IDENTIFICATION OF NON-RESPONDERS TO HER2 INHIBITORS

Abstract

The present invention relates to means and methods for the identification of non-responders to a HER2 inhibitor, whereby one or more mutations in exon 9 of Phosphoinositol-3 kinase (PIK3CA) indicate non-responsiveness.

Description

[0001] The present invention relates to means and methods for the identification of non-responders to a HER2 inhibitor, whereby one or more mutations (mutational SNPs) in exon 9 of Phosphoinositol-3 kinase (PIK3CA) indicate non-responsiveness.

[0002] The HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0003] EGFR, encoded by the erbB1 gene, has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-.alpha.), by the same tumor cells resulting in receptor activation by an autocrine stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther. 64:127-154 (1994). Monoclonal antibodies directed against the EGFR or its ligands, TGF-.alpha. and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research 44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905 (1995).

[0004] The second member of the HER family, p185.sup.neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point mutation analogous to that in the neu proto-oncogene has been reported for human tumors. Overexpression of HER2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, among others, King et al., Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol., 6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988); Williams et al. Pathobiology 59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31 (1993)).

[0005] Antibodies directed against the rat p185.sup.neu and human HER2 protein products have been described. Drebin and colleagues have raised antibodies against the rat neu gene product, p185.sup.neu See, for example, Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and WO94/22478. Drebin et al. Oncogene 2:273-277 (1988) report that mixtures of antibodies reactive with two distinct regions of p185.sup.neu result in synergistic anti-tumor effects on neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.

[0006] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe the generation of a panel of HER2 antibodies which were characterized using the human breast tumor cell line SK-BR-3. Relative cell proliferation of the SK-BR-3 cells following exposure to the antibodies was determined by crystal violet staining of the monolayers after 72 hours. Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize HER2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-.alpha.. See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2 antibodies discussed in Hudziak et at are further characterized in Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993); Pictras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5 (1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et al. Oncogene 15:1385-1394 (1997).

[0007] A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAh HER2, Trastuzumab or Herceptin.TM.; U.S. Pat. No. 5,821,337) is clinically active in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumab received marketing approval from the Food and Drug Administration Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein.

[0008] Other HER2 antibodies with various properties have been described in Tagliabue et a. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

[0009] Homology screening has resulted in the identification of two other HER receptor family members: HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP Pat. Appln. No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al., Nature, 366:473475 (1993)). Both of these receptors display increased expression on at least some breast cancer cell lines.

[0010] The HER receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of HER ligands (Earp et al. Breast Cancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by six different ligands; epidermal growth factor (EGF), transforming growth factor alpha (TGF-.alpha.), amphiregulin, heparin binding epidermal growth factor (HB-EGF), betacellulin and epiregulin (Groenen et al. Growth Factors 11:235-257 (1994)). A family of heregulin proteins resulting from alternative splicing of a single gene are ligands for HER3 and HER4. The heregulin family includes alpha, beta and gamma heregulins (Holmes et at, Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiation factors (NDFs), glial growth factors (GGFs); acctylcholinc receptor inducing activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review, see Groenen et al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev. 47:51-85 (1995). Recently three additional HER ligands were identified; neuregulin-2 (NRG-2) which is reported to bind either HER3 or HER4 (Chang et al. Nature 387 509-512 (1997); and Carraway et al Nature 387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et at PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4 (Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin and epiregulin also bind to HER4.

[0011] While EGF and TGF.alpha. do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and results in transphosphorylation of HER2 in the heterodimer. Dimerization and/or transphosphorylation appears to activate the HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3 is co-expressed with HER2, an active signaling complex is formed and antibodies directed against HER2 are capable of disrupting this complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of HER3 for heregulin (HRG) is increased to a higher affinity state when co-expressed with HER2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996) with respect to the HER2-HER3 protein complex. HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8 (1994)).

[0012] To target the HER signaling pathway, rhuMAb 2C4 (Pertuzumab) was developed as a humanized antibody that inhibits the dimerization of HER2 with other HER receptors, thereby inhibiting ligand-driven phosphorylation and activation, and downstream activation of the RAS and AKT pathways. In a phase I trial of Pertuzumab as a single agent for treating solid tumors, 3 subjects with advanced ovarian cancer were treated with pertuzumab. One had a durable partial response, and an additional subject had stable disease for 15 weeks. Agus et al. Proc Am Soc Clin Oncol 22: 192, Abstract 771 (2003).

[0013] Also antibody variant compositions are described in the art. U.S. Pat. No. 6,339,142 describes a HER2 antibody composition comprising a mixture of anti-HER2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variant(s) is less than about 25%. Trastuzumab is the exemplified HER2 antibody. Reid et al. Poster presented at Well Characterized Biotech Pharmaceuticals conference (January, 2003) "Effects of Cell Culture Process Changes on Humanized Antibody Characteristics" describes an unnamed, humanized IgG1 antibody composition with N-terminal heterogeneities due to combinations of VHS signal peptide, N-terminal glutamine, and pyroglutamic acid on the heavy chain thereof. Harris et al. "The Ideal Chromatographic Antibody Characterization Method" talk presented at the IBC Antibody Production Conference (February, 2002) reports a VHS extension on the heavy chain of E25, a humanized anti-IgE antibody. Rouse et al. Poster presented at WCBP "Glycoprotein Characterization by High Resolution Mass Spectrometry and Its Application to Biopharmaceutical Development" (Jan. 6-9, 2004) describes a monoclonal antibody composition with N-terminal heterogeneity resulting from AHS or HS signal peptide residues on the light chain thereof. In a presentation at IBC Meeting (September, 2000) "Strategic Use of Comparability Studies and Assays for Well Characterized Biologicals," Jill Porter discussed a late-eluting form of ZENAPAX.TM. with three extra amino acid residues on the heavy chain thereof. US2006/0018899 describes a composition comprising a main species pertuzumab antibody and an amino-terminal leader extension variant, as well as other variant forms of the pertuzumab antibody.

[0014] Patent publications related to HER antibodies include: U.S. Pat. No. 5,677,171, U.S. Pat. No. 5,720,937, U.S. Pat. No. 5,720,954, U.S. Pat. No. 5,725,856, U.S. Pat. No. 5,770,195, U.S. Pat. No. 5,772,997, U.S. Pat. No. 6,165,464, U.S. Pat. No. 6,387,371. U.S. Pat. No. 6,399,063, US2002/0192211A1, U.S. Pat. No. 6,015,567, U.S. Pat. No. 6,333,169, U.S. Pat. No. 4,968,603, U.S. Pat. No. 5,821,337, U.S. Pat. No. 6,054,297, U.S. Pat. No. 6,407,213, U.S. Pat. No. 6,719,971, U.S. Pat. No. 6,800,738. US2004/0236078A1, U.S. Pat. No. 5,648,237. U.S. Pat. No. 6,267,958, U.S. Pat. No. 6,685,940, U.S. Pat. No. 6,821,515, WO98/17797, U.S. Pat. No. 6,127,526, U.S. Pat. No. 6,333,398, U.S. Pat. No. 6,797,814, U.S. Pat. No. 6,339,142, U.S. Pat. No. 6,417,335, U.S. Pat. No. 6,489,447, WO99/31140, US2003/0147884A1, US20030170234A1, US2005/0002928A1, U.S. Pat. No. 6,573,043, US2003/0152987A1, WO99/48527, US2002/0141993A1, WO01/00245, US2003/0086924, US2004/0013667A1, WO00/69460, WO01/00238, WO01/15730, U.S. Pat. No. 6,627,196B1, U.S. Pat. No. 6,632,979B1, WO01/00244, US2002/0090662A1, WO01/89566, US2002/0064785, US2003/0134344, WO 04/24866, US2004/0082047, US2003/0175845A1, WO03/087131, US2003/0228663, WO2004/008099A2, US2004/0106161, WO2004/048525, US2004/0258685A1, U.S. Pat. No. 5,985,553, U.S. Pat. No. 5,747,261, U.S. Pat. No. 4,935,341, U.S. Pat. No. 5,401,638, U.S. Pat. No. 5,604,107. WO 87/07646, WO 89/10412, WO 91/05264, EP 412,116 B1, EP 494,135 B1, U.S. Pat. No. 5,824,311. EP 444,181 B1, EP 1,006,194 A2, US 2002/0155527A1, WO 91/02062, U.S. Pat. No. 5,571,894, U.S. Pat. No. 5,939,531, EP 502,812 B1, WO 93/03741, EP 554,441 B1, EP 656,367 A1, U.S. Pat. No. 5,288,477, U.S. Pat. No. 5,514,554, U.S. Pat. No. 5,587,458, WO 93/12220, WO 93/16185, U.S. Pat. No. 5,877,305, WO 93/21319, WO 93/21232, U.S. Pat. No. 5,856,089, WO 94/22478, U.S. Pat. No. 5,910,486, U.S. Pat. No. 6,028,059, WO 96/07321, U.S. Pat. No. 5,804,396, U.S. Pat. No. 5,846,749, EP 711,565, WO 96/16673, U.S. Pat. No. 5,783,404, U.S. Pat. No. 5,977,322, U.S. Pat. No. 6,512,097, WO 97/00271, U.S. Pat. No. 6,270,765, U.S. Pat. No. 6,395,272, U.S. Pat. No. 5,837,243, WO 96/40789, U.S. Pat. No. 5,783,186, U.S. Pat. No. 6,458,356, WO 97/20858, WO 97/38731, U.S. Pat. No. 6,214,388, U.S. Pat. No. 5,925,519, WO 98/02463, U.S. Pat. No. 5,922,845, WO 98/18489, WO 98/33914, U.S. Pat. No. 5,994,071, WO 98/45479, U.S. Pat. No. 6,358,682 B1, US 2003/0059790, WO 99/55367, WO 01/20033, US 2002/0076695 A1, WO 00/78347, WO 01/09187. WO 01/21192, WO 01/32155, WO 01/53354, WO 01/56604, WO 01/76630, WO02/05791, WO 02/11677, U.S. Pat. No. 6,582,919, US2002/0192652A1, US 2003/0211530A1, WO 02/44413, US 2002/0142328, U.S. Pat. No. 6,602,670 B2, WO 02/45653, WO 02/055106, US 2003/0152572, US 2003/0165840. WO 02/087619, WO 03/006509, WO03/012072, WO 03/028638, US 2003/0068318, WO 03/041736, EP 1,357,132, US 2003/0202973, US 2004/0138160, U.S. Pat. No. 5,705,157, U.S. Pat. No. 6,123,939, EP 616,812 B1, US 2003/0103973, US 2003/0108545, U.S. Pat. No. 6,403,630 B1, WO 00/61145, WO 00/61185, U.S. Pat. No. 6,333,348 B1, WO 01/05425, WO 01/64246, US 2003/0022918, US 2002/0051785 A1, U.S. Pat. No. 6,767,541, WO 01/76586, US 2003/0144252, WO 01/87336, US 2002/0031515 A1, WO 01/87334, WO 02/05791, WO 02/09754, US 2003/0157097, US 2002/0076408, WO 02/055106, WO 02/070008, WO 02/089842 and WO 03/86467.

[0015] Patients treated with the HER2 antibody Trastuzumab/Herceptin.TM. are selected for therapy based on HER2 protein overexpression/gene amplification; see, for example, WO99/31140 (Paton et al.), US2003/0170234A1 (Hellmann, S.), and US2003/0147884 (Paton et al.); as well as WO01/89566, US2002/0064785, and US2003/0134344 (Mass et al.). See, also, US2003/0152987, Cohen et al., concerning immunohistochcmistry (IHC) and fluorescence in situ hybridization (FISH) for detecting HER2 overexpression and amplification. WO2004/053497 and US2004/024815A1 (Bacus et al.), as well as US 2003/0190689 (Crosby and Smith), refer to determining or predicting response to Trastuzumab therapy. US2004/013297A1 (Bacus et al.) concerns determining or predicting response to ABX0303 EGFR antibody therapy. WO2004/000094 (Bacus et al.) is directed to determining response to GW572016, a small molecule, EGFR-HER2 tyrosine kinase inhibitor. WO2004/063709, Amler et al., refers to biomarkers and methods for determining sensitivity to EGFR inhibitor, erlotinib HCl. US2004/0209290, Cobleigh et al., concerns gene expression markers for breast cancer prognosis.

[0016] Patients treated with pertuzumab (a HER2 dimerisation inhibitor described herein below in more detail) can be selected for therapy based on HER activation or dimerization. Patent publications concerning pertuzumab and selection of patients for therapy therewith include: WO01/00245 (Adams et al.); US2003/0086924 (Sliwkowski, M.); US2004/0013667A1 (Sliwkowski, M.); as well as WO2004/008099A2, and US2004/0106161 (Bossenmaier et al.).

[0017] Herceptin.TM./Trastuzumab is indicated in the art for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 protein or have HER 2 gene amplification:

[0018] a) As monotherapy for the treatment of those patients who have received at least two chemotherapy regimens for their metastatic disease. Prior chemotherapy must have included at least an anthracycline and a taxane unless patients are unsuitable for these treatments. Hormone receptor positive patients must also have received hormonal therapy, unless patients are unsuitable for these treatments,

[0019] b) In combination with paclitaxel for the treatment of those patients who have not received chemotherapy for their metastatic disease and for whom an anthracycline is not suitable and

[0020] c) In combination with docetaxel for the treatment of those patients who have not received chemotherapy for their metastatic disease.

[0021] Herceptin.TM./Trastuzumab can also be used as adjuvant treatment in early breast cancer. Herceptin.TM./Trastuzumab is also approved for the treatment of patients with HER2-positive early breast cancer following surgery, chemotherapy (neoadjuvant (i.e. before surgery) or adjuvant), and radiotherapy (if applicable). In addition Herceptin in combination with capecitabine or 5-fluorouracil and cisplatin is indicated for the treatment of patients with HER2 positive locally advance or metastatic adenocarcinoma of the stomach or gastroesophageal junction who have not received prior anti-cancer treatment for their metastatic disease.

[0022] In the art, the treatment of breast cancer patients with Herceptin.TM./Trastuzumab is, for example, recommended and routine for patients having HER2-positive cancer. HER2-positive cancer is present if a high HER2 (protein) expression level detected by immunohistochemical methods (e.g. HER2 (+++)) or HER2 gene amplification detected by in-situ-hybridization (e.g. ISH positive, like a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell or ratio of .gtoreq.2.0 for the number of HER2 gene copies to the number of signals for CEP17.) or both is found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites.

[0023] The NEOSPHERE study (Neoadjuvant Study of Pertuzumab and Herceptin in an Early Regimen Evaluation) is a randomized multicentre, international Phase II study that was conducted in 78 centres worldwide (except the USA) in 417 women with newly diagnosed HER2-positive early, inflammatory or locally advanced breast cancer who had never received Herceptin. Prior to surgery (neoadjuvant treatment) these women were randomized to four study arms. The primary endpoint was complete tumour disappearance at time of surgery (pathological complete response, pCR) and the results were:

[0024] pCR of 29.0 percent for Herceptin and docetaxel

[0025] pCR of 45.8 percent for Herceptin, pertuzumab and docetaxel

[0026] pCR of 16.8 percent for Herceptin and pertuzumab

[0027] pCR of 24.0 percent for pertuzumab and docetaxel

[0028] The data shows that the two antibodies plus docetaxel given in the neoadjuvant setting prior to surgery significantly improved the rate of complete tumour disappearance (pathological complete response rate, pCR, of 45.8 percent) in the breast by more than half compared to Herceptin plus docetaxel (pCR of 29.0 percent), p=0.014. The study is described in detail e.g. in Lancet Oncol. 2012 January; 13(1):25-32. doi: 10.1016/S1470-2045(11)70336-9. Epub 2011 Dec. 6, which is incorporated by reference herein in its entirety. Core biopsies (tumor tissue) from 387 patients were used for biomarker analyses.

