Group of B7H3 monoclonal antibodies and medical application thereof
1. A panel of B7H3 monoclonal antibodies or antigen-binding fragments thereof, comprising a heavy chain and a light chain, wherein
The CDR1 amino acid sequence of the heavy chain is selected from SEQ ID NO: 29. 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107; what is needed is
The CDR2 amino acid sequence of the heavy chain is selected from SEQ ID NO: 30. 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108; what is needed is
The CDR3 amino acid sequence of the heavy chain is selected from SEQ ID NO: 31. 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109; what is needed is
The CDR1 amino acid sequence of said light chain is selected from the group consisting of SEQ ID NO: 32. 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110; what is needed is
The CDR2 amino acid sequence of said light chain is selected from the group consisting of SEQ ID NO: 33. 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111; what is needed is
The CDR3 amino acid sequence of said light chain is selected from the group consisting of SEQ ID NO: 34. 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112; it is composed of
The heavy and light chains of the antigen-binding fragment of (a) comprise amino acid sequences spanning CDR1 to CDR3 of the heavy and light chains, respectively, of the antibody.
2. The panel of B7H3 monoclonal antibodies or antigen-binding fragments thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 1. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27; the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 2. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28.
3. The panel of B7H3 monoclonal antibodies or antigen-binding fragments thereof according to claim 2, wherein the heavy and light chains are humanized; the amino acid sequence of the humanized heavy chain variable region is selected from SEQ ID NO: 113. 115, 117, 119, 121, 123; the amino acid sequence of the humanized light chain variable region is selected from SEQ ID NO: 114. 116, 118, 120, 122, 124.
4. The panel of B7H3 monoclonal antibodies or antigen-binding fragments thereof according to claim 2, wherein the heavy and light chains are humanized; the amino acid sequence of the humanized heavy chain variable region is SEQ ID NO: 113; the amino acid sequence of the humanized light chain variable region is SEQ ID NO: 114.
5. use of a panel of B7H3 monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1-4 in the preparation of a medicament for: the medicine for regulating the activity of B7H3 or the level of B7H3, the medicine with obvious endocytosis (selectively poisoning or inhibiting tumors and the like through toxin coupling), or obvious antibody-dependent cellular cytotoxicity (ADCC), or the medicine for blocking the action of B7H3 in immunosuppression so as to enhance the immunity of the organism, the medicine for promoting T lymphocytes, or the medicine for improving the production of cytokines such as IFN-gamma in the T lymphocytes.
6. A monoclonal antibody conjugate comprising a monoclonal antibody according to claim 1 to 4, wherein the monoclonal antibody is the panel of B7H3 monoclonal antibodies or antigen-binding fragments thereof, and a conjugate moiety selected from one or more of the group consisting of a radionuclide, a drug, a toxin, a cytokine receptor fragment, an enzyme, fluorescein, and biotin.
7. Use of a monoclonal antibody conjugate as claimed in claim 6 for the preparation of a medicament comprising: a drug for regulating the activity of B7H3 or the level of B7H3, a drug with obvious endocytosis (tumor can be selectively killed or inhibited by toxin coupling), a drug with obvious antibody-dependent cellular cytotoxicity (ADCC), or a drug for blocking the action of B7H3 in immunosuppression so as to enhance the immunity of the organism, a drug for promoting T lymphocytes or a drug for improving the production of cytokines such as IFN-gamma in the T lymphocytes.
8. Use of a monoclonal antibody conjugate as claimed in claim 6 for the preparation of a medicament for the prophylactic and/or therapeutic and/or adjunctive treatment of tumours.
Technical Field
B7H3, also known as CD276, is a type I transmembrane protein (Chapoval a.i., et al., (2001), nat. immunol.2: 269). In mice, B7H3 is encoded by chromosome 9; and in humans, by chromosome 15. B7H3 has 20-27% amino acid homology with other B7 family ligands (Sun m., et al., (2002), j. immunol.168: 6294; los m., (2010), clin.dev. immunol.2010: 683875). In terms of protein composition and structure, murine B7H3 has only 2 IgB7H3 types (consisting of an extracellular pair of IgV and IgC); whereas human B7H3 has 4 IgB7H3 types, almost identical IgV-IgC tandem repeats (Sun m., et al., (2002), j.immunol 1.168: 6294; Steinberger p., et al., (2004), j.immunol.172: 2352). Although mRNA from B7H3 is present in some normal tissues, such as liver, small intestine, pancreas, testis, heart and colon, B7H3 protein is relatively rare in normal tissues (Greenwald R.J., et al., (2005), Annu. Rev. Immunol.23: 515-48). This difference between B7H3 mRNA and protein may reflect the presence of a post-transcriptional tight regulatory mechanism (Hofmeyer k.a., et al., (2008), proc.natl.acad.sci.105: 10277; Calabro l., et al., (2011), j. Cell physiol.226: 2595). Overall, B7H3 is present only in some non-immune fibroblasts, endothelial cells and osteoblasts, as well as some immune cells, such as B cells, T cells, monocytes, dendritic cells or NK cells; expression of B7H3 can be induced by CSF or lipopolysaccharide stimulation of granulocyte-macrophages (Suh W.K., et al., (2003), nat. Immunol.4: 899; Chapoval A.I., et al., (2001), nat. Immunol.2: 269; Greenwald R.J.et al., (2005), Annu. Rev. Immunol.23: 515).
B7H3 belongs to an important immune checkpoint protein. Although preliminary studies have shown that B7H3 stimulates T cells, it may to some extent promote proliferation of CD4+ and CD8+ T cells, enhance cytotoxicity of T lymphocytes and stimulate production of interferon-gamma (IFN- γ) (Chapoval a.i., et al., (2001), nat. immunol.2: 269), and deletion of B7H3 may reduce chronic rejection of allogeneic heart transplantation (Wang l.et al., (2005), eur.j.immunol.35: 428). However, more studies have shown that B7H3 has significant immunosuppressive effects. For example, B7H3 significantly inhibited the activation of T cells by CD3 antibodies or allogeneic DC cells, while B7H3 blocking antibodies were effective in reversing this inhibition (Prasad d.v.r., et al., (2004), j.immunol.173: 2500). Structural information of the B7H3 protein revealed that the FG loop in the B7H3 IgV domain may play a critical role in T cell suppression (Vigdorovich v., et al., (2013), Structure 21: 707). In addition to T cells, B7H3 may also have inhibitory effects on natural killer cells (NK) (Castriconi r., et al., (2004), proc.natl.acad.sci.101: 12640). A possible mechanism involves regulatory T cells (Tregs) inducing expression of Dendritic Cells (DC) B7H3 such that stimulation of T lymphocytes by DC cells is inhibited (Mahnke k., et al., (2007), eur.j.immunol.37: 2117). In mouse models, mice lacking B7H3 develop more severe airway inflammation and also cause some autoimmune diseases such as experimental autoimmune-dependent encephalomyelitis to occur earlier (Suh w.k., et al., (2003), nat. immunol.4: 899).
In many malignancies, the expression level of B7H3 is quite high, such as melanoma (Wang j., et al., (2013), j. invest.dermotol.133: 2050), leukemia (Hu y., et al., (2015), Hematology 20: 187; Sun j., et al., (2014), oncotarget therms.7: 1979), prostate cancer (Zang x., et al., (2007), proc.natl.acad.sci.104: 19458), ovarian cancer (Zang x., et al., (2010), mod.pathol.23: 1104) and pancreatic cancer (Chen y., et al., (2014), onco.targetters therms.7: 1465-72) and the like. Although B7H3 mRNA was present in both normal and tumor tissues, B7H3 protein was only present at higher levels in tumor cells; this difference may be related to the miRNA-29 regulatory mechanism, as in normal or tumor tissues and Cancer cell lines, and studies have shown that miRNA-29 is inversely related to B7H3 protein levels (Xu H., et al., (2009), Cancer res.69: 6275). In neoplastic patients, aberrant expression of B7H3 is closely associated with poor prognosis, increased levels of tumor grade and metastasis, resistance to therapy, and decreased overall survival in neoplastic patients (Picarda E., et al., (2016), Clin. cancer Res.22: 3425).
