used to respectively extract 22-19 CAR, 19-22-CAR, 19×22 CAR and 22×19 CAR expression plasmids and packaging plasmids PRSV-Rev, pMDlg-PRRE and pMD.2G. Each CAR plasmid and packaging plasmid (four plasmids) were transfected with a PEI transfection reagent (Polyscience) in a ratio of 12.2: 4.11: 8.75: 3.5 (refer to the instructions of the PEI transfection reagent for specific methods). A fresh culture medium was replaced 12 hours after transfection, virus supernatant was collected in 24 hours and 48 hours, respectively, centrifuged at 4° C. by 3000 rpm for 15 minutes, and filtered through a 0.45 .Math.m filter, and then virus supernatant was ultracentrifuged at 4° C. by 50000 g for 1.5 hours to concentrate for 10 folds, and then stored at -80° C.

[0101] 2. Preparation of the T cells: 10 ml of fresh healthy human peripheral blood was obtained, and T cells were purified using RosetteSep T cell enrichment Cocktail (Stemcell) and Ficoll-Paque PLUS (GE Healthcare) (for specific steps, follow the instructions of RosetteSep T cell enrichment Cocktail). Anti-CD3/CD28 magnetic beads (Gibco) were added according to the ratio of cells: magnetic beads=1:1, and cultured for 24 hours to obtain T cells before transfection.

[0102] 3. Lentivirus-infected T cells and culture of post-infected T cells: viral supernatant was taken out from -80° C. freezer, thawed at room temperature, 1×10.sup.6 T cells were transfected with 100 .Math.l of viral supernatant, and Polybrene was added till a final concentration reached 8 .Math.g/ ml. The transfected T cells were centrifuged at 32° C. at a speed of 1800 rpm for 1.5 hours, and then cultured in an incubator with 5% CO.sub.2 at 37° C.

[0103] 4. Detection of the positive rate of CAR-modified T cells by flow cytometry: the above culture T cells were collected and labeled with a rabbit anti-mouse IgG F(ab′).sub.2 antibody, the F(ab′).sub.2 and GFP positive T cells were analyzed by flow cytometry. The results are shown in FIG. 3A and FIG. 3B. It is observed from the figures that the positive rate of CAR-T is 70% or above.

Experimental Example 1: The Killing Effect of 22-19 CAR, 19-22-CAR, 19×22 CAR and 22×19 CAR Lentivirus-Modified T Cells on Leukemia Cells

1. Expression Levels of CD22 in Hematological Tumor Cell Lines

[0104] Namalwa and MV4-11 cell lines were purchased from ATCC in the United States. MV4-11-CD19 and MV4-11-CD22 were monoclonal cell lines screened after the MV4-11 cell lines were infected with CD19 and CD22, respectively. 5×10.sup.5 cells were collected from each of the cultured cell lines, and washed twice with PBS, a PE conjugated anti-human CD19 monoclonal antibody and an APC conjugated anti-human CD22 monoclonal antibody (Biolegend) were stained respectively, PE-isotype and APC-isotype were used as control groups, and then incubated on ice for 30 minutes. The expression levels of CD19 and CD22 in various cell lines were detected by flow cytometry, and the results are shown in FIG. 4A and FIG. 4B, where FIG. 4A was the positive rate of CD19 antigen expression; FIG. 4B was the positive rate of CD22 antigen expression; and the CD19 positive rates of Namalwa and MV4-11-CD19 were both 95% or above, the CD22 positive rates of Namalwa and MV4-11-CD22 were both 95% or above, while wild-type MV4-11 hardly expressed CD19 and CD22.