[0029] However, not all patients having HER2-positive cancer or cancer cells respond to treatment with a HER2 inhibitor. Therefore, efforts have been made in the art to identify non-responding patients that may be excluded from treatment. Barbereschi (Clin Cancer Res 2007, 13:6064-6069) investigated the association of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) mutations on exon 9 and 20 with pathologic features and clinical outcome in breast cancer patients treated with chemotherapy and/or hormone therapy. Berns (Cancer Cell (2007) 12, 395-402) describes that the presence of PIK3CA mutations (inter alia in exon 9 and 20) was associated with poor prognosis after trastuzumab therapy. Also Razis (Breast Cancer Res Treat (DOI 10.1007/s10549-011-1572-5) investigates the association of PIK3CA mutations (in exon 9 and 20) with efficacy of trastuzumab therapy and describes that these mutations were associated with shorter median time to progression. Dave (2011, J Clinical Oncology 29, 166) also find that activating mutations in the PIK3CA conferred resistance to Trastuzumab.

[0030] Thus, the technical problem underlying the present invention is the provision of means and methods for identifying a patient or a group of patients with HER2-positive cancer who are non-responsive to a treatment with a HER2 inhibitor, in particular to a treatment with a HER2 antibody such as Trastuzumab or Pertuzumab.

[0031] The technical problem is solved by provision of the embodiments characterized in the claims.

[0032] Accordingly, the present invention relates to a method for identifying a non-responder to a HER2-inhibitor, said method comprising evaluating the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinosirol-3 kinase (PIK3CA or p110.alpha.) in a sample from a patient with HER2-positive cancer; and whereby the presence of one or more mutations in exon 9 indicates non-responsiveness of said patient to said HER2 inhibitor.

[0033] In the present invention, it was surprisingly found that mutations in exon 9 of Phosphoinositol-3 kinase (PIK3CA) are indicative for non-responsiveness of a patient with HER2-positive cancer to a HER2 inhibitor, such as Trastuzumab and, in particular Pertuzumab. In contrast, as found herein, mutations in exon 20 were not predictive for non-responsiveness. In other words, it was unexpectedly found that the evaluation only of mutations in exon 9 of Phosphoinositol-3 kinase (PIK3CA) is sufficient for a highly reliable determination of non-responders to treatment with HER2 inhibitors, in particular anti-HER2 antibodies. Therefore, exon 9 mutations are, preferably, the only PIK3CA mutations evaluated in the methods of the present invention. In accordance with the invention, solely the presence of mutations in exon 9 of PIK3CA is determined/evaluated/measured, i.e. the presence of mutations in other parts (e.g. like exon 20) of the PIK3CA gene or coding sequence are not evaluated or determined. The invention is, thus, based on the surprising finding that the determination/evaluation of solely (only) mutations (like mutational SNPs) in exon 9 of PIK3CA is enough for a reliable read-out whether a patient will or will not respond to treatment with a HER2 inhibitor. The evaluation of such mutations and/or SNPs is described herein below in more detail and exemplified in the examples. None of the documents discussed above discloses or proposes the use of only (solely) PIK3CA mutations (or mutational SNPs) in exon 9 for identifying non-responders to therapy with HER2 inhibitors.

[0034] Exemplary mutations/SNPs in exon 9 of PIK3CA that can be determined/evaluated in the herein provided method for identification of non-responders to HER2-inhibitors are those where the mutation results in a change in the amino acid sequence at position 542 and 545 of the full length protein sequence of PIK3CA as shown in SEQ ID NO: 2. In the wild-type protein sequence of PIK3CA the amino acid at position 542 and 545 is "E". In the mutant forms of PIK3CA to be determined herein, the wild type "E" at these positions is replaced by the amino acid "K" ("E542K" or "E545K"), amino acid "A" ("E545A") or amino acid "G" ("E545G"). These changes at amino acid level are also reflected in mutations at the nucleic acid level (like mutational SNPs) and corresponding mutated triplets (codons) to be determined/detected/evaluated are given herein further below.

[0035] The non-responders identified by the herein provided means and methods may be subject to other treatments than treatment with a HER2 inhibitor; for example, they may advantageously be treated with compounds other than HER2 inhibitors. The term "non-responder" as used herein can refer to an individual/patient/subject that is less likely to respond to a treatment using a HER2 inhibitor (like pertuzumab or trastuzumab). "Less likely to respond" as used herein refers to a decreased likeliness that a pathological complete response (pcR) will occur in a patient treated with a HER2 inhibitor. In cases where (with the methods of the present invention) it was assessed that the subject is a "non-responder" or is "less likely to respond", said subject is to receive phosphoinositol-3 kinase-targeted agents. Such agents are known in the art and comprise, but are not limited to fused pyrimidine derivatives as disclosed in U.S. Pat. No. 8,022,205 B2 or fused pyrrolopyrimidine derivatives as disclosed in WO2009/099163.

[0036] The sample to be evaluated can be obtained from a patient with HER2-positive cancer. The HER2-positive cancer may assessed be breast cancer, such as early-stage breast cancer. However, the method of identifying non-responders provided herein can be applied to a wide range of HER2-positive cancers, like gastric cancer, colon cancer, lung cancer and the like. In a preferred embodiment, the HER2 inhibitor is an anti-HER2 antibody, like pertuzumab or trastuzumab. Preferably, the patient is a human.

[0037] Accordingly, this invention relates to a method for identifying a non-responder to a HER2-inhibitor, said method comprising detecting/measuring the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) in a sample from a patient with HER2-positive cancer; and whereby the presence of one or more mutations in exon 9 indicates non-responsiveness of said patient to said HER2 inhibitor.

[0038] The present invention relates to a method for identifying a non-responder to a HER2-inhibitor, said method comprising the steps

[0039] (a) obtaining a sample from a patient with HER2-positive cancer,

[0040] (b) evaluating the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) in said sample; whereby the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or pi 110.alpha.) indicates non-responsiveness of said patient to said HER2 inhibitor.

[0041] As mentioned, it has been found herein that the presence of one or more mutations in exon 9 of the catalytic subunit of PI3K i.e. PIK3CA indicates non-responsiveness to a HER2 inhibitor. The following provides some background information on PIK3CA and the family of Phosphatidylinositol 3-kinase to which it belongs; the mutations in exon 9 of the catalytic subunit of PI3K are explained in more detail further below. The mutation may be the replacement or exchange (substitution) of one or more amino acids as compared to the wild-type sequence of exon 9 of Phosphoinositol-3 kinase catalytic subunit (PIK3CA). Corresponding nucleic acid sequences and amino acid sequences of wild-type PIK3CA are shown in SEQ ID NO. 1 and SEQ ID NO: 2, respectively, and in FIG. 7. As used herein, the term "PIK3CA" refers to the catalytic subunit of Phosphoinositol-3 kinase (PI3K), isoform alpha, also refereed to as p110alpha. The terms "PIK3CA", "catalytic subunit of Phosphoinositol-3 kinase isoform alpha" or, short, "p110alpha"/"p110.alpha." are used interchangeably herein. "PIK3CA" is the term recommended and commonly used in the art; however, the entire protein is also known as PI3K. Phosphatidylinositol 3-kinase (PI3K) is composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. The protein encoded by the PIK3CA gene represents the catalytic subunit of PI3K, which uses ATP to phosphorylate PtdIns, PtdIns4P and PtdIns(4.5)P2 (i.e. this catalytic subunit is "PIK3CA" as defined and used herein). This gene has been found to be oncogenic and has been implicated in a variety of cancers.

[0042] Phosphoinositol-3 kinase belongs to the family of Phosphatidylinositol 3-kinases (PI 3-kinases or "PI3Ks"). This is a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. In response to lipopolysaccharide, PI3Ks phosphorylate p65, inducing anandamide synthesis to inhibit NF-.kappa.B activation. This is under the control of FAAH limiting the ability of LPS to increase AEA levels and is also inhibited by wortmannin and cannabidiol, one of the only natural compounds to inhibit FAAH. The phosphoinositol-3-kinase family is divided into three different classes: Class I, Class II, and Class III. The classifications are based on primary structure, regulation, and in vitro lipid substrate specificity.

[0043] The following table provides an overview of the human genes/proteins of Phosphatidylinositol 3-kinases family members. PIK3CA is highlighted in bold.

TABLE-US-00001 group gene protein synonyms class 2 PIK3C2A PI3K, class 2, alpha polypeptide PI3K-C2.alpha. PIK3C2B PI3K, class 2, beta polypeptide PI3K-C2.beta. PIK3C2G PI3K, class 2, gamma polypeptide PI3K-C2.gamma. class 3 PIK3C3 PI3K, class 3 Vps34 class 1 PIK3CA PI3K, catalytic, alpha polypeptide p110-.alpha. catalytic PIK3CB PI3K, catalytic, beta polypeptide p110-.beta. PIK3CG PI3K, catalytic, gamma polypeptide p110-.gamma. PIK3CD PI3K, catalytic, delta polypeptide p110-.delta. class 1 PIK3R1 PI3K, regulatory subunit 1 (alpha) p85-.alpha. regulatory PIK3R2 PI3K, regulatory subunit 2 (beta) p85-.beta. PIK3R3 PI3K, regulatory subunit 3 (gamma) p55-.gamma. PIK3R4 PI3K, regulatory subunit 4 p150 PIK3R5 PI3K, regulatory subunit 5 p101 PIK3R6 PI3K, regulatory subunit 6 p87

[0044] PIK3CA and its genetic variants to be used in the herein provided methods for identifying non-responders to HER2-inhibitors and their use in screening methods for responsiveness to treatment with a compound are described, for example, in WO 2011/031861, WO 2005/091849 and WO 2011/060380.

[0045] In context of the present invention, the mutation (mutational SNP) to be determined/assessed in accordance with the present invention may be a mutation in the codon encoding an amino acid at position 542 and/or 545 of the full-length amino acid sequence of Phosphatidylinositol-3 kinase (PIK3CA) (see e.g. SEQ ID NO. 2 of FIG. 7). The mutation may comprise one or more of the mutations E542K, E545K, E545A and E545G (i.e. mutations/SNP in exon 9 encoding the amino acid K, A or G at position 542 or 545 of the amino acid sequence of Phosphatidylinositol-3 kinase (PIK3CA) instead of wildtype E. The term E542K, E545K, E545A and E545G as used herein refer to amino acid substitutions at a given position of the amino acid sequence of wild type PIK3CA. Corresponding nucleic acid sequences (codons/triplets) encoding the amino acid at positions 542 and 545 in wild type and mutant PIK3CA genes/coding sequences are described below. In accordance with internationally accepted nomenclature, the term "E542K" refers to a substitution/replacement of amino acid "E" at position 542 of the amino acid sequence of wild type PIK3CA by amino acid "K". The same explanation applies, mutatis mutandis, to "E545K", "E545A" and "E545G". These mutations are well known in the art and corresponding mutated sequences can be retrieved from the respective databases like Uniprot. Based on the herein provided teaching, the presence of these mutations can readily be determined by a person skilled in the art. Preferably, the mutation is determined on a nucleic acid level as described below and exemplified in the examples. The mutations E542K E545K, E545A and E545G are also illustrated in the herein described sequences. For example, nucleic acid and amino acid sequences of mutation (mutational SNP) E542K are shown in SEQ ID NO. 17 and SEQ ID NO. 18, respectively; nucleic acid and amino acid sequences of mutation E545K are shown in SEQ ID NO: 19 and SEQ ID NO. 20, respectively; nucleic acid and amino acid sequences of mutation E545A are shown in SEQ ID NO: 21 and SEQ ID NO. 22, respectively, and nucleic acid and amino acid sequences of mutation E545G are shown in SEQ ID NO: 23 and SEQ ID NO. 24, respectively.

[0046] Methods for the determining/evaluation assessed/measured of the presence of the mutations are described herein and provided in the examples. Exemplary, non-limiting methods to be used are methods for sequencing of nucleic acids (e.g. Sanger di-deoxy sequencing), "next generation" sequencing methods, single molecule sequencing, methods enabling detection of variant alleles/mutations, such as Real-time PCR, PCR-RFLP assay (see Cancer Research 59 (1999), 5169-5175), mass-spectrometric genotyping (e.g. MALDI-TOF), HPLC, enzymatic methods and SSPC (single strand conformation polymorphism analysis; see Pathol Int (1996) 46, 801-804).

[0047] Such methods may include enzymatic amplification of DNA or cDNA fragments using oligonucleotides specifically hybridizing to exon 9 (or parts thereof) of the PIK3CA gene by PCR. Given that mutations in exon 9 of the PIK3CA gene are to be evaluated, such amplifications may be carried out in one or two reactions when employing RNA or genomic DNA. The resulting PCR products may be subjected to either conventional Sanger-based dideoxy nucleotide sequencing methods or employing parallel sequencing methods ("next generation sequencing") such as those marketed by Roche (454 technology), Illumina (Solexa technology) or ABI (Solid technology). Mutations may be identified from sequence reads by comparison with publicly available gene sequence data bases. Alternatively, mutations may be identified by incorporation of allele-specific probes that can either be detected using enzymatic detection reactions, fluorescence, mass spectrometry or others; see Vogeser (2007) Dtsch Arztebl 104 (31-32), A2194-200.

[0048] Paraffin-embedded clinical material as well as fresh frozen tissue may be used in the detection of these mutations. Detection may comprise a histolopathology review of the sample to be tested to see whether tumour tissue is present. The following table shows exemplary nucleic acid sequences of the mutations (mutational SNPs) to be determined in accordance with the present invention; any other point mutation(s) that result in an amino acid change at position 542 and/or 545 (or position 546, like the E545D mutation having the sequence "gat"/"T" mutation") of full-length the amino acid sequence of PIK3CA can be included in the assessment in accordance with the present invention.

TABLE-US-00002 gaa codon/triplet encoding wild type E542 gag codon/triplet encoding wild type E545 aaa codon/triplet encoding mutant E542K ((E >> K) aag codon/triplet encoding mutant E545K (E >> K), gcg codon/triplet encoding mutant E545A (E >> A), ggg codon/triplet encoding mutant E545G(E >> G),

[0049] Accordingly, the term "mutation E542K in exon 9 of Phosphoinositol-3 kinase (PIK3CA)" as used herein may refer to a codon/triplet (like aaa) encoding amino acid K at position 542 of the full-length amino acid sequence of PIK3CA (the wild-type sequence thereof is shown in FIG. 7 and SEQ ID NO: 2). The term "mutation E545K in exon 9 of Phosphoinositol-3 kinase (PIK3CA)" as used herein may refer to a codon/triplet (like aag) encoding amino acid K at position 545 of the full-length amino acid sequence of PIK3CA (the wild-type sequence thereof is shown in FIG. 7 and SEQ ID NO: 2). The term "mutation E545A in exon 9 of Phosphoinositol-3 kinase (PIK3CA)" as used herein may refer to a codon/triplet (like gcg) encoding amino acid A at position 545 of the full-length amino acid sequence of PIK3CA (the wild-type sequence thereof is shown in FIG. 7 and SEQ ID NO: 2). The term "mutation E545G in exon 9 of Phosphoinositol-3 kinase (PIK3CA)" as used herein may refer to a codon/triplet (like ggg) encoding amino acid G at position 545 of the full-length amino acid sequence of PIK3CA (the wild-type sequence thereof is shown in FIG. 7 and SEQ ID NO: 2).

[0050] The following exemplary test may be used.

[0051] The PCR amplification of isolated DNA and mutation detection procedures for the PIK3CA mutation detection test are summarized below.

[0052] Each standard 50-.mu.L amplification reaction targeting one of the Exons 7, 9 or 20 included 100 ng genomic DNA, dNTPs (including dUTP), 0.05 U/.mu.L Z05, DNA polymerase, 0.04 U/.mu.L uracil-DNA glycosylase (UNG), and 200-400 nM forward and reverse primer (Table 1), 75-200 nM mutant and wild-type specific probes (Table 2). Amplification was performed in the Cobas.RTM. 4800 analyzer using the following temperature profile: 5 min at 50.degree. C. followed by 55 cycles of 95.degree. C. for 10 sec and 63.degree. C. for 50 sec, followed by a single round of 40.degree. C. for 2 min and 25.degree. C. for 10 sec (melting curve analysis). Fluorescence data was collected during each amplification cycle and during the final melting curve analysis.