The inhibition of T cells, NK cells and DC cells by B7H3 significantly promotes the immune escape of tumor cells; in addition, B7H3 also has important effects on tumor cell proliferation, migration, invasion, angiogenesis, epithelial-to-mesenchymal transition (EMT), cancer dryness and Warburg effects, and tumor cell resistance. An increase in B7H3 promotes the expression of Bcl-2 and Bcl-x 1, enhancing the anti-apoptotic activity of tumor cells through activation of the JAK2-STAT3 signaling pathway (Zhang t., et al., (2015), World j. gastroenterol.21: 1804). Meanwhile, activation of AKT, ERK, and JAK2/STAT3 pathways can induce expression of protein factors involved in tumor cell metastasis, including MMP2, MMP9, CXCR4, and the like, thereby enhancing tumor cell migration and invasiveness (Tekle c., et al., (2013), int.j Cancer 130: 2282; Li y., et al., (2017), Oncotarget 8: 71725; Wang l., et al., (2013), PLoS One 8: e 70689; Liu f., et al., (2015), mol.med.rep.12: 5455). Several studies have shown that B7H3 of tumor endothelial cells can also induce activation of NF- κ B based on TLR 4-dependent mechanisms, resulting in a significant increase in the expression levels of VEGF and IL-8 (Tekle c., et al., (2012), int.j. cancer 130: 2282), thereby promoting tumor invasion and angiogenesis (Ferrara n., et al., (2002), nat.rev. cancer 2: 795). On the other hand, B7H3 may also play a role in regulating the activity of tumor cells EMT and tumor stem cells by decreasing E-cadherin and increasing the expression of N-cadherin, vimentin, CD133 and CD44 (Jiang B., et al., (2016), Oncotarget 731755). Since B7H3 can activate STAT3 signaling, thereby increasing the expression of hexokinase 2, B7H3 also has the effect of promoting aerobic glycolysis in tumor cells (Shi t., et al., (2019), Cell Death dis.10: 308). Meanwhile, B7H3 inhibits the activity of the transcription factor NRF2, so that the levels of Reactive Oxygen Species (ROS) and HIF1a are increased, thereby further enhancing the aerobic glycolysis of tumor cells and promoting the growth of the tumor cells (Lim S., et al., (2016), cancer Res.76: 2231). More and more studies have shown that inhibition or reduction of B7H3 expression can increase tumor Cell response to certain drugs, such as drugs that inhibit DNA replication (including alkylating drugs) and drugs that inhibit PI3K/Akt/mTOR and Ras/Raf/MEK signaling proteins (Flem-Karlsen k., et al., (2017), Pigment Cell Melanoma res.30: 467; Kasten b.b., et al., (2017), nuclear.med.biol.47: 23; Liu H., et al., (2011), mol.cancer ther.10: 960).
Tumor growth was significantly inhibited by transplantation of tumor cells into B7H 3-deficient mice or treatment of tumor mice with B7H3 antibody (Cai d., et al., (2020), Cell. mol.immunol.17: 227; Lee y.h., et al., (2017), Cell res.27: 1034), suggesting that blocking B7H3 signaling could be useful in tumor therapy. Furthermore, by combination with anti-PD-1 antibodies, a double block of B7H3 and PD-1 was produced, producing a synergistic antitumor effect (Lee Y.H., et al., (2017), Cell Res.27: 1034). Due to the significant difference in B7H3 expression levels between normal and tumor tissues, tumor cells can be effectively killed by ADCC effect or toxin coupling of the B7H3 antibody without causing too large an effect on normal tissues (Koenig s., et al., (2014), Medicographia 36: 285). In summary, based on current research information, B7H3 is a promising target for cancer therapy.
Disclosure of Invention
The invention obtains a group of anti-B7H 3 monoclonal antibodies by a hybridoma technology, and the antibodies have higher affinity of B7H3, obvious endocytosis (can selectively poison or inhibit tumors by coupling with toxin), obvious antibody dependent cytotoxicity (ADCC) or can effectively block immunosuppression caused by B7H 3; the invention successfully carries out humanized transformation on 6 candidate antibodies. The antibody has wide application prospect in the preparation of related medicines for inhibiting cancer cells, regulating the action and level of B7H3 and enhancing the immunity of organisms, in particular to medicines for treating cancers.
The invention provides a murine or humanized B7H3 antibody or functional fragment, which comprises a heavy chain sequence and a light chain sequence. The amino acid sequence information of the heavy chain variable region and the light chain variable region of the anti-human B7H3 murine antibody is as follows: the amino acid sequences of the heavy chain variable region and the light chain variable region of 7F5 are respectively SEQ ID NO: 1. 2; the amino acid sequences of the 9C8 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 3. 4; the amino acid sequences of the heavy chain variable region and the light chain variable region of 5B6 are respectively SEQ ID NO: 5. 6; the amino acid sequences of the heavy chain variable region and the light chain variable region of 7C9 are respectively SEQ ID NO: 7. 8; the amino acid sequences of the heavy chain variable region and the light chain variable region of 2A9 are respectively SEQ ID NO: 9. 10; the amino acid sequences of the 4F11 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 11. 12; the amino acid sequences of the 15A2 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 13. 14; the amino acid sequences of the heavy chain variable region and the light chain variable region of 7B7 are respectively SEQ ID NO: 15. 16; the amino acid sequences of the heavy chain variable region and the light chain variable region of 7E6 are respectively SEQ ID NO: 17. 18; the amino acid sequences of the heavy chain variable region and the light chain variable region of 2E10 are respectively SEQ ID NO: 19. 20; the amino acid sequences of the 2F12 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 21. 22; the amino acid sequences of the 2F7 heavy chain variable region and the light chain variable region are respectively SEQ ID NO: 23. 24; the amino acid sequences of the heavy chain variable region and the light chain variable region of 13A2 are respectively SEQ ID NO: 25. 26; the amino acid sequences of the heavy chain variable region and the light chain variable region of 14B3 are respectively SEQ ID NO: 27. 28.
The amino acid sequence information of the CDR1, CDR2 and CDR3 of the heavy chain and the light chain of the antibody is as follows: the amino acid sequences of the 7F5 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 29. 30, 31, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 32. 33, 34; the amino acid sequences of the 9C8 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 35. 36 and 37, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 38. 39, 40; the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of 5B6 are respectively SEQ ID NO: 41. 42, 43, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 44. 45, 46; the amino acid sequences of the 7C9 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 47. 48 and 49, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 50. 51, 52; 2A9 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 53. 54 and 55, and the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 56. 57, 58; the amino acid sequences of the 4F11 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 59. 60 and 61, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 62. 63, 64; 15A2 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 65. 66 and 67, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 68. 69, 70; the amino acid sequences of the 7B7 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 71. 72, 73, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 74. 75, 76; the amino acid sequences of the 7E6 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 77. 78, 79, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 80. 81, 82; the amino acid sequences of the 2E10 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 83. 84, 85, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 86. 87, 88; 2F12 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 89. 90, 91, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 92. 93, 94; 2F7 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 95. 96 and 97, and the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 98. 99, 100; 13A2 heavy chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 101. 102, 103, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 104. 105, 106. The amino acid sequences of the 14B3 heavy chain CDR1, CDR2 and CDR3 are respectively SEQ ID NO: 107. 108 and 109, wherein the light chain CDR1, CDR2 and CDR3 amino acid sequences are respectively SEQ ID NO: 110. 111, 112.
Furthermore, the anti-human B7H3 antibody or fragment is engineered to be a humanized antibody.