2. Detection of Residual Tumor Cells by Flow Cytometry After Co-Culture of CAR-Modified T Cells With Namalwa, MV4-11-CD19, MV4-11-CD22 and Wild-Type MV4-11 Cell Lines

[0105] The above cells were inoculated into a 24-well culture plate at 2×10.sup.5 cells per well, and CAR-T cells of 2.5×10.sup.4 (E:T=1:8), 5×10.sup.4 (E:T=1:4), and 1×10.sup.5 (E: T=1:2), 2×10.sup.5 (E:T=1:1), and 4×10.sup.5 (E:T=2:1) were added, respectively, empty vector-T cells were used as a control group, and co-cultured in an incubator. The co-cultured Namalwa, MV4-11-CD19, MV4-11-CD22 and wild-type MV4-11 cells were stained with PE conjugated anti-human CD19 monoclonal antibody and the APC conjugated anti-human CD22 monoclonal antibody (Biolegend), and PE-Cy7 anti-human CD3 monoclonal antibody (Biolegend) were used to label the T cells, and the residual cells were tested by flow cytometry. The results are shown in FIG. 5A to FIG. 5L. 1) Compared with vector-T, 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T could significantly kill CD19.sup.+CD22.sup.+ Namalwa target cells (FIG. 5A and FIG. 5B). 2) Compared with vector-T and 22 CAR-T, 19 CAR-T, 19-22 CAR-T and 22-19 CAR-T could significantly kill MV4-11-CD19 target cells (FIG. 5C and FIG. 5D). 3) Compared with vector-T and 19 CAR-T, 22 CAR-T, 19-22 CAR-T and 22-19 CAR-T could significantly kill MV4-11-CD22 target cells (FIG. 5E and FIG. 5F). 4) Similar to vector-T, 19-22 CAR-T and 22-19 CAR-T had no killing effect on CD19/CD22 double negative MV4-11 target cells (FIG. 5G and FIG. 5H). 5) Compared with vector-T, both 19×22 CAR-T and 22×19 CAR-T could significantly kill the MV4-11-CD19 target cells and the MV4-11-CD22 target cells (FIG. 5I). 6) Similar to vector-T, 19×22 CAR-T and 22×19 CAR-T had no killing effect on the CD19/CD22 double-negative MV4-11 target cells (FIG. 5J). 7) Compared with vector-T, 1922-BB CAR-T, 1922-28B CAR-T, 1922-B28 CAR-T and 1922-2828 CAR-T could significantly kill the MV4-11-CD19 target cells and the MV4-11-CD22 target cells (FIG. 5K). 8) Similar to vector-T, 1922-BB CAR-T, 1922-28B CAR-T, 1922-B28 CAR-T and 1922-2828 CAR-T had no killing effect on the CD19/CD22 double negative MV4-11 target cells (FIG. 5L). The results proved that 19-22 CAR-T, 22-19 CAR-T, 19×22 CAR-T, 22×19 CAR-T, 1922-BB CAR-T, 1922-28B CAR-T, 1922-B28 CAR-T and 1922-2828 CAR-T had obvious dual-target advantages, and had efficient and specific killing effects on CD19.sup.+, CD22.sup.+ and CD19.sup.+/CD22.sup.+ target cells. In the above experiments, the killing effects of 22-19 CAR-T and 22×19 CAR-T were more significant.

3. Degranulation Assays to Analyze the Activation of CAR-Modified T Cells

[0106] CAR-T and vector-T cells were co-cultured with Namalwa, MV4-11-CD19, MV4-11-CD22 and wild-type MV4-11 cell lines respectively according to the E:T ratio of 1:1, and an anti-CD107a antibody and monensin were added to the co-culture system; and the expression level of CD107a on the surface of GFP.sup.+ cells was tested by flow cytometry after 4h. The results are shown in FIG. 6A and FIG. 6B. 1) After co-culture with Namalwa cells (CD19.sup.+CD22.sup.+), the degranulation levels of 19 CAR-T, 22 CAR-T, 19-22 CAR-T, 22-19 CAR-T, 19×22 CAR-T and 22×19 CAR-T groups were all 10% or above, and the degranulation level of vector-T was 2% or below. 2) After co-culture with MV4-11-CD19 cells, the degranulation levels of 19 CAR-T, 19-22 CAR-T, 22-19 CAR-T, 19×22 CAR-T and 22×19 CAR-T groups were all 25% or above, while the degranulation levels of vector-T and 22 CAR-T were 2% or below; 3) after co-culture with target cells MV4-11-CD22, the degranulation levels of 22 CAR-T, 19-22 CAR-T, 22-19 CAR-T, 19×22 CAR-T and 22×19 CAR-T groups were all 15% or above, while the degranulation levels of vector-T and 19 CAR-T were 2% or below. 4) After co-culture with wild-type MV4-11, the degranulation levels of vector-T, 19 CAR-T, 22 CAR-T, 19-22 CAR-T, 19×22 CAR-T and 22×19 CAR-T were all 2% or below, demonstrating that 19-22 CAR-T, 22-19 CAR-T, 19×22 CAR-T and 22×19 CAR-T could be significantly and specifically activated by CD19.sup.+ or CD22.sup.+ target cells.