[0053] The following exemplary primers/primer pair may be used in the method for identifying a non-responder to a HER2-inhibitor provided herein, wherein the non-responder has a mutation in exon 9 of PIK3CA.

TABLE-US-00003 TABLE 1 Primer Sequences for the PIK3CA Mutation Detection Test Primer Sequence 5' to 3' 542/545 Forward primer UAAAAUUUAUUGAGAAUGUAUUUGCTTTTTC (SEQ ID NO: 25) PIK3CA-9F13 542/545 Reverse primer TCCATTTTAGCACTTACCTGTGAC (SEQ ID NO: 26) PIK3CA-9R01 Key: U = 5-propynyl dU

[0054] The following exemplary probes may be used in the method for identifying a non-responder to a HER2-inhibitor provided herein, wherein the non-responder has a mutation in exon 9 of PIK3CA.

TABLE-US-00004 TABLE 2 Probe Sequence for the PIK3CA Mutation Detection Test Probe Sequence 5' to 3' 542 WT Probe FTTTCAQAGAGAGGAUEUEGUGUAGAAAUUGEP (SEQ ID NO: 27) 542 542K Mutation Probe LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (SEQ ID NO: 28) 545 WT Probe OCTGCTCAGTQAUUUIAGAGAGAGGATCTCGTGTP (SEQ ID NO: 29) 545 545K Mutation Probe JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (SEQ ID NO: 30) 545 545A Mutation Probe FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (SEQ ID NO: 31) 545 545G Mutation Probe LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (SEQ ID NO: 32) Key: F = FAM Reporter Dye, J = JA270 Reporter Dye, O = CY5.5 Reporter Dye, L = HEX Reporter Dye, U = 5-propynyl dU, E = 5-methyl dC, I = deoxyinosine, Z = 7-deaza dG, Q = BHQ2 Quencher Dye, P = 3' Phosphate

[0055] In accordance with the present invention, the presence of one or more mutations in exon 9 of PIK3CA may be evaluated/determined/measured in the herein provided method for identifying a non-responder to HER2 inhibitors (the terms "evaluating", "determining" and "measuring" can be used interchangeably in context of the present invention). In one embodiment, the presence of only one of the mutations is evaluated/determined. Accordingly, the presence of only one of the E542K, E545K, E545A and E545G mutations of PIK3CA (i.e. the nucleic acids in exon 9 of PIK3CA encoding the amino acid at these positions in the full length amino acid sequence of PIK3CA) may be evaluated, i.e. only E542K, only E545K, only E545A or only E5450. The methods of the present invention may also comprise the subsequent evaluation of the presence of two or more of these mutations in any order. For example, the evaluation of the presence of E542K may be followed by the evaluation of E545K (or vice versa) which may be followed by the evaluation of the presence of E545A, which may be followed by the evaluation of the presence of E545G. Other possible orders of evaluation are easily conceivable by a person skilled in the art and contemplated herein.

[0056] The mutations may be evaluated in combination/simultaneously. Again, any combination is envisaged. For example, the presence of E542K and E545K is evaluated; or the presence of E542K and E545A; or the presence of E542K and E545G is evaluated. Other combinations are easily conceivable. The evaluation of a combination of these mutations may be followed or preceded by the evaluation of the presence of one other mutation or a combination of other mutations.

[0057] As mentioned, the present invention provides for means to determine whether an individual/patient with HER2-positive cancer (i.e. suffering from, suspected to suffer from or being prone to suffer from HER2-positive cancer) will respond to treatment with a HER2 inhibitor or will not respond to treatment to a HER2 inhibitor. This assessment may be advantageously done before the start of treatment with the HER2 inhibitor. Even if a patient has been treated with a HER2 inhibitor, a person skilled in the art can determine whether a person showed no response after the treatment with the HER2 inhibitor. For example, a non-response to an inhibitor may be reflected in an increased suffering from cancer, such as an increased growth of a cancer/tumor and/or increase in the size of a tumor, the (increase in) the formation of metastases or a increase in the number or size of metastases. A non-response may also be the development of a tumor or metastases, for example after resection of a tumor, in the shortening of time to disease progression, or in the increase in the size of (a) tumor(s) and/or (a) metastases, for example in neoadjuvant therapy.

[0058] In accordance with the methods provided in the present invention a patient group can be identified that does not respond to treatment with HER2 inhibitors, like Pertuzumab or Trastuzumab. It has been found herein that some individuals with HER2 positive cancer or cancer cells do not adequately respond to treatment with a HER2 inhibitor, if the patients have mutations in exon 9 of PIK3CA. In one embodiment of the present invention, at least 80%, 90%, 95% or more of the patient group identified by the herein provided method do not respond to treatment with a HER2 inhibitor. That means that at least 80% of the identified individuals having the herein described mutation(s) in exon 9 of PIK3CA will not respond to the treatment with the herein defined HER2 inhibitors, like Pertuzumab or Trastuzumab.

[0059] As the skilled artisan fully appreciates a positive test for one or more mutations in exon 9 of PIK3CA in a sample of a patient with HER2-positive cancer does not indicate that the patient will not respond to treatment with absolute certainty. However, by the herein provided methods sub-groups of patients are identified that have a lower chance of response (=show a lower response rate) to a treatment with a HER2 inhibitor like pertuzumab or trastuzumab as compared to the sub-groups of patients not having these mutations in exon 9 of PIK3CA. With other words the determination of a presence of one or more mutations in exon 9 of PIK3CA indicates (=is indicative for) that the patient has a lower chance (=probability, likelihood) to respond to treatment with a HER2 inhibitor, as compared to a patient having no mutation in exon 9 of PIK3CA (wild type PIK3CA). Preferably, the response is pathologic complete response (pCR). The term "pCR" as used herein refers to the absence of invasive cancer cells in tissue like breast tissue or absence of invasive tumor cells in tissue like breast tissue and/or lymph nodes. pCR is commonly used as an endpoint in neoadjuvant treatment such as in breast cancer treatment.

[0060] The term "HER2-positive cancer" as used herein refers to a cancer/tumorous tissue etc. which comprises cancer cells which have higher than normal levels of HER2. Examples of HER2-positive cancer include HER2-positive breast cancer and HER2-positive gastric cancer. For the purpose of the present invention, "HER2-positive cancer" has an immunohistochemistry (IHC) score of at least 2+ and/or an in situ hybridization (ISH) amplification ratio .gtoreq.2.0 (i.e. is ISH-positive). Accordingly, HER2-positive cancer is present if a high HER2 (protein) expression level detected e.g. by immunohistochemical methods and/or HER2 gene amplification detected by in-situ-hybridization (ISH) positive, like a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell or ratio of .gtoreq.2.0 for the number of HER2 gene copies to the number of signals for CEP17.) is found in samples obtained from die patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites. In one embodiment "HER2-positive cancer" has an immunohistochemistry (IHC) score of HER2(3+) and/or is ISH positive.

[0061] The expression level of HER2 may be detected by an immunohistochemical method, whereas said HER2 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH). Corresponding assays and kits are well known in the art, for protein expression assays as well as for the detection of gene amplifications. Alternatively other methods like qRT-PCR might be used to detect levels of HER2 gene expression.

[0062] The expression level of HER2 can, inter alia, be detected by an immunohistochemical method. Such methods are well known in the art and corresponding commercial kits are available. Exemplary kits which may be used in accordance with the present invention are, inter alia, HerceptTest.TM. produced and distributed by the company Dako or the test called Ventana Pathway.TM.. The level of HER2 protein expression may be assessed by using the reagents provided with and following the protocol of the HercepTest.TM.. A skilled person will be aware of further means and methods for determining the expression level of HER2 by immunohistochemical methods; see for example WO 2005/117553. Therefore, the expression level of HER2 can be easily and reproducibly determined by a person skilled in the art without undue burden. However, to ensure accurate and reproducible results, the testing must be performed in a specialized laboratory, which can ensure validation of the testing procedures.

[0063] The expression level of HER2 can be classified in a low expression level, an intermediate expression level and a high expression level. It is preferred in context of this invention that HER2-positive disease is defined by a strong expression level of HER2 (e.g. HER2(3+) by THC), for example determined in a sample of a cancer patient.

[0064] The recommended scoring system to evaluate the IHC staining patterns which reflect the expression levels of HER2 designated herein HER2(0), HER2(+), HER2(++) and HER2(+++), is as follows:

TABLE-US-00005 Staining HER2 Intensity overexpression Score Staining Pattern assessment 0 No staining is observed or membrane staining is observed negative in <10% of the tumor cells 1+ A faint/barely perceptible membrane staining is detected negative in >10% of the tumor cells. The cells are only stained in part of their membrane. 2+ A weak to moderate complete staining is detected in >10 weak to moderate % of the tumor cells. overexpression. 3+ A strong complete membrane staining is detected in >10 strong % of the tumor cells. overexpression.

[0065] The terms HER2(+), HER2(++) and HER2(+++) used herein are equivalent to the terms HER2(1+), HER2(2+) and HER2(3+). A "low protein expression level" used in context of this invention corresponds to a 0 or 1+ score ("negative assessment" according to the table shown herein above), an "weak to moderate protein expression level" corresponds to a 2+ score ("weak to moderate overexpression", see the table above) and a "high protein expression level" corresponds to a 3+ score ("strong overexpression", see the table above). As described herein above in detail, the evaluation of the protein expression level (i.e. the scoring system as shown in the table) is based on results obtained by immunohistochemical methods. As a standard or routinely, the HER-2 status is, accordingly, performed by immunohistochemistry with one of two FDA-approved commercial kits available; namely the Dako Herceptest.TM. and the Ventana Pathway.TM.. These are semi-quantitative assays which stratify expression levels into 0 (<20,000 receptors per cell, no expression visible by IHC staining), 1+(.about.100,000 receptors per cell, partial membrane staining, <10% of cells overexpressing HER-2), 2+(.about.500,000 receptors per cell, light to moderate complete membrane staining, >10% of cells overexpressing HER-2), and 3+(.about.2,000,000 receptors per cell, strong complete membrane staining, >10% of cells overexpressing HER-2).

[0066] Alternatively, further methods for the evaluation of the protein expression level of HER2 may be used, e.g. Western Blots, ELISA-based detection systems and so on.

[0067] A HER2-positive cancer may also be diagnosed by assessing the gene amplification status of HER2. HER2-positive cancer is, accordingly, diagnosed if this assessment by ISH is positive. In accordance with this assessment, a HER2-positive cancer may, inter alia, relate to an average HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell (for those test systems without an internal centromere control probe) or to a HER2/CEP17 ratio of >=2.0 (for those test systems using an internal chromosome 17 centromere control probe). In other words, the HER2-positive cancer may, inter alia, relate to a HER2 gene copy number greater than 4. The amplification level of the HER2 gene may easily be identified by in situ hybridization (ISH) like fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH). These methods are known to the skilled artisan. The principles of these methods can be deduced from standard text books. Commercial kits for the determination of the HER2 gene amplification status by in situ hybridization are available.

[0068] The HER2-positive cancer may be breast cancer or gastric cancer. Further, the HER2-positive cancer may be ovarian cancer, lung cancer, colorectal cancer, kidney cancer, bone cancer, bone marrow cancer, bladder cancer, skin cancer, prostate cancer, esophagus cancer, salivary gland cancer, pancreas cancer, liver cancer, head and neck cancer, CNS (especially brain) cancer, cervix cancer, cartilage cancer, colon cancer, genitourinary cancer, gastrointestinal tract cancer, pancreas cancer, synovium cancer, testis cancer, thymus cancer, thyroid cancer and uterine cancer. In one embodiment the breast cancer is early-stage breast cancer, as also assessed in the appended example.

[0069] The sample to be assessed in accordance with the herein provided methods for identification a non-responder to a HER2 inhibitor may comprise non-diseased cells and/or diseased cells, i.e. non-cancerous cells and/or cancerous cells however the content of cancerous cells among non cancerous cells should be higher than 50%. The sample may also (or even solely) comprise cancer/tumor cell(s), such as breast cancer/tumor cell(s). The term "sample" shall generally mean any biological sample obtained from a patient's tumor. The sample may be a tissue resection or a tissue biopsy. The sample may also be a metastatic lesion or a section of a metastatic lesion or a blood sample known or suspected to comprise circulating tumor cells. In accordance with the above, the biological sample may comprise cancer cells and to a certain extent i.e. less than 50% non-cancer cells (other cells). The skilled pathologist is able to differentiate cancer cells from normal tissue cells. Methods for obtaining tissue biopsies, tissue resections and body fluids and the like from mammals, such as humans, are well known in the art.

[0070] As mentioned, the sample is obtained from a patient with HER2-positive cancer as defined above. For example, the sample may be obtained from a tumorous tissue, (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s) suspected of being HER2-positive tumour, like a breast tumor and the like. A person skilled in the art is in the position to identify such tumors and/or individuals/patients suffering from corresponding cancer using standard techniques known in the art and methods disclosed herein. Generally, said tumor cell or cancer cell may be obtained from any biological source/organism, particularly any biological source/organism, suffering from the above-mentioned cancer. In context of this invention particular useful cells are, preferably, human cells. These cells can be obtained from e.g. biopsies or from biological samples. The tumor/cancer/tumor cell/cancer cell is a solid tumor/cancer/tumor cell/cancer cell. In accordance with the above, the cancer/tumor cell may be a breast cancer/tumor cell or said sample comprises a cancer/tumor cell, such as a breast cancer/tumor cell. In line with the above, said tumor/cancer may be a breast tumor/cancer.

[0071] The method for identifying a non-responder to a HER2-inhibitor provided herein may further comprise obtaining a sample of tissue from a patient with HER2-positive cancer prior to said step of identifying. The tissue may be cancerous tissue. The method may further comprise adjusting the treatment of the patient in response to the presence of said one or more mutations in exon 9.

[0072] The identification of non-responders allows for the treatment of patients that do not have the mutations in exon 9 of PIK3CA, as these patients respond well to treatment with HER2 inhibitor(s), such as Pertuzumab. Accordingly, the present invention relates in one embodiment to an inhibitor of HER2 for use in treating a patient with HER2-positive cancer, whereby the cancer has been found to be PIK3CA mutation-negative in Exon 9 of PIK3CA. Also the use of an inhibitor of HER2 for the preparation of a pharmaceutical composition for the treatment of a HER2-positive cancer patient is envisaged, whereby the cancer has been found to be PIK3CA mutation-negative.

[0073] Further, the present invention relates to the use of a HER2 inhibitor to treat a HER2-positive cancer patient by administering the HER2 dimerization inhibitor in an amount effective to treat the cancer, provided the cancer has been found to be PIK3CA mutation-negative. Accordingly, a method for the treatment of a HER2-positive cancer patient is provided which comprises administering the HER2 dimerization inhibitor in an amount effective to treat the cancer, provided the cancer has been found to be PIK3CA mutation-negative. The term "PIK3CA mutation-negative" as used herein means that the mutations are not present (absent). As mentioned above, the PIK3CA mutation comprises or consists of preferably one or more mutations in exon 9 of Phosphoatidylinositol-3 kinase (PIK3CA) as defined herein above. Preferably, the patient is a human.

[0074] In one embodiment of the present invention, the HER2 inhibitor is to be administered as a single anti-tumor agent. In a further embodiment, the inhibitor may be administered in form of a combination therapy, such as chemotherapy, an anti-hormonal therapy and/or another HER2 targeted agent/another HER2 targeted therapy in addition.