The amino acid sequence of the heavy chain variable region of the anti-human B7H3 humanized antibody 2A9 is SEQ ID NO: 113, wherein the light chain variable region amino acid sequence is SEQ ID NO: 114, and a carrier; the amino acid sequence of the heavy chain variable region of the humanized antibody 4F11 is SEQ ID NO: 115 and the amino acid sequence of the light chain variable region is SEQ ID NO: 116; the amino acid sequence of the heavy chain variable region of the humanized antibody 9C8 is SEQ ID NO: 117 having the variable light chain amino acid sequence of SEQ ID NO: 118; the amino acid sequence of the heavy chain variable region of the humanized antibody 15A2 is SEQ ID NO: 119, and the amino acid sequence of the light chain variable region is SEQ ID NO: 120 of a solvent; the amino acid sequence of the heavy chain variable region of the humanized antibody 5B6 is SEQ ID NO: 121, and the light chain variable region amino acid sequence thereof is SEQ ID NO: 122; the amino acid sequence of the heavy chain variable region of the humanized antibody 7B7 is SEQ ID NO: 123, having the amino acid sequence of the light chain variable region of SEQ ID NO: 124.
an expression vector comprising a nucleic acid molecule of the above antibody.
A pharmaceutical composition comprising the above antibody or a functional fragment thereof, and a pharmaceutically acceptable carrier.
The antibody or functional fragment thereof, nucleic acid molecules, expression vectors, host cells and pharmaceutical compositions are used for preparing B7H3 immune function medicines.
The recombinant B7H3 receptor protein prepared by a mammalian cell expression system is used as an antigen, and after a mouse is immunized, spleen cells of the mouse are fused with myeloma cells to obtain hybridoma cells. A plurality of monoclonal hybridoma cell strains are obtained by cloning and screening a large number of hybridoma cells for a plurality of times. The hybridoma cell lines can secrete monoclonal antibodies (figures 1 and 2) which are specifically combined with B7H3 receptors, part of the monoclonal antibodies have obvious endocytosis effects by cells (figures 3 and 4), part of the monoclonal antibodies have effects of promoting human immune cells to secrete cytokines such as IFN-gamma (figure 5), and part of the monoclonal antibodies have obvious effects of mediating ADCC (figures 6,7 and 8), wherein part of the monoclonal antibodies can be used for future ADC, ADCC and B7H3 antagonist development. Further, genes encoding light and heavy Chain variable regions of the antibody were cloned by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and a humanized antibody was constructed by a complementary-determining region grafting (CDR-grafting) method. In vitro functional experiments show that the humanized B7H3 antibody can specifically bind to a B7H3 receptor protein (figure 9 and figure 10), and simultaneously has obvious endocytosis effect by cells (figure 11) and obvious poisoning effect on Calu-6 tumor cells (figure 12), ADCC (figure 13), ADCC (figure 14) on Jurkat-B7H3 and PC9 cells by NK92MI-hCD16 cells after coupling MMAE toxin, and the effect of promoting the production and secretion of cytokines such as IFN-gamma by human immune cells (figure 15). The positive control antibodies employed in this patent are from patents US8802091 and WO2017180813 Al.
Drawings
FIG. 1 (a-b): determination of the binding Properties of the B7H3 hybridoma monoclonal antibody to the B7H3-hFc protein by ELISA
FIG. 2 (a-b): determination of the binding Properties of the B7H3 hybridoma monoclonal antibody to PC9 cells by FACS method
FIG. 3 (a-b): activity of B7H3 hybridoma antibody endocytosis by Jurkat-B7H3 cells by FACS
FIG. 4 (a-b): activity of B7H3 hybridoma antibody endocytosis by PC9 cells was measured by FACS
FIG. 5 (a-b): promotion of IFN-gamma secretion from human PBMC by B7H3 hybridoma antibody
FIG. 6 (a-b): reporter gene assay for ADCC mediated by B7H3 hybridomas or chimeric antibodies on Jurkat-B7H3 cells
FIG. 7 (a-b): reporter gene method was used to test ADCC mediated by some B7H3 chimeric antibodies on PC9 tumor cells
FIG. 8: reporter Gene assay to test ADCC on DLD1 tumor cells mediated by some B7H3 chimeric antibodies
FIG. 9: determination of the binding Properties of the humanized B7H3 monoclonal antibody to B7H3-His protein by ELISA
FIG. 10: binding characteristics of humanized B7H3 monoclonal antibody to B7H3 protein in PC9 cells were determined by FACS method
FIG. 11: effect of humanized B7H3 monoclonal antibody on endocytosis by PC9 cells was measured by FACS
FIG. 12: poisoning effect of humanized B7H3 antibody ADC on Calu-6 tumor cells
FIG. 13 (a-b): reporter Gene assay for ADCC of humanized B7H3 antibody on Jurkat-B7H3 and PC9 cells
FIG. 14 (a-b): humanized B7H3 antibody mediates ADCC killing of NK92MI-hCD16 cells against Jurkat-B7H3 and PC9 cells
FIG. 15: the humanized B7H3 monoclonal antibody has the effect of promoting IFN-gamma production and secretion of human PBMC cells.
Detailed Description
Example 1
Mouse immunization and cell fusion with anti-B7H 3 antibody production
After fully emulsifying human B7H3(NCBI Reference Sequence: NM-001024736.2) extracellular domain and mFc fusion protein (B7H3-ECD-mFc) as antigens with an equal volume of complete Freund's adjuvant (Sigma, Cat. No.: F5581), 6-8 week-old Balb/c mice (purchased from Showa New drug research center, Ltd.) were immunized subcutaneously at an antigen immunization dose of 50. mu.g/mouse. Mice were then immunized subcutaneously three times every 2 weeks after full emulsification with the same dose of antigen with incomplete freund's adjuvant (Sigma, cat. No.: F5506). After three immunizations, the serum titer of the mice was measured, and 3 days before the fusion, the mice were boosted by intraperitoneal injection. Using PEG Hybri-Max (Sigma, Cat. No.:7181) as a fusion agent, mouse spleen cells and SP2/0 cells were mixed at a ratio of 4:1, and the fused cells were added to a 96-well plate (1X 10)5One/well) containing 0.1mL of 1 XHAT (Invitrogen, Cat. No.:21060-017) medium per well. 0.1mL of HT (Invitrogen, Cat. No.: 11067-one 030) medium was added on day 3, the medium in the 96-well plate was aspirated off on day 7, and 0.2 mL of fresh HT medium was added. Supernatants were harvested on day 9 for various screens and tests.
Example 2
Antigen binding of hybridoma antibodies and subcloning
Binding assays for hybridoma antibodies include ELISA and FACS, and subcloning using limiting dilution:
1) screening for B7H3 binding positive clones by ELISA: a96-well ELISA plate (Corning, Cat. No.:9018) was coated with 50. mu. L B7H3-hFc (final concentration: 2. mu.g/mL) overnight at room temperature; after washing 3 times with washing buffer (PBS + 0.05% Tween20), blocking buffer (PBS + 2% BSA (Sigma, cat. No.: V90093)) was added for incubation at room temperature for 1 hour, and the ELISA plate was washed 3 times with washing buffer; adding hybridoma supernatant, incubating at room temperature for 1 hr, and washing for 3 times; 100 μ L of 10000-fold diluted HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.:31432) was added to each well, incubated at room temperature for 1 hour in the dark, and washed 3 times; adding 100 μ L of TMB (Cat. No.: ES-002) into each well, incubating at room temperature for 2 min, and adding 100 μ L/well of stop buffer (2N H)2SO4) The color reaction was stopped and the OD450 of each well was read with a microplate reader (Tecan Spark).
2) FACS was used to screen B7H3 for binding positive clones: 50 μ L of hybridoma supernatant or purified hybridoma antibody positive for the above assay was mixed with 50 μ L of 293T-B7H3 cells (2X 10)5One/well), added to a 96-well U-bottom cell plate, incubated at 4 ℃ for 1 hour, washed twice with FACS buffer (PBS + 3% FCS), added with 400-fold dilution of PE-labeled goat-anti-mouse secondary antibody (Biolegend, cat. No.:405307), incubated at 4 ℃ for 30 minutes protected from light, washed twice with FACS buffer and centrifuged, and the cell signal value on the PE channel was detected with BD Accuri C6 flow cytometer.
3) Subcloning: subcloning by limiting dilution method-subcloning the polyclonal hybridoma cells positive in the ELISA and FACS assays described above, followed by repeated screening by ELISA and FACS methods to obtain positive hybridoma monoclonals.