4. ELISA to Detect the Levels of Cytokines IFN-γ, TNF-α and IL-2 in Co-Culture Supernatant of Lymphoma Cell Lines and CAR-T Cells

[0107] Namalwa, MV4-11-CD19, MV4-11-CD22 and wild-type MV4-11 cell lines were inoculated in 24-well plates at 2×10.sup.5 cells per well, and CAR-T and vector-T cells were added at 2×10.sup.5 cells to each well, the culture medium was supplemented to 1 ml, and co-cultured in an incubator for 24 hours. Human IFN-γ, TNF-α and IL-2 ELISA assay kits (R&D) were used to test the cytokine secretion level in the co-culture supernatant (see instructions of the ELISA assay kits for specific steps). The results are shown in FIG. 7A to FIG. 7C. 1) After co-culture with CD19.sup.+CD22.sup.+ Namalwa cells, the release levels of all three cytokines were significantly higher in 19 CAR-T, 19-22 CAR-T and 22-19 CAR-T groups compared with that of vector-T cells, the secretion levels of TNF-α were significantly higher in the 19-22 CAR-T and 22-19 CAR-T groups than in the 19 CAR-T group, and the secretion levels of IL-2 were significantly higher in the 19-22 CAR-T and 22-19 CAR-T groups than in a 22 CAR-T group. 2) After co-culture with MV4-11-CD19, the release levels of the three cytokines were significantly higher in the 19 CAR-T, 19-22 CAR-T, and 22-19 CAR-T groups than in the vector-T and 22 CAR-T groups. 3) After co-culture with MV4-11-CD22, the release levels of TNF-α and IFN-.sub.Y were significantly higher in the 22 CAR-T, 19-22 CAR-T, and 22-19 CAR-T groups than in the vector-T and 19 CAR-T groups; while it could be seen that the secretion levels of IL-2 were higher in the 19-22 CAR-T and 22-19 CAR-T groups than in the 22 CAR-T group. 4) After co-culture with wild-type MV4-11, the release levels of all three cytokines were low in vector-T, 19 CAR-T, 22 CAR-T, and 19-22 CAR-T. It demonstrated that 19-22 CAR-T and 22-19 CAR-T could specifically activate CD19.sup.+, CD22.sup.+ and CD19.sup.+CD22.sup.+ target cells and release cytokines, and exhibited higher release levels of cytokines than 19 CAR-T or 22 CAR-T.

5. Expression Levels of CD19 and CD22 in Bone Marrow Mononuclear Cells (BMMCs) of ALL Patients

[0108] Patient samples were obtained from the Institute of Hematology and Blood Diseases Hospital, CAMS, and informed consent was obtained from the patients. The BMMCs were separated by Ficoll gradient centrifugation, 5×10.sup.5 cells were collected and washed twice with PBS, and then incubated with PE conjugated anti-human CD19 monoclonal antibody, APC conjugated anti-human CD22 monoclonal antibody (Biolegend), PE-isotype and APC-isotype (control groups) on ice for 30 minutes. The proportion and intensity of the BMMCs expressing CD19 and CD22 in each patient were analyzed using flow cytometry, as shown in FIG. 8, where P1-P4 represent patients No. 1-4.