[0075] The chemotherapy may be docetaxel, anthracycline/taxane chemotherapy, therapy with an anti-metabolite agents, therapy with an anti-hormonal compound, therapy with an anti-estrogen, therapy with a tyrosine kinase inhibitor, therapy with a raf inhibitor, therapy with a ras inhibitor, therapy with a dual tyrosine kinase inhibitor, therapy with taxol, therapy with taxane, therapy with doxorubicin, therapy with adjuvant (anti-) hormone drugs and/or therapy with cisplatin and the like. In accordance with the present invention, the HER2 inhibitor may be administered by any one of a parenteral route, oral route, intravenous route, subcutaneous route, intranasal route or transdermal route. Further, the HER2 inhibitor may be employed in a neoadjuvant or adjuvant setting. Accordingly, said HER2 inhibitor may be administered to a patient in need of such a treatment and having the herein defined biomarker status before, during or after a surgical intervention/resection of the cancerous tissue. Therefore, the present invention is useful in neoadjuvant therapy, i.e. the treatment with the herein defined HER2 inhibitor (like Pertuzumab or Trastuzumab) given to the herein defined cancer patient group prior to surgery, as well as in adjuvant therapy. Again, the patient group of the present invention to be treated by the means and methods provided herein (in particular with Pertuzumab) are cancer patients without one or more mutations in exon 9 of PIK3CA. The attending physician may modify, change or amend the administration schemes for the HER2 inhibitor in accordance with his/her professional experience.

[0076] In one embodiment, the HER2 inhibitor is a HER dimerization/signaling inhibitor or an inhibitor of HER2 shedding. The HER dimerization inhibitor may be a HER2 dimerization inhibitor. HER dimerization inhibitor inhibits HER heterodimerization or HER homodimerization. The HER dimerization inhibitor may be an anti-HER antibody.

[0077] The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Also human and humanized as well as CDR-grafted antibodies are comprised.

[0078] The HER antibody may bind to a HER receptor selected from the group consisting of EGFR, HER2 and HER3. Preferably, the antibody binds to HER2. In one embodiment, the anti HER2 antibody may bind to domain II of HER2 extracellular domain. In another embodiment, the antibody may bind to a junction between domains I, II and III of HER2 extracellular domain. Most preferably, the anti HER2 antibody is Pertuzumab.

[0079] For the purposes herein, "Pertuzumab" and "rhuMAb 2C4", which are used interchangeably, refer to an antibody comprising the variable light and variable heavy domains (amino acid sequences thereof shown in SEQ ID Nos. 5 and 6, respectively, as depicted in FIG. 2). The variable light and variable heavy domains of variant 574/Pertuzumab are also shown in FIG. 2 (amino acid sequences thereof shown in SEQ ID Nos. 7 and 8, respectively, as depicted in FIG. 2). Where Pertuzumab is an intact antibody, it preferably comprises an IgG1 antibody; in one embodiment comprising the light chain amino acid sequence in it preferably comprises the light chain and heavy chain amino acid sequences in SEQ ID Nos. 11 and 12, respectively, as shown in FIG. 3A/3B and 5A/5B (FIG. 5A/5B show the light chain and heavy chain amino acid sequences of a variant Pertuzumab, SEQ ID NOs: 15 and 16, respectively). The heavy chain amino acid sequences of Pertuzumab as shown in SEQ ID NO: 12 (FIG. 3B) may optionally comprise an additional amino acid "K" at position 449 at the C-terminus. The antibody is optionally produced by recombinant Chinese Hamster Ovary (CHO) cells. The terms "Pertuzumab" and "rhuMAb 2C4" herein cover biosimilar versions of the drug with the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Pertuzumab. Again, corresponding sequences are shown in FIGS. 2 to 5.

[0080] In a further embodiment, the inhibitor of HER shedding is a HER2 shedding inhibitor. The inhibitor of HER shedding may inhibit HER heterodimerization or HER homodimerization. Said inhibitor of HER shedding may be an anti-HER antibody. The anti-HER antibody may bind to a HER receptor selected from the group consisting of EGFR, HER2 and HER3. Preferably, the antibody binds to HER2. In one embodiment, the HER2 antibody binds to sub-domain IV of the HER2 extracellular domain. Preferably, the HER2 antibody is Herceptin.TM./Trastuzumab.

[0081] For the purposes herein, "Trastuzumab" and "rhuMAb4D5-8", which are used interchangeably, refer to an antibody comprising the variable light domains and variable heavy domains (amino acid sequences thereof are shown in FIG. 4 in SEQ ID NO: 13 and 14, respectively; the domain is indicated by arrows). Where Trastuzumab is an intact antibody, it preferably comprises an IgG1 antibody; in one embodiment comprising the light chain amino acid sequence of SEQ ID NO: 13 and the heavy chain amino acid sequence of SEQ ID NO: 14 as shown in FIG. 4. The antibody is optionally produced by Chinese Hamster Ovary (CHO) cells. The terms "Trastuzumab" and "rhuMAb4D5-8" herein cover biosimilar versions of the drug with the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Trastuzumab.

[0082] The HER2 positive cancer to be treated may be breast cancer, such early stage breast cancer. The term "early-stage breast cancer" as used herein refers to breast cancer that has not spread beyond the breast or the axillary lymph nodes. Such cancer is generally treated with neoadjuvant or adjuvant therapy. "Neoadjuvant therapy" refers to systemic therapy given prior to surgery. "Adjuvant therapy" refers to systemic therapy given after surgery. Also other HER2 positive cancer types like gastric cancer can be treated in accordance with the present invention. In one embodiment, the treatment is neoadjuvant or adjuvant therapy of the early-stage breast cancer.

[0083] The pharmaceutical composition to be employed in the medical uses of the present invention, will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of the pharmaceutical composition for purposes herein is thus determined by such considerations.

[0084] The skilled person knows that the effective amount of pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. For example, if said compound is a (poly)peptide or protein the total pharmaceutically effective amount of pharmaceutical composition administered parenterally per dose will be in the range of about 1 .mu.g protein/kg/day to 10 mg protein/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg protein/kg/day, and most preferably for humans between about 0.01 and 1 mg protein/kg/day.

[0085] The following administration may be employed in respect of Pertuzumab:

[0086] A dosing regimen of pertuzumab administered every 3 weeks to patients in Phase II studies (TOC2689g, RO16934) using a fixed 840 mg loading dose (equivalent to 12 mg/kg for a 70 kg patient) for treatment cycle I followed by a fixed 420 mg "maintenance" dose (equivalent to 6 mg/kg) for subsequent treatment cycles resulted in steady-state serum trough concentrations of approximately 60 .mu.g/mL by the second treatment cycle. A dose based on body-surface area or weight was not superior to a fixed dose, supporting the continued use of a fixed dose of pertuzumab in female patients with locally advanced, inflammatory or early stage HER2-positive breast cancer, metastatic breast cancer and ovarian cancer.

[0087] If given continuously, the pharmaceutical composition is typically administered at a dose rate of about 1 .mu.g/kg/hour to about 50 g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art. Pharmaceutical compositions of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.

[0088] Pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0089] The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

[0090] For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

[0091] Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. The carrier may be a parenteral carrier, such as a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[0092] The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0093] The components of the pharmaceutical composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.

[0094] Another embodiment of the present invention relates to the use of a nucleic acid or antibody capable of detecting a mutation in exon 9 of PIK3CA for identifying a non-responder to a HER2-inhibitor in accordance with the herein provided methods. The oligonucleotide(s) may be about 15 to 100 nucleotides in length.

[0095] Accordingly, the present invention relates in one embodiment to a forward primer having the sequence 5'-UAAAAUUUAUUGAGAAUGUAUUUGCTTTTITC-3' (SEQ ID NO: 25). This forward primer can be used in amplification of exon 9 or a part thereof encoding the mutant triplet which encodes position 542 and/or 545 of the herein described mutant exon 9 of PIKC3A. In a further embodiment, the present invention relates to a reverse primer having the sequence 5'-TCCATTTTAGCACTTACCTGTGAC-3' (SEQ ID NO: 26). This reverse primer can also be used in amplification of exon 9 or a part thereof encoding the mutant triplet which encodes position 542 and/or 545 of the herein described mutant exon 9 of PIKC3A. The present invention provides a primer pair of the forward primer having the sequence 5'-UAAAAUUUAUUGAGAAUGUAUUUGCTTTTTC-3' (SEQ ID NO: 25) and the reverse primer having the sequence 5'-TCCATTTTAGCACTTAACCTGTGAC-3' (SEQ ID NO: 26).

[0096] In a further embodiment, the present invention relates to probe(s)/probe sequence(s) for evaluating/determining the presence of one or more mutations in exon 9 of PIK3CA. In one embodiment, the present invention relates to a probe having the sequence 5'-FTTTCAQAGAGAGGAUEUEGUGUAGAAAUUGEP-3' ("542 WT Probe") (SEQ ID NO: 27). In one embodiment, the present invention relates to a probe having the sequence 5'-LATTTTQOAGAOAGOAUEUEOUGUAGAAAUUGEUUP-3' (542K Mutation Probe) (SEQ ID NO: 28). In one embodiment, the present invention relates to a probe having the sequence 5'-OCTGCTCAGTQAUUUIAAGAGAGGATCTCGTGTP-3' (545 WT Probe) (SEQ ID NO: 29). In one embodiment, the present invention relates to a probe having the sequence 5'-JAATCACTAAGQAGGAGAAAGAIUUUEUAUGGAGUEP-3' (545K Mutation Probe) (SEQ ID NO: 30). In one embodiment, the present invention relates to a probe having the sequence 5'-FCTOCGCQGGAGAAAGAUUUUEUAUGOAGUEAP-3' (545A Mutation Probe) (SEQ ID NO: 31). In one embodiment, the present invention relates to a probe having the sequence 5'-LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP-3' (545G Mutation Probe) (SEQ ID NO: 32). In this context, the following abbreviations used: F=FAM Reporter Dye, J=JA270 Reporter Dye, O=CY5.5 Reporter Dye, L=HEX Reporter Dye, U=5-propynyl dU, E=5-methyl dC, I=deoxyinosine, Z=7-deaza dG, Q=BHQ2 Quencher Dye, P=3' Phosphate

[0097] A person skilled in the art is, based on his general knowledge and the teaching provided herein, in the position to identify and/or prepare further oligo- or polynucleotide(s) for use in the present methods. In particular these oligo- or polynucleotides may be used as probe(s) in the detection methods described herein. A skilled person will know, for example, computer programs which may be useful for the identification of corresponding probes to be used herein. For example, the PIK3CA coding sequence (SEQ ID NO: 1) may be used in this context. Exemplary nucleic acid sequences are available on corresponding databases, such as the NCBI database (www.ncbi.nlm.nih.gov/sites/entrez).

[0098] The present invention also relates to a kit useful for carrying out the herein provided methods, the kit comprising a nucleic acid or an antibody capable of detecting a mutation in exon 9 of PIK3CA. Also envisaged herein is the use of the herein described kit for carrying out the herein provided methods.

[0099] For example, said kit may comprise (a) compound(s) required for specifically determining the one or more mutations in exon 9 of PIK3CA. Moreover, the present invention also relates to the use of (a) compound(s) required for specifically determining the presence of one or more mutations in exon 9 of PIK3CA for the preparation of a kit for carrying out the methods or uses of this invention.

[0100] On the basis of the teaching of this invention, the skilled person knows which compound(s) is (are) required for specifically determining the presence of one or more mutations in exon 9 of PIK3CA. Particularly, such compound(s) may be (a) (nucleotide) probe(s), (a) primer(s) (pair(s)), (an) antibody(ies) and/or (an) aptamer(s) specific to the mutation described herein. The kit (to be prepared in context) of this invention may be a diagnostic kit.

[0101] The kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to determine one or more mutations in exon 9 of PIK3CA i.e. (how) to diagnose non-responsiveness to a HER2 inhibitor. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.

[0102] The kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention. For example, such substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the presence of a mutation in exon 9 of PIK3CA.

[0103] The present invention relates to a method of detecting mutations in the human PI3KCA nucleic acid in a sample comprising:

[0104] (a) contacting the nucleic acid in the sample with at least one mutation-specific oligonucleotide from Table 2;

[0105] (b) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0106] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid.

TABLE-US-00006

[0106] TABLE 2 Probe Sequences for the PIK3CA Mutation Detection Test Probe Sequence 5' to 3' 542 WT Probe FTTTCAQAGAGAGGAUEUEGUGUAGAAAUUGEP (SEQ ID NO: 27) 542 542K Mutation Probe LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (SEQ ID NO: 28) 545 WT Probe OCTGCTCAGTQAUUUIAGAGAGAGGATCTCGTGTP (SEQ ID NO: 29) 545 545K Mutation Probe JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (SEQ ID NO: 30) 545 545A Mutation Probe FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (SEQ ID NO: 31) 545 545G Mutation Probe LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (SEQ ID NO: 32) Key: F = FAM Reporter Dye, J = JA270 Reporter Dye, O = CY5.5 Reporter Dye, L = HEX Reporter Dye, U = 5-propynyl dU, E = 5-methyl dC,, I = deoxyinosine, Z = 7-deaza dG, Q = BHQ2 Quencher Dye, P = 3' Phosphate

[0107] The present invention relates to a method of detecting mutations in the human PI3KCA nucleic acid in a sample comprising:

[0108] (a) contacting the nucleic acid in the sample with one or more of the following mutation-specific oligonucleotides:

TABLE-US-00007

[0108] (542 542K Mutation Probe); (SEQ ID NO: 28) LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (545 545K Mutation Probe); (SEQ ID NO: 30) JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (545 545A Mutation Probe); (SEQ ID NO: 31) FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP and/or (545 545G Mutation Probe); (SEQ ID NO: 32) LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP



[0109] (b) incubating the sample under conditions allowing hybridization of the one or more oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0110] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid.

[0111] The method may further comprise, prior to detection is step (c), contacting the nucleic acid in the sample with at least one oligonucleotide from Table 1 and generating an amplification product containing the target sequence within the PI3KCA nucleic acid.

TABLE-US-00008 TABLE 1 Primer Sequences for the PIK3CA Mutation Detection Test Primer Sequence 5' to 3' 542/545 Forward primer UAAAAUUUAUUGAGAAUGUAUUUGCTTTTTC (SEQ ID NO: 25) PIK3CA-9F13 542/545 Reverse primer TCCATTTTAGCACTTACCTGTGAC (SEQ ID NO: 26) PIK3CA-9R01 Key: U = 5-propynyl dU

[0112] The present invention relates to a method of detecting mutations in the human PI3KCA nucleic acid in a sample comprising:

[0113] (a) contacting the nucleic acid in the sample with at least one mutation-specific oligonucleotide from Table 2;

[0114] (b) (i) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0115] (ii) contacting the nucleic acid in the sample with at least one oligonucleotide from Table 1

[0116] (iii) generating an amplification product containing the target sequence within the PI3KCA nucleic acid;

[0117] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid.

[0118] In accordance with the above, the present invention provides a method of detecting mutations in the human PI3KCA nucleic acid in a sample comprising:

[0119] (a) contacting the nucleic acid in the sample with one or more of the following mutation-specific oligonucleotides:

TABLE-US-00009

[0119] (542 542K Mutation Probe); (SEQ ID NO: 28) LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (545 545K Mutation Probe); (SEQ ID NO: 30) JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (545 545A Mutation Probe); (SEQ ID NO: 31) FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP and/or (545 545G Mutation Probe) (SEQ ID NO: 32) LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP



[0120] (b) (i) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0121] (ii) contacting the nucleic acid in the sample with one or both of the following oligonucleotides:

[0122] UAAAAUUUAUUGAGAAUGUAUUUGCITTTTTC (SEQ ID NO: 25) (5421545 Forward primer PIK3CA-9F13) and/or TCCATTTAGCACTTACCTGTGAC (SEQ ID NO: 26) (542/545 Reverse primer PIK3CA-9R01);

[0123] (iii) generating an amplification product containing the target sequence within the PI3KCA nucleic acid;

[0124] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid.

[0125] The present invention provides a method of determining whether a patient having a malignant tumor is likely to respond to a HER2-inhibitor, comprising:

[0126] (a) contacting the nucleic acid in the sample from the patient with the oligonucleotide from Table 2;

[0127] (b) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0128] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid, wherein the presence of the mutation indicates that the patient is likely to respond to the HER2 inhibitor.