Antibody purification, concentration determination and endotoxin determination:
positive monoclonal hybridoma cells were cultured in 50mL serum-free medium (Invitrogen, Cat. No.:12045-076) for 8-9 days, and the supernatant was harvested by centrifugation. The monoclonal antibody was purified by Protein A affinity chromatography, the purified antibody sample was concentrated by changing the solution in an ultrafiltration centrifuge tube (Millipore, Cat. No.: ACS500024), the Protein concentration was measured by BCA method, and the endotoxin content of the purified antibody sample was measured by limulus reagent (Tachypleus tridentatus, Inc., Xiamen).
The binding ability of the purified antibody samples to B7H3 was tested by ELISA and FACS and the results are shown in FIG. 1(a-B), FIG. 2(a-B) and Table 1. The selected hybridoma antibodies all have higher affinity.
TABLE 1 binding force of B7H3 hybridoma monoclonal antibody and B7H3 antigen EC50 value
Example 3
Testing of the endocytosis and B7H3 blocking of hybridoma monoclonal antibodies
Intracellular phagocytosis assay for antibodies: in a 96-well plate, 50. mu.L of 2X10 was added per well6[ 50 μ L of labeled B7H3 antibody,/ml of PC9 cells, 96-well cell culture plates were incubated at 4 ℃ for 1 hour, washed with FACS buffer 2 times, then added with secondary antibody labeled with pH-dependent fluorescent dye CypHer5E (GE, Cat. No.: PA15401) and Goat anti-mFc (Jackson, Cat. No.:115 and 005 and 071), incubated at 4 ℃ for 0.5 hour, washed 2 times, then added with medium (1640+ 10% FBS), placed in an incubator for incubation for 3 hours, centrifuged to remove the medium, resuspended in PBS at pH9.0, and then subjected to detection of CypHer5E signal of PC9 cells by BD C6 flow cytometer to calculate the efficiency of B7H3 antibody into the interior of the cells. As shown in fig. 3(a-d) and fig. 4(a-d), Jurkat-B7H3 and PC9 cells had significant endocytic activity against B7H3 antibodies, including 15a2,5B6,7C9, 2F7,7B7,2E10,4F11, and the like.
The effect of B7H3 hybridoma antibodies on cytokine secretion by human PBMC cells was tested: PBMC cells (TPCS, Cat. No.: PB025C) resuspended in complete medium (RPMI1640+ 10% FCS) were added to 96 wells (Corning, Cat. No.: 3799), 40ng/mL OKT3(eBioscience, Cat. No.:16-0037-85) was added, and incubated at 37 ℃ for 72 hours. After counting activated PBMC cells, resuspension with complete medium (2.5X 10)5cells/mL). In a 96-well plate, 100. mu.L of PBMC cells, 50. mu.L of B7H3 antibody at various concentrations (starting at 20. mu.g/mL,10-fold serial dilution), a 96-well cell culture plate (Corning, cat. No.:3599) was incubated at 37 ℃ in a 5% CO2 incubator for 48 hours, and the supernatant was collected. Using IFN-gamma ELISA kit (R)&D Systems, Cat. No. DY285) to detect the concentration of the cytokine. As shown in fig. 5, the B7H3 antibodies including 5B6, 2F12, 2F7, 15a2, 13a2, 14B3, 4F11, 2a9 and the like can significantly promote IFN- γ secretion from PBMC cells.
Example 4
Cloning and sequencing of B7H3 antibody variable region genes
The B7H3 monoclonal hybridoma cell line was lysed with TRIzon (Cwbiotech, Cat. No.: CW0580) to extract total RNA from the hybridoma cells. RNA from hybridoma cells was reverse transcribed into cDNA using HiFi Script cDNA Synthesis kit (Cwbiotech, Cat. No.: CW 2569). The variable region genes of the heavy and light chains of the Antibody were amplified by PCR using cDNA as a template and degenerate primers (Kettleborough et al, (1993), Eur J Immunology 23: 206-211; Strebe, et al, (2010), Antibody Engineering 1: 3-14). After ligation of the PCR amplification products to the T/A vector, DH5a competent cells were transformed, plated and cultured overnight at 37 ℃. The monoclonal antibody is selected from the culture plate, amplified, extracted and used to determine the gene sequence of the antibody. The Complementarity Determining Regions (CDRs) and framework regions of the antibody were analyzed based on its gene sequence. The B7H3 antibody sequence number is shown in Table 2, and the specific sequence information is shown in the sequence table.
TABLE 2B 7H3 antibody sequence number description
Example 5
ADCC Activity assay for hybridoma monoclonal antibodies
The ADCC activity of the antibody was tested by the Reporter gene (Reporter) method: 293T-B7H3, PC9 or DLD1 tumor cells are taken as target cells, Jurkat-mCD16.2-NF-kB or Jurkat-hCD16-NF-kB cells are taken as effector cells, and the mCD16.2 or hCD16 mediated activation signals of transcription factor NF-kB are utilized to screen ADCC positive clones. 50 μ L of hybridoma antibody positive in the ELISA and FACS assays described above was combined with 25 μ L of 293T-B7H3, PC9 or DLD1 tumor cells (7.5X 10)4One/well) were mixed, added to a 96-well plate, and 25. mu.L of Jurkat-mCD6.2-NF-kB cells (2.5X 10) were added4One/well), mixed well and incubated in an incubator at 37 ℃ for 4 hours. mu.L of preheated Bright-Glo solution (Promega, Cat. No.: E2620) was added to each well, left at room temperature in the dark for 3 minutes, and the Luminescence (Luminescence) signal value of each sample was measured with a microplate reader (Tecan Spark).
The mouse IgG1 antibody had no ADCC activity due to the hybridoma antibody. After gene sequencing, we confirmed that 9 of the 14 hybridoma antibodies were based on mouse IgG1 and 5 were based on IgG2b or IgG2a (see table 3). FIG. 6a shows the results of an ADCC reporter gene assay for hybridoma antibodies targeting cells based on Jurkat-B7H 3. As expected, 5 IgG2b or IgG2 a-based hybridoma antibodies had ADCC activity, while 9 were mouse IgG 1-based and were not active. Thus, we substituted Fc for 9 hybridoma antibodies based on mouse IgG1 to become chimeric antibodies based on human IgG1, as shown in FIG. 6 b. Figures 7 and 8 reflect the results of ADCC reporter assays based on PC9 or DLD1 tumor cells as targeting cells, in the form of chimeric antibodies based on human IgG 1. The test results of ADCC reporter genes show that the ADCC activity is relatively strong and comprises 9C8, 5B6,7C9, F5, 7E6, 2A9, 4F11 and the like.
TABLE 3 Fc types of B7H3 hybridoma antibodies
Abs
2A9
13A2
4F11
9C8
5B6
7E6
7F5
2F7
7C9
2E10
7B7
14B3
2F12
15A2
Fc
IgG2b
IgG2b
IgG1
IgG1
IgG1
IgG1
IgG1
IgG2b
IgG1
IgG2a
IgG2b
IgG1
IgG1
IgG1
Example 6
Humanized B7H3 antibodies 2a9, 4F11, 9C8, 15a2,5B6 and 7B7
By combining various characteristics of hybridoma antibodies, 6B 7H3 antibodies including 2A9, 4F11, 9C8, 15A2,5B6 and 7B7 are selected and subjected to humanized modification.