6. Detection of Residual Tumor Cells by Flow Cytometry After Co-Culture of CAR-Modified T Cells With BMMCs From ALL Patients

[0109] BMMCs were seeded in 24-well culture plates at 4×10.sup.5 cells/well, 1×10.sup.5 (E:T=1:4) of CAR-modified T cells were added respectively, empty vector-T cells were used as control group, and then co-cultured in an incubator for 48 h. The co-cultured cells were then stained with PE conjugated anti-human CD19 monoclonal antibody and APC conjugated anti-human CD22 monoclonal antibody (Biolegend) for detection of residual leukemic cells from ALL patients, and APC-Cy7 conjugated anti-human CD3 monoclonal antibody (Biolegend) for detection of T cells, and analyzed by flow cytometry. The results are shown in FIG. 9, which demonstrated that after co-culture of CAR-T with CD19.sup.+/CD22.sup.+ BMMCs from patients 1-4 for 48 hours, only 3.03%, 0.24%, 1.34%, and 2.87% of CD19.sup.+/CD22.sup.+ cells could survive in 19-22 CAR-T treatment group, 4.10%, 0.45%, 1.68%, and 3.89% of CD19.sup.+/CD22.sup.+ cells in 22-19 CAR-T treatment group, respectively, while 46.58%, 63.95%, 23.76%, and 44.88% of CD19.sup.+/CD22.sup.+ cells remained in the control group. From above results, it suggested that CAR-T had a killing effect on CD19.sup.+/CD22.sup.+ leukemic primary BMMCs.

7. Degranulation Assays to Analyze Activation of CAR-Modified T Cells After Co-Cultured With BMMCs From ALL Patients

[0110] CAR-T and vector-T cells were co-cultured with BMMCs of patients 1-4 according to the E:T ratio of 1:1, and an anti-CD107a antibody and monensin were added to co-culture systems; and the expression level of CD107a on the surface of CD3.sup.+ cells was tested by flow cytometry after 4 h. The results are shown in FIG. 10, which revealed that after co-cultured with BMMCs from patients 1-4, the activation percentages of 19-22 CAR-T cells were 35.53%, 40.37%, 31.37% and 30.20%, respectively; and the activation percentages of 22-19 CAR-T cells were 51.21%, 33.04%, 18.01% and 50.23%, respectively; while the activation percentages of vector T-cells were only 3.07%, 1.73%, 3.43%, and 2.40%, respectively. From the above results, it showed that there was a significant difference in the activation of CAR-T and vector-T by cocultured with BMMCs of ALL patients.

8. Effects of CAR-Modified T Cells in a CD19.SUP.+./CD22.SUP.+ Lymphoma Mouse Model

[0111] Female NOD/SCID mice at 6-8 weeks were used and randomly divided into vector-T and CAR-T treatment groups, and 5×10.sup.6 Namalwa cells were inoculated intravenously; and 1×10.sup.7 vector-T cells or CAR-T cells were intravenously administered on day 5, day 9 and day 12 after transplantation (see FIG. 11A), respectively. Compared with the control group, the mice did not experience significant weight loss due to CAR-T injection, suggesting that CAR-T treatment had no significant toxic side effects on the mice (see FIG. 11B). The median survival times of vector-T group, 19-22 CAR-T group and 22-19 CAR-T group were 25, 36 and 37 days, respectively. The tumor load plots are shown in FIG. 11C, and the tumor loads of the CAR-T groups were significantly lower than that of the vector-T group. The survival curves are shown in FIG. 11D. By calculating the differences of survival times between CAR-T and vector-T treatment groups, it was found that both 19-22 CAR-T and 22-19 CAR-T could significantly prolong the survival times of the mice, and there was a significant statistical difference (p=0.0011) compared with the control group. According to the median survival and tumor load outcome plots, it was found that 22-19 CAR-T had a slightly better treatment effect than that of 19-22 CAR-T.

[0112] Female NOD/SCID mice at 6-8 weeks were used and randomly divided into two groups, and 2.5×10.sup.6 Namalwa cells were inoculated intravenously; and 1×10.sup.7 vector-T cells or CAR-T cells were administered intravenously on day 5, 9 and 12 after transplantation (see FIG. 12A), respectively. The survival curves are shown in FIG. 12B. By calculation the difference of the survival times between the two groups, it was found that 22×19 CAR-T group could significantly prolong the survival time of the mice, and had a significant statistic difference (p=0.0014) compared with that of control group.

[0113] The above are only the preferred embodiments of the present invention. It should be noted that those ordinarily skilled in the art can also make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be regarded as falling within the protection scope of the present invention.