TABLE-US-00010

[0128] TABLE 2 Probe Sequences for the PIK3CA Mutation Detection Test Probe Sequence 5' to 3' 542 WT Probe FTTTCAQAGAGAGGAUEUEGUGUAGAAAUUGEP (SEQ ID NO: 27) 542 542K Mutation Probe LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (SEQ ID NO: 28) 545 WT Probe OCTGCTCAGTQAUUUIAGAGAGAGGATCTCGTGTP (SEQ ID NO: 29) 545 545K Mutation Probe JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (SEQ ID NO: 30) 545 545A Mutation Probe FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (SEQ ID NO: 31) 545 545G Mutation Probe LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (SEQ ID NO: 32) Key: F = FAM Reporter Dye, J = JA270 Reporter Dye, O = CY5.5 Reporter Dye, L = HEX Reporter Dye, U = 5-propynyl dU, E = 5-methyl dC, I = deoxyinosine, Z = 7-deaza dG, Q = BHQ2 Quencher Dye, P = 3' Phosphate

[0129] The method may further comprise, prior to detection in step (c), contacting the nucleic acid in the sample with at least one oligonucleotide from Table 1 and generating an amplification product containing the target sequence within the PI3KCA nucleic acid.

TABLE-US-00011 TABLE 1 Primer Sequences for the PIK3CA Mutation Detection Test Primer Sequence 5' to 3' 542/545 Forward primer UAAAAUUUAUUGAGAAUGUAUUUGCTT PIK3CA-9F13 TTTC (SEQ ID NO: 25) 542/545 Reverse primer TCCATTTTAGCACTTACCTGTGAC PIK3CA-9R01 (SEQ ID NO: 26) Key: U = 5-propynyl dU

[0130] The present invention provides a method of determining whether a patient having a malignant tumor is less likely to respond to a HER2-inhibitor, comprising:

[0131] (a) contacting the nucleic acid in the sample from the patient with one or more of the following mutation-specific oligonucleotides:

TABLE-US-00012

[0131] (SEQ ID NO: 28) LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (542 542K Mutation Probe); (SEQ ID NO: 30) JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (545 545K Mutation Probe); (SEQ ID NO: 31) FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (545 545A Mutation Probe); and/or (SEQ ID NO: 32) LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (545 545G Mutation Probe);



[0132] (b) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0133] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid, wherein the presence of the mutation indicates that the patient is less likely to respond to the HER2 inhibitor.

[0134] The present invention relates to a method of determining whether a patient having a malignant tumor is less likely to respond to a HER2-inhibitor, comprising:

[0135] (a) contacting the nucleic acid in the sample from the patient with one or more of the following mutation-specific oligonucleotides:

TABLE-US-00013

[0135] (SEQ ID NO: 28) LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (542 542K Mutation Probe); (SEQ ID NO: 30) JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (545 545K Mutation Probe); (SEQ ID NO: 31) FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (545 545A Mutation Probe); and/or (SEQ ID NO: 32) LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (545 545G Mutation Probe);



[0136] (b) (i) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0137] (ii) contacting the nucleic acid in the sample with one or both of the following oligonucleotides:

TABLE-US-00014

[0137] (SEQ ID NO: 25) UAAAAUUUAUUGAGAAUGUAUUUGCTTTTTC (542/545 Forward primer PIK3CA-9F13) and/or (SEQ ID NO: 26) TCCATTTTAGCACTTACCTGTGAC (542/545 Reverse primer PIK3CA-9R01);



[0138] (iii) generating an amplification product containing the target sequence within the PI3KCA nucleic acid;

[0139] (c) detecting the hybridization thereby detecting the presence of the mutation in the PI3KCA nucleic acid, wherein the presence of the mutation indicates that the patient is less likely to respond to the HER2 inhibitor.

[0140] The present invention provides a method for identifying a non-responder to a HER2-inhibitor, said method comprising

[0141] a) detecting the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) nucleic acid in a sample from an individual,

[0142] (b)) identifying the patient as less likely to respond to a HER2 inhibitor if the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) nucleic acid is detected.

TABLE-US-00015

[0142] TABLE 2 Probe Sequences for the PIK3CA Mutation Detection Test Probe Sequence 5' to 3' 542 WT Probe FTTTCAQAGAGAGGAUEUEGUGUAGA AAUUGEP (SEQ ID NO: 27 542 542K Mutation Probe LATTTTQGAGAGAGGAUEUEGUGUAG AAAUUGEUUP (SEQ ID NO: 28) 545 WT Probe OCTGCTCAGTQAUUUIAGAGAGAGGA TCTCGTGTP (SE) ID NO: 29) 545 545K Mutation Probe JAATCACTAAGQAGGAGAAAGAUUUU EUAUGGAGUEP (SEQ ID NO: 30) 545 545A Mutation Probe FCTGCGCQGGAGAAAGAUUUUEUAUG GAGUEAP (SEQ ID NO: 31) 545 545G Mutation Probe LCCTGCCCQGTGAUUUIAGAGAGAGG ATCTCGP (SEQ ID NO: 32) Key: F = FAM Reporter Dye, J = JA270 Reporter Dye, O = CY5.5 Reporter Dye, L = HEX Reporter Dye, U = 5-propynyl dU, E = 5-methyl dC,, I = deoxyinosine, Z = 7-deaza dG, Q = BHQ2 Quencher Dye, P = 3' Phosphate

[0143] The term "non-responder" as used herein can refer to a "patient who is less likely to respond" "Less likely to respond" as used herein refers to a decreased likeliness that a pathological complete response (pcR) will occur in a patient treated with a HER2 inhibitor.

[0144] The present invention relates to a method for identifying a non-responder to a HER2-inhibitor, said method comprising detecting the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) nucleic acid by

[0145] (a) contacting the nucleic acid in the sample from a patient with HER2-positive cancer with one or more of the following mutation-specific oligonucleotides:

TABLE-US-00016

[0145] (SEQ ID NO: 28) LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (542 542K Mutation Probe); (SEQ ID NO: 30) JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (545 545K Mutation Probe); (SEQ ID NO: 31) FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (545 545A Mutation Probe); and/or (SEQ ID NO: 32) LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (545 545G Mutation Probe);



[0146] (b) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PIK3CA nucleic acid;

[0147] (c) detecting hybridization

[0148] (d) identifying the patient as less likely to respond to a HER2 inhibitor if the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) nucleic acid is detected.

[0149] The method can further comprise, prior to detection in step (c), contacting the nucleic acid in the sample with at least one oligonucleotide from Table 1 and generating an amplification product containing the target sequence within the PI3KCA nucleic acid.

TABLE-US-00017 TABLE 1 Primer Sequences for the PIK3CA Mutation Detection Test Primer Sequence 5' to 3' 542/545 Forward primer UAAAAUUUAUUGAGAAUGUAUUUGCTT PIK3CA-9F13 TTTC (SEQ ID NO: 25) 542/545 Reverse primer TCCATTTTAGCACTTACCTGTGAC PIK3CA-9R01 (SEG ID NO: 26) Key: U = 5-propynyl dU

[0150] The present invention relates to a method for identifying a non-responder to a HER2-inhibitor, said method comprising detecting the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) nucleic acid by

[0151] (a) contacting the nucleic acid in the sample from a patient with HER2-positive cancer with one or more of the following mutation-specific oligonucleotides:

TABLE-US-00018

[0151] (SEQ ID NO: 28) LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (542 542K Mutation Probe); (SEQ ID NO: 30) JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (545 545K Mutation Probe); (SEQ ID NO: 31) FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (545 545A Mutation Probe); and/or (SEQ ID NO: 32) LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (545 545G Mutation Probe);



[0152] (b) (i) incubating the sample under conditions allowing hybridization of the oligonucleotide to the target sequence within the PI3KCA nucleic acid;

[0153] (ii) contacting the nucleic acid in the sample with one or both of the following oligonucleotides:

TABLE-US-00019

[0153] (SEQ ID NO: 25) UAAAAUUUAUUGAGAAUGUAUUUGCTTTTTC (542/545 Forward primer PIK3CA-9F13) and/or (SEQ ID NO: 26) TCCATTTTAGCACTTACCTGTGAC (542/545 Reverse primer PIK3CA-9R01);



[0154] (iii) generating an amplification product containing the target sequence within the PI3KCA nucleic acid;

[0155] (c) detecting hybridization

[0156] (d) identifying the patient as less likely to respond to a HER2 inhibitor if the presence of one or more mutations in exon 9 of the catalytic subunit of Phosphoinositol-3 kinase (PIK3CA or p110.alpha.) nucleic acid is detected.

[0157] The present invention is further illustrated by reference to the following non-limiting figures and examples.

[0158] The Figures show:

[0159] FIG. 1.

[0160] FIG. 1 provides a schematic of the HER2 protein structure, and amino acid sequences for Domains I-IV (SEQ ID Nos. 1-4, respectively) of the extracellular domain thereof.

[0161] FIG. 2.

[0162] FIGS. 2A and 2B depict alignments of the amino acid sequences of the variable light (V.sub.L) (FIG. 2A) and variable heavy (V.sub.H) (FIG. 2B) domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 5 and 6, respectively); V.sub.L and V.sub.H domains of variant 574/Pertuzumab (SEQ ID Nos. 7 and 8, respectively), and human V.sub.L and V.sub.H consensus frameworks (hum .kappa.1, light kappa subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 9 and 10, respectively). Asterisks identify differences between variable domains of Pertuzumab and murine monoclonal antibody 2C4 or between variable domains of Pertuzumab and the human framework. Complementarity Determining Regions (CDRs) are in brackets.

[0163] FIG. 3.

[0164] FIGS. 3A and 3B show the amino acid sequences of Pertuzumab light chain (FIG. 3A; SEQ ID NO. 11) and heavy chain (FIG. 3B; SEQ ID No. 12). CDRs are shown in bold. Calculated molecular mass of the light chain and heavy chain are 23,526.22 Da and 49,216.56 Da (cysteines in reduced form). The carbohydrate moiety is attached to Asn 299 of the heavy chain.

[0165] FIG. 4.

[0166] FIGS. 4A and 4B show the amino acid sequences of Trastuzumab light chain (FIG. 4A; SEQ ID NO. 13) and heavy chain (FIG. 4B; SEQ ID NO. 14), respectively. Boundaries of the variable light and variable heavy domains are indicated by arrows.

[0167] FIG. 5.

[0168] FIGS. 5A and 5B depict a variant Pertuzumab light chain sequence (FIG. 5A; SEQ ID NO. 15) and a variant Pertuzumab heavy chain sequence (FIG. 5B; SEQ ID NO. 16), respectively.

[0169] FIG. 6.

[0170] FIG. 6 shows Results of PIK3CA mutational analyses. PIK3CA mutations were in general associated with decreased sensitivity to HER2-targeted therapy in NeoSphere (The NeoSphere study is described in detail e.g. in Lancet Oncol. 2012 January; 13(1):25-32. doi: 10.1016/S1470-2045(11)70336-9. Epub 2011 Dec. 6). Analyses per Exon i.e. Exons 7, 9 and 20 was carried out to explore in more detail the impact of specific mutations. Exon 9 mutations: Out of 28 mutations detected across the 4 arms, 26 were found to be in the non-responder group. Exon 20 mutations had little impact on pCR. Too few exon 7 mutations to draw conclusions. TH=Patients treated with docetaxel (75.fwdarw.100 mg/m.sup.2) and trastuzumab (8.fwdarw.6 mg/kg), THP=Patients treated with docetaxel (75.fwdarw.4100 mg/m.sup.2), trastuzumab (8.fwdarw.6 mg/kg) and pertuzumab (840.fwdarw.4420 mg), HP=Patients treated with trastuzumab (8.fwdarw.6 mg/kg) and pertuzumab (840.fwdarw.420 mg), TP=Patients treated with docetaxel (75.fwdarw.100 mg/m.sup.2) and pertuzumab (840.fwdarw.420 mg).

[0171] FIG. 7.

[0172] FIG. 7 shows the PIK3CA gene and the protein sequence aligned. Exon9 is annotated with ***. The wild-type triplets encoding positions E542 and E545 of the wild-type amino acid sequence are indicated in bold letters.

EXAMPLE

Identification of Non-Responders to HER2 Inhibitors by Determining the Presence of Mutations in Exon 9 of PIK3CA

[0173] Each standard 50-.mu.L amplification reaction targeting one of the Exons 7, 9 or 20 included 100 ng genomic DNA, dNTPs (including dNTPs), 0.05 U/.mu.L Z05, DNA polymerase, 0.04 U/.mu.L uracil-DNA glycosylase (UNG), and 200-400 nM forward and reverse primer (Table 1), 75-200 nM mutant and wild-type specific probes (Table 2). Amplification was performed in the Cobas.RTM. 4800 analyzer using the following temperature profile: 5 min at 50.degree. C. followed by 55 cycles of 95.degree. C. for 10 sec and 63.degree. C. for 50 sec, followed by a single round of 40.degree. C. for 2 min and 25.degree. C. for 10 sec (melting curve analysis). Fluorescence data was collected during each amplification cycle and during the final melting curve analysis.

TABLE-US-00020 TABLE A Primer Sequences for the PIK3C4 Mutation Detection Test Primer Sequence 5' to 3' Codon 420 Forward Primer UUUUGGGGAAGAAAAGUGUUUUG PIK3CA-7F03 AA (SEQ ID NO: 33) Codon 420 Reverse primer GATTCAAAGCCATTTTTCCAGAT PIK3CA-7R04 ACTAGA (SEQ ID NO: 34) Codon 542/545 Forward UAAAAUUUAUUGAGAAUGUAUUU primer PIK3CA-9F13 GCTTTTTC (SEQ ID NO: 25) Codon 542/545 Reverse TCCATTTTAGCACTTACCTGTGAC primer PIK3CA-9R01 (SEQ ID NO: 26) Codon 1047 Forward primer GAGGCTTTGGAGTATTTCATGAA PIK3CA-20F01 (SEQ ID NO: 35) Codon 1047 Reverse primer CCAATCCATTTTTGTTGTCCA PIK3CA-20R01 (SEQ ID NO: 36) Key: U = 5-propynyl dU

TABLE-US-00021 TABLE B Probe Sequences for the PIK3CA Mutation Detection Test Probe Sequence 5' to 3' Codon 420 WT Probe JCAATGGACAGQGUUEEUUAAAAAAEAAAGAAAAAUAUUP (SEQ ID NO: 37) Codon 420 420R Mutation Probe OGAACACCQTCCAUUGGEAUGGGGAAAUAUAAAP (SEQ ID NO: 38) Codon 542 WT Probe FTTTCAQAGAGAGGAUEUEGUGUAGAAAUUGEP (SEQ ID NO: 27) Codon 542 542K Mutation Probe LATTTTQGAGAGAGGAUEUEGUGUAGAAAUUGEUUP (SEQ ID NO: 28) Codon 545 WT Probe OCTGCTCAGTQAUUUIAGAGAGAGGATCTCGTGTP (SEQ ID NO: 29) Codon 545 545K Mutation Probe JAATCACTAAGQAGGAGAAAGAUUUUEUAUGGAGUEP (SEQ ID NO: 30) Codon 545 545A Mutation Probe FCTGCGCQGGAGAAAGAUUUUEUAUGGAGUEAP (SEQ ID NO: 31) Codon 545 545G Mutation Probe LCCTGCCCQGTGAUUUIAGAGAGAGGATCTCGP (SEQ ID NO: 32) Codon 1047 WT Probe FTZCACATCQTZZTZZCTZZACAACAAP (SEQ ID NO: 39) Codon 1047 1047R Mutation Probe LGACGTQCAUEAUUEAUUUGUUUEAUGP (SEQ ID NO: 40) Codon 1047 1047L Mutation Probe JGCACTTCATGQTGGCTGGACAACAAAAAP (SEQ ID NO: 41) Codon 1047 1047Y Mutation Probe OACCATGATATQCAUEAUUEAUUUGUUUEP (SEQ ID NO: 42) Key: F = FAM Reporter Dye, J = JA270 Reporter Dye, O = CY5.5 Reporter Dye, L = HEX Reporter Dye, U = 5-propynyl dU, E = 5-methyl dC,, I = deoxyinosine, Z = 7-deaza dG, Q = BHQ2 Quencher Dye, P = 3' Phosphate

[0174] FIG. 6 shows Results of PIK3CA mutational analyses. PIK3CA mutations were in general associated with decreased sensitivity to HER2-targeted therapy in NeoSphere. Analyses per exon i.e. exons 7, 9 and 20 was carried out to explore in more detail the impact of specific mutations. For exon 9 mutations, out of 28 mutations detected across the 4 arms, 26 were found to be in the non-responder group. Exon 20 mutations had little impact on pCR. There were too few exon 7 mutations to draw conclusions.