And performing humanized transformation on the B7H3 antibody by adopting a complementary determinant grafting method. First, human germline antibody (germline antibody) sequences with the highest homology to the light and heavy chain variable region sequences of murine 2a9, 4F11, 9C8, 15a2,5B6 and 7B7 antibodies, respectively, were searched in the IMGT database. The embryo line selected from the humanized 2A9 antibody light chain variable region is IGKV3-11 x 01, and the humanized heavy chain variable region is IGHV1-2 x 02. The 4F11 antibody light chain variable region humanized selected embryo line is IGKV3-11 x 01, and the heavy chain variable region humanized selected IGHV1-2 x 02. The 9C8 antibody light chain variable region humanized selected germ line is IGKV3-11 x 01, and the heavy chain variable region humanized selected IGHV1-46 x 01. The embryo line selected from the humanized 15A2 antibody light chain variable region is IGKV3-11 x 01, and the heavy chain variable region is selected from IGHV1-8 x 01. The humanized selected germ line of the 5B6 antibody light chain variable region is IGKV1-39 x 01, and the humanized selected heavy chain variable region is IGHV1-69 x 02. The 7B7 antibody light chain variable region humanized selected germ line is IGKV1-39 x 01, and the heavy chain variable region humanized selected IGHV3-21 x 01. The CDR regions of the murine antibody are retained and the framework region (framework) sequences of the murine antibody are replaced with the framework region sequences of the human germline antibody. Establishing a structural model of the murine antibody, comparing the amino acids of each site in the framework regions of the humanized antibody and the corresponding murine antibody one by one, if the adopted human amino acid sequence of a certain site of the framework region does not cause the damage or change of the space structure of the CDR region, using the human amino acid sequence of the site, or else using the corresponding murine sequence (namely, carrying out reversion to the murine sequence) at the site.
According to the structural simulation, the 24 th Ala of the humanized heavy chain of the 2A9 antibody is back mutated into Thr, the 48 th Met is back mutated into Ile, the 67 th Val is back mutated into Ala, the 69 th Met is back mutated into Leu, the 71 th Arg is back mutated into Val, and the 73 th Thr is back mutated into Lys. The 46 th Leu of the humanized light chain of the 2A9 antibody is back mutated into Arg, the 47 th Leu is back mutated into Trp, the 48 th Ile is back mutated into Val, and the 71 th Phe is back mutated into Tyr. The 24 th Ala of the humanized heavy chain of the 4F11 antibody is back mutated into Thr, the 48 th Met is back mutated into Ile, the 67 th Val is back mutated into Ala, the 69 th Met is back mutated into Leu, the 71 th Arg is back mutated into Val, and the 73 th Thr is back mutated into Lys. The 46 th Leu of the humanized light chain of the 4F11 antibody is back mutated into Arg, the 47 th Leu is back mutated into Trp, the 48 th Ile is back mutated into Val, and the 71 th Phe is back mutated into Tyr. The method comprises the following steps of carrying out reverse mutation on 48 th Met of a humanized heavy chain of a 9C8 antibody to Ile, carrying out reverse mutation on 67 th Val to Ala, carrying out reverse mutation on 69 th Met to Leu, carrying out reverse mutation on 71 th Arg to Val, and carrying out reverse mutation on 73 th Thr to Lys. The 2 nd Ile of the humanized light chain of the 9C8 antibody is back mutated into Thr, the 46 th Leu is back mutated into Arg, the 47 th Leu is back mutated into Trp, and the 71 th Phe is back mutated into Tyr. The 48 th Met of the humanized heavy chain of the 15A2 antibody is back mutated into Ile, the 67 th Val is back mutated into Ala, the 69 th Met is back mutated into Leu, the 71 th Arg is back mutated into Ala, and the 73 th Thr is back mutated into Lys. The 36 th Tyr of the humanized light chain of the 15A2 antibody is back mutated into Phe, the 47 th Leu is back mutated into Trp, the 49 th Tyr is back mutated into His, the 58 th Ile is back mutated into Phe, and the 71 th Phe is back mutated into Tyr. The 27 th Gly of the humanized heavy chain of the 5B6 antibody is subjected to reverse mutation to Tyr, the 30 th Ser is subjected to reverse mutation to Ile, the 48 th Met is subjected to reverse mutation to Ile, the 67 th Val is subjected to reverse mutation to Ala, and the 69 th Ile is subjected to reverse mutation to Leu. The 46 th Leu of the humanized light chain of the 5B6 antibody is back mutated into Arg, the 47 th Leu is back mutated into Pro, the 66 th Gly is back mutated into Ala, the 71 th Phe is back mutated into Tyr, and the 98 th Phe is back mutated into Ile. The Ser at position 49 of the humanized heavy chain of the 7B7 antibody was back mutated to Ala. The 2 nd Ile of the humanized light chain of the 7B7 antibody is subjected to reverse mutation to Ser, and the 48 th Ile is subjected to reverse mutation to Val.
The amino acid sequence numbers of the variable regions of the heavy chain and the light chain of the humanized antibody are respectively SEQ ID NO: 113 and SEQ ID NO: 114. the amino acid sequence numbers of the variable regions of the heavy chain and the light chain of the 4F11 humanized antibody are respectively SEQ ID NO: 115 and SEQ ID NO: 116. the variable region amino acid sequence numbers of the heavy chain and the light chain of the 9C8 humanized antibody are SEQ ID NOs: 117 and SEQ ID NO: 118. 15a2 humanized antibody heavy and light chain variable region amino acid sequence numbers are SEQ ID NOs: 119 and SEQ ID NO: 120. 5B6 humanized antibody heavy and light chain variable region amino acid sequence numbers of SEQ ID NO: 121 and SEQ ID NO: 122. 7B7 humanized antibody heavy and light chain variable region amino acid sequence numbers of SEQ ID NO: 123 and SEQ ID NO: 124. the above humanized antibody was constructed as IgG1 subtype. The amino acid sequence information for the variable regions of the heavy and light chains of the humanized antibodies is shown in Table 4.
Nucleic acid sequences encoding the light and heavy chains of the humanized antibodies 2a9, 4F11, 9C8, 15a2,5B6 and 7B7 were synthesized and inserted into the expression vector pcdna3.1. 200mL of 293 cells (cell density 1X 10) were co-transfected with 0.1mg of antibody light chain and 0.1mg of antibody heavy chain expression plasmid6/mL), shaking the flask at 37 ℃ for 6 days, centrifuging and collecting the supernatant, purifying the humanized antibody with Protein A, and detecting the activity of the purified humanized antibody.
Table 4. sequence listing of humanized antibodies to B7H 3.
Example 7
Binding of humanized B7H3 antibodies to B7H3
The binding activity of the humanized B7H3 antibody sample to the B7H3 protein was measured by ELISA and FACS, which are shown in example 2. The results of the assay of humanized antibodies hu2A9, hu4F11, hu5B6, hu9C8, hu15A2 and hu7B7 are shown in Table 5-6 and FIGS. 9-10. In general, the antibodies of the invention, after humanization, retain the high antigen affinity effect prior to humanization, particularly hu15a2, hu7B7, and hu5B 6.
TABLE 5 detection of binding of humanized B7H3 antibody to B7H3-mFc protein by ELSIA
TABLE 6 binding of humanized B7H3 antibody to cell surface B7H3 as determined by FACS
Example 8
Detection of cell endocytosis of humanized B7H3 antibody
The specific measurement method is described in example 3. The results are shown in table 7 and fig. 11; the PC9 cells had significant endocytosis of humanized B7H3 antibody. Compared to positive controls (MGA 018 from macrogenetics and 8H9 from MSKCC), the antibodies of the invention were much more potent than 8H9 for endocytosis; in addition, hu15A2 and hu7B7 also outperform MGC 018.
TABLE 7 endocytosis of humanized B7H3 antibody by PC9 tumor cells
Example 9
Poisoning effect of humanized B7H3 antibody (B7H3-ADC) conjugated with toxin on Calu-6 tumor cells
Vc-MMAE was conjugated to the B7H3 antibody by chemical conjugation. The B7H3 antibody was reduced with TCEP at a molar ratio of 3:1, incubated at 37 ℃ for 45 minutes and the TCEP was removed by ultrafiltration. Resuspend the antibody with 0.5ml PBS, add 6 times mole number of Vc-MMAE of B7H3 antibody, incubate at 4 ℃ for 120 minutes, remove unconjugated Vc-MMAE by ultrafiltration, detect the antibody concentration for use. 100 μ l of PC9 (5000 cells per well) and 100 μ l of gradient diluted B7H3-ADC were added to a 96-well plate. After incubation at 37 ℃ for 5 days, x. mu.l of CellTiter-Glo was added to each well, the fluorescence intensity was measured, and the ability of the antibody to inhibit tumor cell proliferation was calculated. The results are shown in fig. 12 and table 8, and the humanized B7H3 antibody has significant toxic effect on Calu-6 tumor cells after being coupled with MMAE toxin; has stronger activity than a MacroGenics positive control (MGC 018-MMAE).
TABLE 8 poisoning effect of conjugates of humanized B7H3 antibody and MMAE on Calu-6 tumor cells
Example 10
Detection of ADCC by humanized B7H3 antibody
The specific measurement method is described in example 5. The results are shown in table 9 and fig. 13, demonstrating that the humanized B7H3 antibody has significant ADCC activity. MGA271(MG-Ab) is an antibody to Macrogenics; in contrast, humanized 9C8, 5B6, 2a9 and 4F11 antibodies had smaller EC50 values, reflecting better ADCC activity.
TABLE 9 ADCC Effect of humanized B7H3 antibody
Example 11
Humanized B7H3 antibody mediates ADCC killing of NK92MI-hCD16 cells against Jurkat-B7H3 and PC9 cells
NK92MI-hCD16 was used to mimic NK cells to evaluate the tumor killing activity of the B7H3 antibody. Mu.l of PC9 stained with CFSE (0.5X 10 per well)5Cells) and 25. mu.l NK92MI-hCD16 (1X10 per well)5Cells) and 50 μ l of the B7H3 antibody was added as a gradient. After 4 hours of incubation at 37 ℃, 5. mu.l of 7-AAD was added to each well, incubated at room temperature for 10 minutes, and the 96-well plate was centrifuged offAfter the supernatant was resuspended, the ratio of CFSE and 7-AAD double positive cells to CFSE positive cells was measured by flow cytometry, and the NK cell killing activity mediated by B7H3 antibody was obtained. The results are shown in fig. 14, and the humanized B7H3 antibodies 9C8, 5B6, 2a9 and 4F11 can obviously cause ADCC killing effect of NK92MI-hCD16 cells on PC9 tumor cells.
Example 12
Promotion of cytokine secretion from human PBMC by humanized B7H3 antibody
The specific measurement method is described in example 3. As shown in FIG. 15, the humanized B7H3 antibodies 5B6, 2A9 and 4F11 significantly promoted IFN-. gamma.secretion from human PBMC cells.
The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.
SEQUENCE LISTING
<110> east China Biotechnology (Suzhou) Ltd
<120> a group of B7H3 monoclonal antibodies and medical application thereof
<130> 2021
<160> 124
<170> PatentIn version 3.5
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Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
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Leu Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Ala Tyr
20 25 30
Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Phe Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ala Ala Val Tyr Tyr Cys
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Thr Arg Ser Ser Thr Arg Leu Arg Pro Phe Asp Tyr Trp Gly Gln Gly
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Thr Thr Leu Thr Val Ser Ser
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Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
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Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
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Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Ser Gly Gln Ser
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Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn
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Leu Glu Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
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Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
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Gly Gly Ile Asn Pro Asn Ser Gly Gly Thr Thr Tyr Asn Leu Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Ala Arg Lys Glu Gly Phe Phe Thr Thr Asp Tyr Trp Gly Gln Gly Thr
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Thr Leu Thr Val Ser Ser
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Gln Thr Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
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His Trp Tyr Arg Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
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Asp Thr Ser Asn Leu Ala Thr Gly Val Pro Val Arg Phe Ser Gly Ser
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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp His Tyr Asn Pro Pro Thr
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Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Thr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Tyr Ile Asn Pro Thr Ser Asp Tyr Ala Asn Phe Asn Gln Lys Phe
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Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Arg Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Ala Arg Trp Gly Thr Thr Val Phe Gln Phe Tyr Phe Asp Tyr Trp Gly
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Gln Gly Thr Thr Leu Thr Val Ser Ser
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Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
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Ala Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
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Asp Ala Ala Thr Tyr Phe Cys Gln His Trp Ser Ser Asp Pro Pro Thr
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Ile Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Glu Val Gln Leu Gln Gln Ser Gly Thr Glu Leu Val Arg Pro Gly Ala
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Leu Val Lys Leu Ser Cys Lys Ala Ser Ala Leu Asn Ile Lys Asp Phe
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Tyr Met His Trp Val Lys Gln Arg Pro Glu Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe
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Gln Gly Lys Ala Ser Ile Thr Ala Asp Ile Ser Ser Ser Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Ser Ser Thr Arg Gln Arg Pro Phe Asp Tyr Trp Gly Gln Gly
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Thr Thr Leu Thr Val Ser Ser
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Asp Ile Val Met Thr Gln Ser His Lys Val Met Ser Thr Ser Val Gly
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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Thr Gly
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Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
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Gly Gly Ile Asn Pro Asn Ser Gly Gly Thr Thr Tyr Asn Glu Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
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Ala Arg Arg Met Pro Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
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Thr Val Thr Val Ser Ser
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Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Val Ser Asn Pro Leu Thr
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Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
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Ile Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
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Gly Gly Ile Asn Pro Asn Thr Gly Gly Ile Thr Tyr Asn Gln Lys Phe
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Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Ser
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Ala Arg Ala Gly Gly Asn Phe Leu Trp Tyr Phe Asp Val Trp Gly Ala
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Gly Thr Thr Val Thr Val Ser Ser
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Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
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Gly Ser Gly Thr Ser Tyr Ser Leu Ala Ile Ser Ser Met Glu Ala Glu
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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Leu Thr
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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala
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Gly Trp Ile Asp Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Phe Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
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Val Arg Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
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Ser Ser
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Gln Ile Leu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
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Glu Lys Val Thr Met Thr Cys Arg Thr Ser Ser Ser Val Ser Tyr Leu
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His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile His
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Asp Thr Ser Asn Leu Ala Ser Gly Phe Pro Ala Arg Phe Ser Gly Ser
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Gly Ser Gly Thr Ser Tyr Ser Leu Ile Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Trp Thr
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Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly
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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