[0175] While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus the scope of the invention should not be limited by the examples described herein, but by the claims presented below.

[0176] The present invention refers to the following nucleotide and amino acid sequences:

[0177] The sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants.

[0178] SEQ ID No. 1:

[0179] Nucleotide sequence encoding homo sapiens phosphoinositide-3-kinase, catalytic, alpha (PIK3CA), (NCBI accession number: NG_012113.1 GI:237858742).

[0180] SEQ ID No. 2:

[0181] Amino acid sequence of homo sapiens phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit. The sequence can be retrieved from the NCBI database under accession number NP_006209.2 GI:54792082 or from Uniprot database under accession number >sp|P42336|.

[0182] Positions 542 and 545 are indicated in bold letters.

TABLE-US-00022 MPPRPSSGELWGIHLMPPRILVECLLPNGMIVTLECLREATLITIKHELF KEARKYPLHQLLQDESSYIFVSVTQEAEREEFFDETRRLCDLRLFQPFLK VIEPVGNREEKILNREIGFAIGMPVCEFDMVKDPEVQDFRRNILNVCKEA VDLRDLNSPHSRAMYVYPPNVESSPELPKHIYNKLDKGQHVVIWVIVSPN NDKQKYTLKINHDCVPEQVIAEAIRKKTRSMLLSSEQLKLCVLEYQGKYI LKVCGCDEYFLEKYPLSQYKYIRSCIMLGRMPNLMLMAKESLYSQLPMDC FTMPSYSRRISTATPYMNGETSTKSLAWVINSALRIKILCATYVNVNIRD IDKIYVRTGIYHGGEPLCDNVNTQRVPCSNPRWNEWLNYDIYIPDLPRAA RLCLSICSVKGRKGAKEEHCPLAWGNINLFDYTDTLVSGKMALNLWPVPH GLEDLLNPIGVTGSNPNKETPCLELEFDWFSSVVKFPDMSVIEEHANWSV SREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSEITEQEKDF LWSHRHYCVTIPEILPKLLLSVKWNSRDEVAQMYCLVKDWPPIKPEQAME LLDCNYPDPMVRGFAVRCLEKYLTDDKLSQYLIQLVQVLKYEQYLDNLLV RFLLKKALTNQRIGHFFFWHLKSENHNKTVSQRFGLLLESYCRACGMYLK HLNRQVEAMEKLINLTDILKQEKKDETQKVQMKFLVEQMRRPDFMDALQG FLSPLNPAHQLGNLRLEECRIMSSAKRPLWLNWENPDIMSELLFQNNEII FKNGDDLRQDMLTLQIIRIMENIWQNQGLDLRMLPYGCLSIGDCVGLIEV VRNSHTIMQIQCKGGLKGALQFNSHTLHQWLKDKNKGEIYDAAIDLFTRS CAGYCVATFILGIGDRHNSNIMVKDDGQLFHIDFGHFLDHKKKKFGYKRE RVPFVLTQDFLIVISKGAQECTKTREFERFQEMCYKAYLAIRQHANLFIN LFSMMLGSGMPELQSFDDIAYIRKTLALDKTEQEALEYFMKQMNDAHHGG WTTKMDWIFHTIKQHALN

[0183] SEQ ID No. 3:

[0184] Nucleotide sequence encoding exon 9 of homo sapiens phosphoinositide-3-kinase, catalytic, alpha (PIK3CA).

TABLE-US-00023 AGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACA GCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGAGCAGG AGAAAGATTTTCTATGGAGTCACAG

[0185] SEQ ID No. 4:

[0186] Amino acid sequence encoded by exon 9 of homo sapiens phosphoinositide-3-kinase, catalytic, alpha (PIK3CA).

[0187] SEQ ID No. 5:

[0188] Amino acid sequence of the variable light (V.sub.L) (FIG. 2A) domain of murine monoclonal antibody 2C4 (SEQ ID Nos. 5 and 6, respectively) as shown in FIG. 2.

[0189] SEQ ID No. 6:

[0190] Amino acid sequence of the variable heavy (V.sub.II) (FIG. 2B) domain of murine monoclonal antibody 2C4 as shown in FIG. 2.

[0191] SEQ ID No. 7:

[0192] Amino acid sequence of the variable light (V.sub.L) (FIG. 2A) domain of variant 574/Pertuzumab as shown in FIG. 2.

[0193] SEQ ID No. 8:

[0194] Amino acid sequence of the variable heavy (V.sub.H) (FIG. 2B) domain of variant 574/Pertuzumab as shown in FIG. 2.

[0195] SEQ ID No. 9:

[0196] human V.sub.L consensus frameworks (hum .kappa.1, light kappa subgroup I; humIII, heavy subgroup III) as shown in FIG. 2.

[0197] SEQ ID No. 10:

[0198] human V.sub.H consensus frameworks (hum .kappa.1, light kappa subgroup I; humIII, heavy subgroup III) as shown in FIG. 2.

[0199] SEQ ID No. 11:

[0200] Amino acid sequences of Pertuzumab light chain as shown in FIG. 3A.

[0201] SEQ ID No. 12:

[0202] Amino acid sequences of Pertuzumab heavy chain as shown in FIG. 3B.

[0203] SEQ ID No. 13:

[0204] Amino acid sequence of Trastuzumab light chain domain as shown in FIG. 4A. Boundaries of the variable light domain are indicated by arrows.

[0205] SEQ ID No. 14:

[0206] Amino acid sequence of Trastuzumab heavy chain as shown in FIG. 4B. Boundaries of the variable heavy domain are indicated by arrows.

[0207] SEQ ID No. 15:

[0208] Amino acid sequence of variant Pertuzumab light chain sequence (FIG. 5A).

[0209] SEQ ID No. 16:

[0210] Amino acid sequence of variant Pertuzumab heavy chain sequence (FIG. 5B).

[0211] SEQ ID No. 17:

[0212] Nucleotide sequence encoding exon 9 of homo sapiens E542K mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). The triplet (codon) encoding the mutant amino acid "K" at position 542 of the full-length amino acid sequence of PIKC3CA (see SEQ ID NO: 2 and FIG. 7) is highlighted in bold letters.

TABLE-US-00024 AGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAA GAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTAAAATC ACTGAGCAGGAGAAAGATTTTCTATGGAGTCACAG

[0213] SEQ ID No. 18:

[0214] Amino acid sequence of homo sapiens E542K mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). Position 542 is highlighted in bold. The mutant shows an increase in lipid kinase activity; oncogenic in vivo; occurs in the interface between the PIK3CA helical domain and the nSH2 (N-terminal SH2) region of the p85 regulatory subunit and may reduce the inhibitory effect of p85; requires interaction with RAS to induce cellular transformation.

TABLE-US-00025 MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA 70 80 TLITIKHELF KEARKYPLHQ LLQDESSYIF VSVTQEAERE 90 100 110 120 EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA 130 140 150 160 IGMPVCEFDM VKDPEVQDFR RNILNVCKEA VOLRDLNSPH 170 180 190 200 SRAMYVYPPN VESSPELPKE IYNKLDKGQI IVVIWVIVSP 210 220 230 240 NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK 250 260 270 280 LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG 290 300 310 320 RMPNLMLMAK ESLYSQLPMD CFTMPSYSRR ISTATPYMNG 330 340 350 360 ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI 370 380 390 400 YHGGEPLCDN VNTQRVPCSN PRWNEWLNYD IYIPDLPRAA 410 420 430 440 RLCLSICSVK GRKGAKEERC PLAWGNINLF DYTDTLVSGK 450 460 470 480 MALNLWPVPH GLEDLLNPIG VTGSNPNKET PCLELEFDWF 490 500 510 520 SSVVKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD 530 540 550 560 NELRENDKEQ LKAISTRDPL S ITEQEKDF LWSHRHYCVT 570 580 590 600 ZPEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME 610 620 630 640 LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLX 650 660 670 680 YEQYLDNLLV RFLLKKALTN QRIGHFFFWH LKSEMHNKTV 690 700 710 720 SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK 730 740 750 760 QEKKDETQKV QMKFLVEQMR RPDFMDALQG FLSPLNPAHQ 770 780 790 800 LGNLRLEECR IMSSAKRPLW LNWENPDIMS ELLFQNNEII 810 820 830 840 FKNGDDLRQD MLTLQIIRIM ENIWQNQGLD LRMLPYGCLS 850 860 870 880 IGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW 890 900 910 920 LKDKNKGEIY DAAIDLFTRS CAGYCVATFI LGIGDRHNSN 930 940 950 960 IMVKDDCQLF HIDFGEFLDH KKKKFGYKRE RVPFVLTQDF 970 980 990 1000 LIVISKGAQE CTKTREFERF QEMCYKAYLA IRQHANLFIN 1010 1020 1030 1040 LFSMMLGSGM PELQSFDDIA YIRKTLALDK TEQEALEYFM 1050 1060 KQMNDAHHGG WTTKMDWIFH TIKQHALN

[0215] SEQ ID No. 19:

[0216] Nucleotide sequence encoding exon 9 of homo sapiens E545K mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). The triplet (codon) encoding the mutant amino acid "K" at position 545 of the full-length amino acid sequence of PIKC3CA (see SEQ ID NO: 2 and FIG. 7) is highlighted in bold letters.

TABLE-US-00026 AGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAA GAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACT AAGCAGGAGAAAGATTTTCTATGGAGTCACAG

[0217] SEQ ID No. 20:

[0218] Amino acid sequence of homo sapiens E545K mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). Position 545 is highlighted in bold. The mutant shows an increase in lipid kinase activity; oncogenic in vivo; occurs in the interface between the PIK3CA helical domain and the nSH2 (N-terminal SH2) region of the p85 regulatory subunit and may reduce the inhibitory effect of p85; requires interaction with RAS to induce cellular transformation; enhances invadopodia-mediated extracellular matrix degradation and invasion in breast cancer

TABLE-US-00027 MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA 70 80 TLITIKHELF KEAPKYPLHQ LLQDESSYIF VSVTQEAERE 90 100 110 120 EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA 130 140 150 160 IGMPVCEFDM VKDPEVQDFR RNILNVCKEA VDLRDLNSPH 170 180 190 200 SRAMYVYPPN VESSPELPKH IYNKLDKCQI IVVIWVIVSP 210 220 230 240 NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK 250 260 270 280 LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG 290 300 310 320 RMPNLMLMAK ESLYSQLPMD CFTMPSYSRR ISTATPYMNG 330 340 350 360 ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI 370 380 390 400 YHGGEPLCDN VNTQRVPCSN PRWNEWLNYD IYIPDLPRAA 410 420 430 440 RLCLSICSVK GRKGAKEEHC PLAWGNINLF DYTDTLVSGK 450 460 470 480 MALNLWPVPH GLEDLINPIG VTGSNPNKET PCLELEFDWF 490 500 510 520 SSVVKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD 530 540 550 560 NELRENDKEQ LKAISTRDPL SEIT QEKDF LWSHRHYCVT 570 580 590 600 IPEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME 610 620 630 640 LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLK 650 660 670 680 YEQYLDNLLV RFLLKKALTN QRIGHFFFWH LKSEMHNKTV 690 700 710 720 SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK 730 740 750 760 QEKKDETQKV QMKFLVEQMR RPDFMDALQG FLSPLNPAHQ 770 780 790 800 LGNLRLEECR IMSSAKRPLW LNWENPDIMS ELLFQNNEII 810 820 830 840 FKNGDDLRQD MLTLQIIRIM ENIWQNQGLD LRMLPYGCLS 850 860 870 880 IGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW 890 900 910 920 LKDKNKGEIY DAAIDLFTRS CAGYCVATFI LGIGDRHNSN 930 940 950 960 IMVKDDGQLF HIDFGEFLDH KKKKFGYKRE RVPFVLTQDF 970 980 990 1000 LIVISKGAQE CTKTREFERF QEMCYKAYLA IRQHANLFIN 1010 1020 1030 1040 LFSMMLGSGM PELQSFDDIA YIRKTLALDK TEQEALEYFM 1050 1060 KQMNDAAHGG WTTKMDWIFH TIKQHALN

[0219] SEQ ID No. 21:

[0220] Nucleotide sequence encoding exon 9 of homo sapiens E545A mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). The triplet (codon) encoding the mutant amino acid "A" at position 545 of the full-length amino acid sequence of PIKC3CA (see SEQ ID NO: 2 and FIG. 7) is highlighted in bold letters.

TABLE-US-00028 AGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAA GAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACT GCGCAGGAGAAAGATTTTCTATGGAGTCACAG

[0221] Amino acid sequence of homo sapiens E545A mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). Position 545 is highlighted in bold.

TABLE-US-00029 MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA 70 80 TLITIKHELF KEARKYPLHQ LLQDESSYIF VSVTQEAERE 90 100 110 120 EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA 130 140 150 160 IGMPVCEFDM VKDPEVQDFR RNILNVCKEA VDLRDINSPH 170 180 190 200 SRAMYVYPPN VESSPELPKH TYNKLDKGQI IVVIWVIVSP 210 220 230 240 NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK 250 260 270 280 LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG 290 300 310 320 RMPNLMLMAK ESLYSQLPMD CFTMPSYSRR ISTATPYMNG 330 340 350 360 ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI 370 380 390 400 YHGGEPLCDN VNTQRVPCSN PRWNEWLMYD IYIPDLPRAA 410 420 430 440 RLCLSICSVK GRKGAKEEHC PLAWGNINLF DYTDTLVSGK 450 460 470 480 MALNLWPVPH GLEDLLNPIG VTGSNPNKET PCLELEFDWF 490 500 510 520 SSVVKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD 530 540 550 560 NELRENDKEQ LKAISTRDPL SEIT QEKDF LWSHRHYCVT 570 580 590 600 IPEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME 610 620 630 640 LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLK 650 660 670 680 YEQYLDNLLV RFLLKKALTN QRIGHFFFWH LKSEMHNKTV 690 700 710 720 SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK 730 740 750 760 QEKKDETQKV QMKFLVEQMR RPDFMDALQG FLSPLNPAHQ 770 780 790 800 LGNLRLEECR IMSSAKRPLW LNWENPDIMS ELLFQNNEII 810 820 830 840 FKNGDDLRQD MLTLQIIRIM ENIWQNQGLD LRMLPYGCLS 850 860 870 880 TGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW 890 900 910 920 LKDKNKGEIY DAAIDLFTRS CAGYCVATFI LGIGDRHNSN 930 940 950 960 IMVKDDGQLF HIDFGHFLDH KKKKFGYKRE RVPFVLTQDF 970 980 990 1000 LIVISKGAQE CTKTREFERE QEMCYKAYLA IRQHANLFIN 1010 1020 1030 1040 LFSMMLGSGM PELQSFDDIA YIRKTLALDK TEQEALEYFM 1050 1060 KQMNDAHHGG WTTKMDWIFH TIKQHALN

[0222] SEQ ID No. 23:

[0223] Nucleotide sequence encoding exon 9 of homo sapiens F5450 mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). The triplet (codon) encoding the mutant amino acid "G" at position 545 of the full-length amino acid sequence of PIKC3CA (see SEQ ID NO: 2 and FIG. 7) is highlighted in bold letters.