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Ala Val Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Ile Tyr Ile Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Thr Pro Asp Asp Thr Ala Leu Tyr Tyr Cys
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Ala Arg His Gly Gly Ala Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
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Asp Ser Leu Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
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Glu Thr Val Thr Ile Thr Cys Arg Gly Ser Glu Ser Val His Ser Tyr
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Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Gln Phe Ser Leu Arg Ile Asn Ser Leu Gln Pro
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Glu Asp Phe Gly Thr Tyr Tyr Cys Gln Gln His Tyr Gly Ser Pro Pro
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Trp Thr Phe Gly Gly Gly Thr Ile Leu Glu Ile Lys
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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val
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Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr
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Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Ser Tyr Tyr Gly Asn Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Gly Tyr Ser Tyr Gly Ser Ser Tyr Arg Pro Asp Arg
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Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Ile Val Ser Ala
115 120 125
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Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly
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Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
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Thr Met His Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Thr Asp Asn Gly Gly Thr Thr Tyr Ser Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Val Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Phe Asn Asn Tyr Gly Pro Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Ala Leu Val Thr Val Ser Ala
115 120
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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Ile Gly
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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Arg Ser Ala
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Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile
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Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ala Arg Phe Thr Gly
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Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Gly Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Phe Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Lys Lys
100 105
<210> 21
<211> 121
<212> PRT
<213> artificial
<400> 21
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Ser Gly Ala
1 5 10 15
Ser Val Asn Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn
20 25 30
Thr Met His Trp Val Lys Leu Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Arg Trp Pro Pro Asp Trp Tyr Phe Asp Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 22
<211> 106
<212> PRT
<213> artificial
<400> 22
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Leu Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 23
<211> 120
<212> PRT
<213> artificial
<400> 23
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Ile Tyr Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gly Ile Lys Pro Ser Asn Gly Asp Thr Asn Phe Asn Ala Lys Phe
50 55 60
Met Asn Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Phe Phe Tyr Gly Ser Pro Tyr Asp Phe Asp Val Trp Gly Ala
100 105 110
Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 24
<211> 107
<212> PRT
<213> artificial
<400> 24
Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ala Val Gly
1 5 10 15
Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Asp Thr His
20 25 30
Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Thr Leu Leu Met
35 40 45
Tyr Gly Thr Ser Asn Arg Phe Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Arg Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 25
<211> 120
<212> PRT
<213> artificial
<400> 25
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Val Met His Trp Val Lys Gln Arg His Gly Gln Ser Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe
50 55 60
Glu Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr
65 70 75 80
Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Met Pro Phe Gly Ile Ser Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 26
<211> 106
<212> PRT
<213> artificial
<400> 26
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Phe Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Ser
35 40 45
Glu Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Gly Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys His Gln Trp Ile Ser Tyr Pro Pro Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 27
<211> 123
<212> PRT
<213> artificial
<400> 27
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp His
20 25 30
Ala Ile His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile
35 40 45
Gly Val Ile Asn Thr Tyr Ser Gly Asn Thr Asn Phe Asn Gln Lys Leu
50 55 60
Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Gly Ala His Ser Ser Gly Tyr Pro Tyr Trp Tyr Phe Asp Val
100 105 110
Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 28
<211> 106
<212> PRT
<213> artificial
<400> 28
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Thr Asn Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Met Lys
100 105
<210> 29
<211> 5
<212> PRT
<213> artificial
<400> 29
Ala Tyr Tyr Ile His
1 5
<210> 30
<211> 17
<212> PRT
<213> artificial
<400> 30
Trp Ile Asp Pro Glu Asn Gly Asn Thr Phe Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 31
<211> 10
<212> PRT
<213> artificial
<400> 31
Ser Ser Thr Arg Leu Arg Pro Phe Asp Tyr
1 5 10
<210> 32
<211> 16
<212> PRT
<213> artificial
<400> 32
Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr
1 5 10 15
<210> 33
<211> 7
<212> PRT
<213> artificial
<400> 33
Gln Met Ser Asn Leu Ala Ser
1 5
<210> 34
<211> 8
<212> PRT
<213> artificial
<400> 34
Ala Gln Asn Leu Glu Leu Tyr Thr
1 5
<210> 35
<211> 5
<212> PRT
<213> artificial
<400> 35
Glu Tyr Thr Ile His
1 5
<210> 36
<211> 17
<212> PRT
<213> artificial
<400> 36
Gly Ile Asn Pro Asn Ser Gly Gly Thr Thr Tyr Asn Leu Lys Phe Lys
1 5 10 15
Gly
<210> 37
<211> 9
<212> PRT
<213> artificial
<400> 37
Lys Glu Gly Phe Phe Thr Thr Asp Tyr
1 5
<210> 38
<211> 10
<212> PRT
<213> artificial
<400> 38
Ser Ala Ser Ser Ser Leu Ser Tyr Ile His
1 5 10
<210> 39
<211> 7
<212> PRT
<213> artificial
<400> 39
Asp Thr Ser Asn Leu Ala Thr
1 5
<210> 40
<211> 9
<212> PRT
<213> artificial
<400> 40
Gln Gln Trp His Tyr Asn Pro Pro Thr
1 5
<210> 41
<211> 5
<212> PRT
<213> artificial
<400> 41
Thr Tyr Thr Ile His
1 5
<210> 42
<211> 17
<212> PRT
<213> artificial
<400> 42
Tyr Ile Asn Pro Thr Ser Asp Tyr Ala Asn Phe Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 43
<211> 12
<212> PRT
<213> artificial
<400> 43
Trp Gly Thr Thr Val Phe Gln Phe Tyr Phe Asp Tyr
1 5 10
<210> 44
<211> 10
<212> PRT
<213> artificial
<400> 44
Arg Ala Ser Gln Ser Leu Ser Tyr Ile Asn
1 5 10
<210> 45
<211> 7
<212> PRT
<213> artificial
<400> 45
Ala Thr Ser Ser Leu Gln Ser
1 5
<210> 46
<211> 9
<212> PRT
<213> artificial
<400> 46
Gln His Trp Ser Ser Asp Pro Pro Thr
1 5
<210> 47
<211> 5
<212> PRT
<213> artificial
<400> 47
Asp Phe Tyr Met His
1 5
<210> 48
<211> 17
<212> PRT
<213> artificial
<400> 48
Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 49
<211> 10
<212> PRT
<213> artificial
<400> 49
Ser Ser Thr Arg Gln Arg Pro Phe Asp Tyr
1 5 10
<210> 50
<211> 11
<212> PRT
<213> artificial
<400> 50
Lys Ala Ser Gln Asp Val Arg Thr Gly Val Ala
1 5 10
<210> 51
<211> 7
<212> PRT
<213> artificial
<400> 51
Ser Ala Phe Tyr Arg Tyr Thr
1 5
<210> 52
<211> 9
<212> PRT
<213> artificial
<400> 52
Gln Gln Tyr Tyr Ser Ile Pro Trp Thr
1 5
<210> 53
<211> 5
<212> PRT
<213> artificial
<400> 53
Glu Tyr Ile Met His
1 5
<210> 54
<211> 17
<212> PRT
<213> artificial
<400> 54
Gly Ile Asn Pro Asn Ser Gly Gly Thr Thr Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 55
<211> 9
<212> PRT
<213> artificial
<400> 55
Arg Met Pro Tyr Trp Tyr Phe Asp Val
1 5
<210> 56
<211> 10
<212> PRT
<213> artificial
<400> 56
Ser Ala Ser Ser Ser Val Ser Tyr Ile His
1 5 10
<210> 57
<211> 7
<212> PRT
<213> artificial
<400> 57
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 58
<211> 9
<212> PRT
<213> artificial
<400> 58
Gln Gln Trp Val Ser Asn Pro Leu Thr
1 5
<210> 59
<211> 5
<212> PRT
<213> artificial
<400> 59
Glu Tyr Ile Met His
1 5
<210> 60
<211> 17
<212> PRT
<213> artificial
<400> 60