TABLE-US-00030 AGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAA GAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACT GGGCAGGAGAAAGATTTTCTATGGAGTCACAG

[0224] SEQ ID No. 24:

[0225] Amino acid sequence of homo sapiens E545G mutant of phosphoinositide-3-kinase, catalytic, alpha (PIK3CA). Position 545 is highlighted in bold.

TABLE-US-00031 MPPRPSSGEL WGIHLMPPRI LVECLLPNGM IVTLECLREA 70 80 TLITIKHELF KEARKYPLHQ LLQDESSYIF VSVTQEAERE 90 100 110 120 EFFDETRRLC DLRLFQPFLK VIEPVGNREE KILNREIGFA 130 140 150 160 IGMPVCEFDM VKDPEVQDFR RNILNVCKEA VDLRDLNSPH 170 180 190 200 SRAMYVYPPN VESSPELPKH IYNKLDKGQI IVVIWVIVSP 210 220 230 240 NNDKQKYTLK INHDCVPEQV IAEAIRKKTR SMLLSSEQLK 250 260 270 280 LCVLEYQGKY ILKVCGCDEY FLEKYPLSQY KYIRSCIMLG 290 300 310 320 RMPNLMLMAK ESLYSQLPMD CFTMPSYSRR ISTATPYMNG 330 340 350 360 ETSTKSLWVI NSALRIKILC ATYVNVNIRD IDKIYVRTGI 370 380 390 400 YHGGEPLCDN VNTQRVPCSN PRWNEWLNYD IYIPDLPRAA 410 420 430 440 RLCLSICSVK GRKGAKEEHC PLAWGNINLF DYTDTLVSGK 450 460 470 480 MALNLWPVPH GLEDLLNFIG VTGSNPNKET PCLELEFDEF 490 500 510 520 SSVVKFPDMS VIEEHANWSV SREAGFSYSH AGLSNRLARD 530 540 550 560 NELRENDKEQ LKAISTRDPL SEIT QEKDF LWSHRHYCVT 570 580 590 600 IPEILPKLLL SVKWNSRDEV AQMYCLVKDW PPIKPEQAME 610 620 630 640 LLDCNYPDPM VRGFAVRCLE KYLTDDKLSQ YLIQLVQVLK 650 660 670 680 YEQYLDNLLV RFLLKKALTN QRIGHFFFWH LKSEMHNKTV 690 700 710 720 SQRFGLLLES YCRACGMYLK HLNRQVEAME KLINLTDILK 730 740 750 760 QEKKDETQKV QMKFLVEQMR RPDFMDALQG FLSPLNPAHQ 770 780 790 800 LGNLRLEECR IMSSAKRPLW LNWENPDIMS ELLFQNNEII 810 820 830 840 FKNGDDLRQD MLTLQIIRIM ENIWQNQGLD LRMLPYGCLS 850 860 870 880 IGDCVGLIEV VRNSHTIMQI QCKGGLKGAL QFNSHTLHQW 890 900 910 920 LKDKNKGEIY DAAIDLFTRS CAGYCVATFI LGIGDRHNSN 930 940 950 960 IMVKDDGQLF HIDFGHFLDH KKKKFGYKRE RVPFVLTQDF 970 980 990 1000 LIVISKGAQE CTKTREFERF QEMCYKAYLA IRQHANLFIN 1010 1020 1030 1040 LFSMMLGSGM PELQSFDDTA YIRKTLALDK TEQEALEYFM 1050 1060 KQMNDAHHGG WTTKMCWIFH TIKQHALN