Gly Ile Asn Pro Asn Thr Gly Gly Ile Thr Tyr Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 61
<211> 11
<212> PRT
<213> artificial
<400> 61
Ala Gly Gly Asn Phe Leu Trp Tyr Phe Asp Val
1 5 10
<210> 62
<211> 10
<212> PRT
<213> artificial
<400> 62
Ser Ala Ser Ser Ser Val Ser Tyr Met His
1 5 10
<210> 63
<211> 7
<212> PRT
<213> artificial
<400> 63
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 64
<211> 9
<212> PRT
<213> artificial
<400> 64
Gln Gln Trp Asn Ser Asn Pro Leu Thr
1 5
<210> 65
<211> 5
<212> PRT
<213> artificial
<400> 65
Ser Tyr Asp Ile Asn
1 5
<210> 66
<211> 17
<212> PRT
<213> artificial
<400> 66
Trp Ile Asp Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210> 67
<211> 5
<212> PRT
<213> artificial
<400> 67
Arg Gly Phe Asp Tyr
1 5
<210> 68
<211> 10
<212> PRT
<213> artificial
<400> 68
Arg Thr Ser Ser Ser Val Ser Tyr Leu His
1 5 10
<210> 69
<211> 7
<212> PRT
<213> artificial
<400> 69
Asp Thr Ser Asn Leu Ala Ser
1 5
<210> 70
<211> 9
<212> PRT
<213> artificial
<400> 70
His Gln Arg Ser Ser Tyr Pro Trp Thr
1 5
<210> 71
<211> 5
<212> PRT
<213> artificial
<400> 71
Ser Tyr Ala Val Ser
1 5
<210> 72
<211> 17
<212> PRT
<213> artificial
<400> 72
Ser Ile Ser Gly Gly Gly Ile Tyr Ile Tyr Tyr Pro Asp Ser Val Lys
1 5 10 15
Gly
<210> 73
<211> 9
<212> PRT
<213> artificial
<400> 73
His Gly Gly Ala Gly Tyr Phe Asp Tyr
1 5
<210> 74
<211> 11
<212> PRT
<213> artificial
<400> 74
Arg Gly Ser Glu Ser Val His Ser Tyr Leu Ala
1 5 10
<210> 75
<211> 7
<212> PRT
<213> artificial
<400> 75
Asn Ala Lys Thr Leu Gln Ser
1 5
<210> 76
<211> 10
<212> PRT
<213> artificial
<400> 76
Gln Gln His Tyr Gly Ser Pro Pro Trp Thr
1 5 10
<210> 77
<211> 5
<212> PRT
<213> artificial
<400> 77
Asp Tyr Ala Met His
1 5
<210> 78
<211> 17
<212> PRT
<213> artificial
<400> 78
Val Ile Asn Ser Tyr Tyr Gly Asn Thr His Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 79
<211> 17
<212> PRT
<213> artificial
<400> 79
Ala Gly Gly Tyr Ser Tyr Gly Ser Ser Tyr Arg Pro Asp Arg Phe Ala
1 5 10 15
Tyr
<210> 80
<211> 11
<212> PRT
<213> artificial
<400> 80
Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala
1 5 10
<210> 81
<211> 7
<212> PRT
<213> artificial
<400> 81
Ala Ala Thr Asn Leu Ala Asp
1 5
<210> 82
<211> 9
<212> PRT
<213> artificial
<400> 82
Gln Gln Leu Tyr Ser Thr Pro Tyr Thr
1 5
<210> 83
<211> 5
<212> PRT
<213> artificial
<400> 83
Glu Tyr Thr Met His
1 5
<210> 84
<211> 17
<212> PRT
<213> artificial
<400> 84
Gly Ile Asn Thr Asp Asn Gly Gly Thr Thr Tyr Ser Gln Lys Phe Lys
1 5 10 15
Asp
<210> 85
<211> 12
<212> PRT
<213> artificial
<400> 85
Ser Phe Asn Asn Tyr Gly Pro Ala Trp Phe Ala Tyr
1 5 10
<210> 86
<211> 11
<212> PRT
<213> artificial
<400> 86
Lys Ala Ser Gln Asp Val Arg Ser Ala Val Ala
1 5 10
<210> 87
<211> 7
<212> PRT
<213> artificial
<400> 87
Trp Ala Ser Thr Arg His Thr
1 5
<210> 88
<211> 8
<212> PRT
<213> artificial
<400> 88
Gln Gln Tyr Ser Ser Tyr Phe Thr
1 5 10
<210> 89
<211> 5
<212> PRT
<213> artificial
<400> 89
Glu Asn Thr Met His
1 5
<210> 90
<211> 17
<212> PRT
<213> artificial
<400> 90
Gly Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys
1 5 10 15
Gly
<210> 91
<211> 12
<212> PRT
<213> artificial
<400> 91
Arg Gly Arg Trp Pro Pro Asp Trp Tyr Phe Asp Val
1 5 10
<210> 92
<211> 10
<212> PRT
<213> artificial
<400> 92
Ser Ala Ser Ser Ser Leu Ser Tyr Met His
1 5 10
<210> 93
<211> 7
<212> PRT
<213> artificial
<400> 93
Asp Thr Ser Lys Leu Ala Ser
1 5
<210> 94
<211> 9
<212> PRT
<213> artificial
<400> 94
Gln Gln Trp Ser Ser Asn Pro Leu Thr
1 5
<210> 95
<211> 5
<212> PRT
<213> artificial
<400> 95
Ser Tyr Tyr Ile Tyr
1 5
<210> 96
<211> 17
<212> PRT
<213> artificial
<400> 96
Gly Ile Lys Pro Ser Asn Gly Asp Thr Asn Phe Asn Ala Lys Phe Met
1 5 10 15
Asn
<210> 97
<211> 11
<212> PRT
<213> artificial
<400> 97
Phe Phe Tyr Gly Ser Pro Tyr Asp Phe Asp Val
1 5 10
<210> 98
<211> 11
<212> PRT
<213> artificial
<400> 98
Lys Ala Ser Glu Asn Val Asp Thr His Val Ser
1 5 10
<210> 99
<211> 7
<212> PRT
<213> artificial
<400> 99
Gly Thr Ser Asn Arg Phe Thr
1 5
<210> 100
<211> 9
<212> PRT
<213> artificial
<400> 100
Gly Gln Ser Tyr Arg Tyr Pro Tyr Thr
1 5
<210> 101
<211> 5
<212> PRT
<213> artificial
<400> 101
Glu Tyr Val Met His
1 5
<210> 102
<211> 17
<212> PRT
<213> artificial
<400> 102
Gly Ile Asn Pro Asn Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu
1 5 10 15
Gly
<210> 103
<211> 11
<212> PRT
<213> artificial
<400> 103
Arg Met Pro Phe Gly Ile Ser Ala Met Asp Tyr
1 5 10
<210> 104
<211> 10
<212> PRT
<213> artificial
<400> 104
Ser Ala Ser Ser Ser Val Ser Tyr Ile His
1 5 10
<210> 105
<211> 7
<212> PRT
<213> artificial
<400> 105
Glu Thr Ser Lys Leu Ala Ser
1 5
<210> 106
<211> 9
<212> PRT
<213> artificial
<400> 106
His Gln Trp Ile Ser Tyr Pro Pro Thr
1 5
<210> 107
<211> 5
<212> PRT
<213> artificial
<400> 107
Asp His Ala Ile His
1 5
<210> 108
<211> 17
<212> PRT
<213> artificial
<400> 108
Val Ile Asn Thr Tyr Ser Gly Asn Thr Asn Phe Asn Gln Lys Leu Lys
1 5 10 15
Gly
<210> 109
<211> 14
<212> PRT
<213> artificial
<400> 109
Gly Ala His Ser Ser Gly Tyr Pro Tyr Trp Tyr Phe Asp Val
1 5 10
<210> 110
<211> 10
<212> PRT
<213> artificial
<400> 110
Arg Ala Ser Ser Ser Val Ser Tyr Met His
1 5 10
<210> 111
<211> 7
<212> PRT
<213> artificial
<400> 111
Ala Thr Ser Asn Leu Ala Ser
1 5
<210> 112
<211> 9
<212> PRT
<213> artificial
<400> 112
Gln Gln Trp Ser Thr Asn Pro Phe Thr
1 5
<210> 113
<211> 118
<212> PRT
<213> artificial
<400> 113
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Ser Gly Gly Thr Thr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Met Pro Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 114
<211> 106
<212> PRT
<213> artificial
<400> 114
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Val Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Val Ser Asn Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 115
<211> 120
<212> PRT
<213> artificial
<400> 115
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Thr Gly Gly Ile Thr Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Gly Asn Phe Leu Trp Tyr Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 116
<211> 106
<212> PRT
<213> artificial
<400> 116
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Leu Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Asn Pro Leu Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 117
<211> 118
<212> PRT
<213> artificial
<400> 117
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gly Ile Asn Pro Asn Ser Gly Gly Thr Thr Tyr Asn Leu Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Lys Glu Gly Phe Phe Thr Thr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 118
<211> 106
<212> PRT
<213> artificial
<400> 118
Glu Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Leu Ser Tyr Ile
20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Asn Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp His Tyr Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 119
<211> 114
<212> PRT
<213> artificial
<400> 119
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Ala Thr Leu Thr Ala Asn Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Arg Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
100 105 110
Ser Ser
<210> 120
<211> 106
<212> PRT
<213> artificial
<400> 120
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Ser Ser Val Ser Tyr Leu
20 25 30
His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp Ile His
35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Phe Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Trp Thr
85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 121
<211> 121
<212> PRT
<213> artificial
<400> 121
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Thr Tyr
20 25 30
Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Thr Ser Asp Tyr Ala Asn Phe Asn Gln Lys Phe
50 55 60
Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Trp Gly Thr Thr Val Phe Gln Phe Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 122
<211> 106
<212> PRT
<213> artificial
<400> 122
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Tyr Ile
20 25 30
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr
35 40 45
Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Ala Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln His Trp Ser Ser Asp Pro Pro Thr
85 90 95
Ile Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 123
<211> 118
<212> PRT
<213> artificial
<400> 123
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ser Ile Ser Gly Gly Gly Ile Tyr Ile Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Gly Gly Ala Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 124
<211> 108
<212> PRT
<213> artificial
<400> 124
Asp Ser Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Gly Ser Glu Ser Val His Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Gly Ser Pro Pro
85 90 95
Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