[0226] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Sequence CWU 1

1

47193190DNAHomo sapiens 1tttcgccctg ttgcccaggt tggtctcaaa ctgctgggct caagtgatcc acccacctca 60gcctcccaaa gtgctgggat tacaggcgtg agccaacaca cctggctgaa aatagtcttt 120tacgaatttc aaagctttga tttctccata agtgcactcc tggtaacttg agaacattac 180acatttgtct cctttctgtg gccagaattg ttttgtattt caagtatgga aaggagcttt 240gccagaatcc tcccgtgcta agtgtaagta gagcagttag actttaacat ggttgtgtga 300tagtaatgac attcatatct gactctaggc aaggcatggc tggaatgaaa aacattgact 360gttaagtgtt ttaaggtgaa cttctcagat actagatctc ttagctcaca tagtggaatt 420ttgaggaaag tacagggttt gctcttcatc tcctaagaaa tgcaatgatg attctttaaa 480gtactgatgg gtcagggagg ggagaaaacc attaaccgag aattgtataa tcagctaaaa 540tattttgcag aaatgaaggc aaaagtaaaa tattctcaaa tgaaggaaaa tgaagggaat 600ttgtcacaag cagactttct ctaaaaagaa tgttaaaatt cttcaggata aagggaaatt 660ataccagaag gaaaaatgga ttttcaggaa tgaagcaaaa cataaatgta aatggatacg 720taaatataaa agattatttt tcctcttgag ttctatatga ctgttgaaag caaaaattat 780aaaaacatct gatggggctt ccatatatat aaatgtaata catatgccaa ctaatgacat 840aaaggtcagg gagtggggta ggagacagtg gtaaatctac acggttgcaa ggcttctttc 900tacattttac ttgaagtggt gtaatagtaa tactaagtag accgcaaaac tttaggtatg 960tatattataa tccatagaat taccaaaaaa ttaccaagaa atatattaaa actacaatat 1020agaaattaaa atggaatatt aaaaattttt caaataatcc acaagaaagc aggaaagtag 1080aaacataaaa acaaaagtat agaggaaaaa cagaaaacaa ataataaaat ggcaagccta 1140aatccatata attacattaa atgtaaatta aacacagcaa ttaaaacaca gaaattgtca 1200gattggaatt tttaaaaaga gatctaatac gtgttgtcta cgagtaactc actttaaata 1260taaaaataga tataaaaaag atgggaaaaa ataccatgga aaaattaatc aaaaaaagct 1320ggactggctg tattaatatc agacaaagta gatttcagaa caaagatatt actaggaata 1380aagagagata gtacagattg attaaaaaga gtcaatgtat caaaacacaa gagttctaaa 1440tgtgtataca tctaacaaca aagcttcaaa acacacaaag taaaaataca cagaagtgaa 1500atgagaaata aagaaatcca caattatatt ttgaaacatc aatgctaaca aaatcatcaa 1560cttggtaaga aactacattc tgcccaacaa tagcaaacaa aatattcttt cataagtgca 1620catgggacat tcaccaagat attctggggc aacacaaatc ttaacttgaa aagaactgaa 1680atcttaaaag tacgtgtgtg tatgtgtgtg tgggtgtatg gttttttttt ttttaagatg 1740gagtctcggt caggcgtggt ggctcacacc tgtaatctca gcactttggg aggccaaggc 1800gggcggatca tgaggtcagg agttcaagac cagcctgacc aatatggtga aaccccgtct 1860ctactaaaaa tacaaaaatt agccgggcgt ggtggcacgt gcctgtagtc ccagctactc 1920ggaaggctga ggcaggagaa tcacttgaac ctgggaggca gaggttgcag tgagccaaga 1980tcgcgccact gcactccagc ctgggtgaca gagcgagact ccatctcaaa aaaaaaaaaa 2040aaaaaaaaaa aacagtgcta catggtaact catatcgtta tctcttggtg tgtttttatt 2100gtttgacttg gcaattttat acatctaaag tatactgcta cataatcaac ttgactctta 2160tttacaaagg acttcaaagc ttagtagtga cttaagcgct attagagaat gtttcggtgc 2220atatttgagt atcagttatg attttcaatg gaaataaacc caaaaggcaa gtaactgtca 2280ttataattct gattattaga atagaacaat agctacatac tggacatttc acctcccgtc 2340tttagaaccc ttaggatgca attatttaat tttgaagtat accatttttt gtttcattta 2400cagcctgaga atcattattt ttttaaaacc ttctttcagc tcctaatcct tgagtgatga 2460gtttggtcat atagatgctt atatcataca gatattttta tatcatatag atattttaaa 2520cactgaataa cattgcaaaa acctttgaaa gcaagatgac acctaagacc aatggcttac 2580atataaggca aacattatca accgtgaatt gaatagtaaa tgctactcct gtaccaatga 2640atggtgtcat gcattcaagt accaggtatg gctttttctg ctatgacaca caacttctta 2700ggggcagata atcacataac aaaaaacata ttatgtaatt agcattttct tattaaaaaa 2760taaattttag gctgggcatg tcggctgaag tctgtaatcc caacactttg ggaggccgag 2820gtagatgaat ctcttgagcc caggagttgg agaccagtct gggcaacgaa gcaagaccct 2880gtctttacaa aaaataaaac atttttaaaa atcacaaata ggccaggtgc ggtggcttac 2940gcctgtaatc ctagcactct gggaagtcga ggggggtgga tcacctgaag tcagaagttt 3000gagaccagcc tgaccaacat ggtgaaaccc cgtctctact aagaaataca aaaattagct 3060ggcatgttgg ctggtgcctg taatcccagc tactcgggag gctgaggcag aagaatcgct 3120tgaacccggg aggcagaggc tgcagtgagc cgagactgca ccgctgaact ccagcctggg 3180ctacagagtg agactctggc tcaaaaaaat aataataata aataaataat aaattttata 3240aggaaaaata tccctagtat atttttcccc accaaggtct acctattaat atctcatata 3300actctgtgct tgaaacacaa tttgggatat attgccttaa aatttacttt gactagcaat 3360tcagtgttcc ttttttaaaa aaatctgtac tctggagtaa cagtgttcta aaactgttga 3420agaacattgg ttcgagaaaa acatttagaa acacaaaccc ctggaatgtg agatgaaaat 3480ccgagctaaa gggagaaaaa ggacgaaaga aagaaaacac agaaaagaaa gaaatacagt 3540caagtgaaac tacgaccaca aagaggaaca gatccatctc atttaccatt ggggtatgga 3600cggacgtgcg gtgttcgaga agactgttat tggtcagagt tttgctctag aggacaagta 3660ggactgtaac atctctagga tgggtcactt cccaagccct ctattactta aaaattcaag 3720gaggaaccga tgctggagta cttgtatctc agacttctaa tcactgctcc tacgcttttc 3780caatattaca acaaaagacc agtaggggga gaaaaacgca cagtaccgaa cccttatcag 3840tagtaatctc aaaagtcaac agattgattt actctcaagc aaacagactt ctaaggtacg 3900cagcaccaag acactacctt gaatcaaatc tatagcctat atgacatttc tgaagtctct 3960gttggcatta cgcgaaaaat cccccacgtc ttctgaatag ttagaattga atcctacaag 4020ctgcttcgaa tcagaattcg atttaaaaaa aaaaatgagg gcatagcaaa aggtctccac 4080gaagtgagtc aaaggactgc agagggctgt gacagtgcat tccgccttcg ggatggtata 4140caacttaaac catgtcggca gaagaacgca cagcaacgct ttgtaaaaag cattctttct 4200attatagaat ccataaccac gctggttagc cactgacagc ggcggttagc caccgcacct 4260cctctcaccc ccgaactaat ctcgtttcct ctatgggtgt aaaagtgaaa taacccactt 4320gctcccaata ttcctttcta tatctctacc ccagctcgcc tgctgctcgt agaaacaaat 4380atactacacg tacgctgtcc taggatgaca caacaccctc actactgcag aagacggatc 4440attaaacaaa cgtcagaaga gcagccccaa ctgtacataa acttcgggcg gaaaagcaag 4500acgcaggcgc agtagcacat attgttaccc tatttgccca ctccctgctc ctcctcgcct 4560caatttcgct tccgcttctt tgcgcatctg cttccggggg attgtaggct ctgcccctcc 4620tcagctctta ccctcttctg ccggaggagg gggggggccg agggggtggg gaagagttcg 4680ttgtttgttt acacgatgtg agcggaaaaa gagaccaata aagtttattc tggaaacaaa 4740aggaaaaaaa aacaggggcg acggagaaag gagtcggggg cgggggcgtg tggcgggggc 4800tagcgaggag agggagcgag aagtagaaag cggcagttcc ggtgccgccg ctgcggccgc 4860tgaggtgtcg ggctgctgct gccgcggccg ctgggactgg ggctggggcc gccggcgagg 4920cagggctcgg gcccggccgg gcagctccgg agcggcgggg gagaggggcc gggaggcggg 4980ggccgtgccg cccgctctcc tctccctcgg cgccgccgcc gccgcccgcg gggctgggac 5040ccgatgcggt tagagccgcg gagcctggaa gagccccgag cgtgagtaga gcgcggactg 5100gccggtagcg ggtgcggtgg gaatggggac cgggtgggtg ccggagactc ccgggcgcgc 5160ccgccgtgtt gggcggaggc tacgggctgg tcgtctccca gcgtcggccc gcggcggagt 5220tcgcctgcga tcgccgcacc ctctccctcg cctgcctctg gcctctccta gctgcagagg 5280cgagggctcg ccgctccctc tcctttctct ctggctgccg cctcgctctt cctttgcttc 5340tactcccagt tttggggacg gggcggctgg aggttgagga agtgcgccca gggccgccgc 5400cccggccgcg gtggctctgt ctcccgcgcc ctgcacgccc acccagcccg cgggtggagg 5460ggctgccgcg aggactgcgg ctcaagggac gcgggcgcga aaccgcgagc ttttcttgag 5520gggctgtcac gcccgaggac tccggccccg ccagcctggc ggccctgcgt ttgcctctta 5580ctgggtttaa atcgcccggc aggggtatgg aaggaacacc gctcccctca cccccgccag 5640ctcccctccc acccttcctt tcgaggttgt tttttccttt tagctgagac aaacgcttag 5700atctttcatt catagacctg agacactgac tcatgtgact tgtctcttta ctaagtgtat 5760tatgtgatgc ctgttccatg gctcttaagt ggctcggaaa ggtagggtag ggcccgggat 5820tgggctatgt aaacaccaga cgttcagccg gcgtgttttt gacctcggct cgaggggttt 5880attttaggat gctaaaggaa tagtgatggt ggccaaccgt gttaccagac ttgggaatcg 5940aaagctattt ttttatgtca ataattgaga ctgtttacat ggttaaataa agggtagggt 6000aaagggagcg caacaagagg gtctttctag aaaacagtct gcatattctt taatgaatta 6060gagttgtgat ttcatcttgg tgaagcttcc tcattccagc actgcgcctt tgtgcacatg 6120caggacctgg gttttcggaa cgatgttttg aatcatagtt tgtgtggccg gggagaaccc 6180cttagaatac ttgggtaggt aaattccagg ttatctcagg ggattatttc tactgcgact 6240tttgaaccac tgagggcaat attggtcata tgttatcctg cttagaaatt gaatttttct 6300gagtatttgc ttacattgca ccgtacaaaa tttgtttatg tgctggattg cgtgttttaa 6360gaaaacttct ggaaaatact caaaataccg tgttgctaag ttagtttgga taaatcaaaa 6420cactttgctt tcaaagcaac tacagtctta acaatttaac gtgtctttgt attcaggttt 6480taagtttttg gtgttttttt tttttccttt taactgaagc actgaggtcc gattgacagc 6540atagatctcc atctaaacat cacatgttct ggaaattttt aattatcaga ttaactgttc 6600tgcagtgtca tcagccaaaa ttatctgaag aggactgtgt gtgtttacgt gtgtgtgtgt 6660gtgtgtgtgt gtgtgtgtgt tggtattttg cttaagtttt ttcatctcac attttgggac 6720aaaataaaac ttcatagctt ttttcaaaag tttcttgtgc tttttcaaac tagagtactt 6780tcctattaaa atcacaaaat agtttattta aaaatgatag catatcgtaa taatagcatc 6840atgggggaaa taacaagaaa gtaaaggaat attaatattg catgattgtg cgtataaagt 6900tttaaagaca ttcctgtcag ttttaaataa ttaaaaatga aacacctgta aatatagaag 6960agtgttttta tttaaggtta aaaaaattta actaaattgg ttatagcatt aaagagatgt 7020tactaaatac aaatattgta cttaaactga aaaatggctt catggaagtg taaattgtca 7080tgggccttga tatatttaaa agggagacta aaagtgcagt ggttttgctt gctctgtgcg 7140tggaacagcc tttctggtgg cagagcttcc ttcctgtcag gataaggtcc tctccatccc 7200aaatcagtat ccccttaaaa aaccaaagaa aaaacccaaa cttataaata atgcacaaca 7260taaagtagac tgtaataact ttttctctaa accagtttac tttgtgtgcc gcttcttata 7320agtacccttt actgaataaa gagttttaaa cagttacgat taccatttaa agggcacttg 7380ctctgttgat ctgtttattt gaagagaggt aacttaatcc catttgatct cttgagcagc 7440tacagtagca ttaaaaggat cagttctcta ttttgttatt ttatatcttg aattccaaga 7500taggggtgat tctttaaatg aagtgtttct taagcattat aaaatatttc atctccagtg 7560aaagtattta caatcttaaa aattattcag ctttagaagt atatgtctta gaatatcatg 7620gtttacattt tattagcaca gttatcaaat agtttttctt aagtattacc ttgagctata 7680ttaacagtgc cgtagacaga gtggtattac tatccctaag aaatggtatt ctaatctgaa 7740gtcagttggc aggtgacaga ctcattgaca aaaccaacat tttccgactc ctttcactgt 7800atcatgctgc gtctaatctc cagcctgttt tttaatagaa ctgaagaaat tagggcccaa 7860ggaggcttaa ttactgaaga tctccatttc ctatatttgc agtaatatct ttatttaatt 7920tcctttacat catgaacagt ggttttcaat cctggctaca cattagaata atctagggag 7980ctttttaaaa gtactctggt ctcatcccca gagatgctca tttaattggt ctgagatgga 8040tcccagttat ctgtgttttg ttttgtttgt tttgaagttc accaggaaat gctaatagaa 8100aaggatgtaa agccctggct gtcaaggacc tgtatatttg gtccttgctt ctttctctag 8160gtttatttct cctccctact cctgcagctc ccttggtctt ttcatactct cctgtgcaaa 8220tacagtttgt acttcctgag acatttgaac actccattct tctttaactg gttgactcct 8280actcatcttt cacatcacct cctttgaaaa gtctagcttg agttaggtat ccctgcttcc 8340tgttcccata gcatcgtgta ctcccactgt catagcatta acctcactgc attgtaattg 8400cctatttctt tgtccatatc ctctaccagg ctgtaaattc catgaggata gggactccta 8460tcttatatac tgtacagtag tattcttatt gccaagcaca gtagcctgta cattacagca 8520cttgactaaa caaataaata tagtagagac agacttcttt aaaacatgta gtgattcata 8580agcaccagtt tttattcttg ccaatttcat ttaatatttt tggtcactaa aaatgtacaa 8640ccctgaaatt gccacacata cgaatttcat ttggggacag ttgagatgtt ataaattgta 8700tagagtggca ttccacctca gacatataca cacacacgca aaaaatgaag attattatca 8760tatatagtac tgcactggaa ataaggagac ttgaattctt acccctgttt caccacaaac 8820atttgggcac gtctcaatct ctttgggtgt catgtataat acagagggat tgtacgaggt 8880gctctacatg gttctcagtg atctagcctt aaaattctgt gagtatcaac agtattggct 8940ttggaatttg aggcaccatg gttcaaaacc tagttcttaa acttactggc aggattactt 9000tggacagatt ttcttatctg taatgggggt gattactatt caacagagtt gttgaattaa 9060atgagataat gtagatccaa gagagttctt gccctggatc aggtgcttaa taaatgttca 9120tttcctttct ccttaggaag tgttagatct catgagtgga cttatctgag ataacagaag 9180gacacagttt atgtaataaa tcctattttc cactttgctg actgttagca ctgcaggtgt 9240gattagggac caaaaaacac tgtgacattt ggtttcagtc tctcagaacc ccattccact 9300gtcaaacacc caggtgttag tcaacctgct tgggaaataa ttatgcctgg aagtttcaac 9360caagagagaa tagcaagtgc caaactattc acacatatat aataatatgc aggagggtac 9420agtctaaact gatgaactta cactgtctcc aaagataagt aaaatcagac ttcatcattc 9480taaaaaaagg aaacaaaatt aaggatgatt tataaaagaa aatccaaaga ttttggtgaa 9540gtctctgttc accaaacctt aaaattcagg agccattatt tagtaatttt caggtgatat 9600aagaaatata ggaaaaattg gtcttctggt caggactaat tccagaacat aggcacaaat 9660aatgttttca atttcagatt agcggttaca gatatcagca gactgaagtt attgatacct 9720ttctctactt gggaactgtg ttttaaaatt taatattgtg ttatatatat cacctttatt 9780tatttggtaa tattgaaagt aaacttacac agaatgtaat acaggttgga catccctaat 9840ctgaaaattt gaaattttat atgctccaaa atctgaaact tcttgagtgc caacatacgg 9900ccataagtgg aaaattctac acctggcatg tttgctttct ggtggtcaaa ggtacacaaa 9960ctttgtttca tgcatataat tatttaaaat actgtattaa attaccttca acctgtgtgt 10020ataaggtgta tgtgaaacat aaatgaattt tatgtttaga cttgggtccc tagtatgtct 10080cattatgtat atgcaaatat tccaaaatct gaaatctgaa atctccagtg ttttggataa 10140gggatactca aaacattgct ctgcagaagt ttcttgttac cagtcttatt tttgtataaa 10200tcaaatttgc ttgcctgagt tttcctttaa gtttatgtga aagtgaattt tataagtaac 10260ttattcgaaa gaaatactgt aataaaaatt aaaaactgtg gatgggggaa actgaagtgt 10320taggatataa aagataaata tgaggcaata tatagttaaa gtctgaggat tcagcttcaa 10380ggctagagat ttccatcatt caaaaagatt attcagctat tggatatact aatattttat 10440cactgtatac cttattcaca tttaattatt atgtgaatca gttgatcctg gttaaggact 10500ttttaggtgt tctttctttc cctctctacc tatacagttt agggagtttt aagttaaaat 10560attccaataa aaagtttgta agtttgtaat aaaagtggtg gcttagagct atctttttct 10620tcaaatagtt cttaaagtga aactgtgtct aatacgtatt agagagctct ggtttagatg 10680cctaaaacgg gtcctcctgg ccagcagacc cagtacctgt ccctggcctg ttaggaaccg 10740ggctgcacag ctggaggtga gcagtggggc cagcattacc acatgtgctc tgcctgttgt 10800cggagcagca gcggcactag attctcatag aaggaccaac cctattgtga actgcacatg 10860caagggatct aggttgcctg ctcctaatga gaatctaatg cctgatgatc tgaggtggaa 10920cagtttcatc cccaaaccat cgcccccaca cttccccaat ccatggaaaa attgtcttcc 10980acgaaactgc tccctggtgc caaaaaagtt ggggaccact ggcctaaggg actaaattcc 11040ccttcggact gctactttgt atatgtatat ggtgggggac agattgtaac acatttttgt 11100gtctttttct atattgatac aataggaatt gtaatgtgtt gcttgctaag taacataatg 11160agataattca aataatcaca agttgcataa aactaggaaa aaatgtgcac atactaatta 11220ttattttaaa tttatattta acagaagcat aatattttat atttggcctg acaatgtact 11280catttctcaa gcatcacttt ggtagttagc ttgataattt aaattgtact catttataag 11340aggttgtgtt ttagcaactt ttgtacatta ttgcgattgt tacaaagggg tctttatgat 11400ggctagcatt gccatttgaa ataaattagg gatttttaaa aatctataag aattcttaca 11460gactatgctt gtcaggtaca cttcggattt taagtatttg tatagtaaag ccaaggaaat 11520cagtgttgtg atgtgaagaa aaaaactgta tagggataga atcattttgc tctaatcata 11580actctcctaa ctcaccagga gttagcaagg cttagtggac gaatatgtct tataagtaca 11640acaaaaatat aaggcactat tatgaagagg attttaggca tatgtttttg aacaggagga 11700gacaatgttg tgtttattgt atcctcagca catactgtag tgtccaaatg taggtgatca 11760gtaagcactg atctttgtga catagaatca tatttagaaa taaaaatact ttcttatatt 11820tctatcagga gccatacgct ttatctgaaa agcaggtaaa ttgctgtgtg ccagtcttat 11880ttgatcattt attgagcaca gatttggccc tttgtattcg tgggttttgc ctccatgggt 11940tccacttacc atggattgaa aatattagga aaaaaaagga tagttatttc tgtactatgc 12000atatacagat tttttttggt cattattccc taaacaaaac agtagaacaa ctatttacat 12060aacatttaca ttgtattaag tattaaaagt aatctagaga tgatttaaaa tatacaggag 12120taggttatat gcatctgcta ccccatttta tataagggac ttgaacctct atggattctg 12180gtatccacag gggtcctaga cccaatctcc aattttccat agataccaag ggataactgt 12240atataagaaa cacttttgta taagttatta actagttgcc tatttctatt gatatttgga 12300gacattttgg caaagattgt taggttacag agaggctgtg tgctgtggtg aaaaaaaatt 12360cgatcacgaa tcagaaaaca cggtttttcc attcatctat taactagctg tttggcctaa 12420gatcttggcc tttcagagct tgtttcttca cctgcaaaat ttctaataca actcccaaat 12480gctgagattc ctagtatgtt aaagcctgtt tttttatttc agggaagagt aagtaaatca 12540ctccctagag agaagacagt ctagctcagt ttgttgtatt ttcagaagct aagatcattt 12600tgtctgcctg tttataaatt tgcacatatt ttaaaggtcc ttgtgtgaaa ttctgtggca 12660gagaaaagct gccattggaa aagaatggag tggaaggatt ttcccccctt ttttgaaacc 12720gttagtgact ttggagaata aattgctaca ggtgtggtca aaattttggt tactggcact 12780gggagcaatt aaatgactgc cagacattga gagtaggtca tctactgcac accagttgcc 12840agggttataa aggccagcag acttgtcaca gcacccttaa tagccactgg ccatcatcct 12900tacttgttaa agacttctag gaaagtatat tccatatttc ccttggtaac tcatccctct 12960aggtggtttt cagaaaatgt gttctaatct tagaaacata aatctgtagg catggtaagc 13020ctcttttttc taggtctatc ctcagttgtg cagataagta tgtggaaaga taacagaaat 13080cattgtattt gatcagaaag cagtgatgtt tatgctattg atttattgtt tttttctgtt 13140aatattcaaa ttaccttcaa cttttgaata ttttttaacc agcacactgc tcaggtaatg 13200agatattatt tagagttatt ttgaggatgt actcattttt caacaagcta agcatatttc 13260tgcagcttgt ttttctttga gagttcttgc ttttccaagt tctttttcct gatggactta 13320tggcaggtct agacctgaaa gtactattgt atttcaaaac aactgactgc ctttagcccc 13380acagtgactg aactgtattt ttttcttagg aggttaatgt ctttggcaag ctaaacatcc 13440ttggaatttt ttgctaagtc agccactatt ttgtgaatat agatttatgg agtatatcat 13500caaattcaaa gcagcattaa tgggaaaatt tgcggaaaaa atattgaaat tagacactac 13560ttacaaactc caatctgaaa tgaaaccata catgacagca gggaaaggaa agaattacat 13620tttggaaccc taacttttat gcatataaat atcaatgcat agtaatgtca ttttaaagtt 13680atatgccttc tgaagctttt cttaaaatct aatggctact attatttgca gttgagacca 13740tgataggcag catcttatgg tatttatgaa agtgatattt gtgttctttt ttctaactat 13800ataccaattg taaaagttga acatgacttt ttcaaagcag tttctatact tgataataac 13860agtggtagta aaactcagaa cagtttaaaa tattagtaat tttacagtgt agagactttt 13920cttcactaga aatgaccagc tttcagattt cttagtgata ttatgagact ctagatttct 13980tcaaaatcat tttgagtaaa tctaccagtg tataactgct aaagcacagt taaatgtatc 14040atgctgtggt cttttttgtt tgtttgtttg gtataaatat cagacatata tgtttgttta 14100ctctaaaact aatttttttt accaatcaaa attgaggcac gtaatctgac aatgggacag 14160gatatttgca gtcaggatag tcttagaaaa gccacaaaat atggtcgatg tacttagact 14220tctttaattt tccttgcctt ttcctaaata tatctcataa tgattaagag catgttctct 14280agagtcagac tatcgagatt actagttttg taactttggg aaagttattt aacctgctaa 14340atattctccg ggtaatacgt atttcttcat ttgtaaatga ggggtaataa ctacctacat 14400cacaatgttg taatgaggat taaataaata atacttgaaa gcatttagta aagtgtgttg 14460tacatagtaa gcactcagta agtatctgtg aactccttga gagtaagaac catgttacat 14520tcatccattt atttgggtgc tttaacatta tgaagtgaat tgaattctgt cccttccaag 14580aatttaccag tggatccgat ttctggatag tgggcaataa ggttgccttc aaacaagttt 14640agagtagttt tcagttcttt acaatactgc ttgcttttta ttcgttaaaa cttttaaaat 14700ttcattctag ctctaggaat ctgaatgcta tttcatacct actcctaagg gaagttagca 14760tatggatatt tagtttaata ggaatgagga attaatggac agaaggtatt aagtaagtga 14820atgtacaatt ttcacatcta gtctgtgctt cctgtggatt gattttccct gtccattcct 14880ggaatcttcg aattcccagc acttatctta atagcactct accattgaag cagttttgtt 14940ttgtgctgtt agatacttcc tttccctgtc ttgtcttttt ttcatgcact aaagacaaga 15000gttgtgtcgt attgtcacat agtccttata ttacagggct

tttaaagaaa tctcacgtcc 15060tgttttcaag ctgcttgtat cttgtttgtc cattctactt gctatgaagt ctttattgaa 15120tataatatta aactcttttg catatcattg tttgttacct aggacactca atggacatgt 15180gtaaacttta caagtcattt atgtaaaaat aaggaaagca accatgtgtt ttaattattt 15240ccatcagttc aatctctaag tctttgcctt taattaccaa acagctatgt ggtgccactg 15300atacgagttt ctgtggtatc ggctacctgg ttttagtact gttgttacta ctttatgtaa 15360atgagactat tcactcatat ttaagtttgt ggaaaatgta tatatgttct tgcgttttag 15420cagttttggc tagactgctc ttgatggtca tgcagggtaa cttacttttc taaagttgat 15480gatttcatgg tctgtatttc tcatatacca ggataatagt aaaatctaca cactttaaaa 15540aactacatat gttgttgtta acatatatgg aggtttcttc agaaattatc tgcgattttt 15600gttgtaattg ttttcattgt tgtgtgcttt taaagaatga cttttattaa gcatgcttcc 15660aagctttttt gtacatgaaa aactgcatat aattttgtct cttttagggg gtgatatgta 15720caatgaaaaa ataacatgac attggattgc cctggggctg accaagagtt tgtttccata 15780gtaagggatt atgactaatt taggcattga agttaagaat ttattactta tttttaaatg 15840acatcacgaa gagttacagc tgaaacccat ggaagaagtt ttcttgattt ttctatatgt 15900acacacaaat gtgtattttg aattcccata aacccaaact ttaatataaa cctgaggagg 15960gggagcatta tggactttgt atatgctgcc tcccttaatc ctcacaataa cacttctgat 16020acatgtgtaa tcacttcatt tccatgatta ggaaaatgaa gtcccacatc aaattaatac 16080agtagaatct cacctcattt attttacatg aaatcaactg tacctactca gtcaaacata 16140aaaacgtgac aaaaataata cagtcatgtg tagctttaca gtggcaatat gttgtgagaa 16200atgcatcatt agatgatttc atcattgtgc agacaccaca gagtgtaccg acacaaactt 16260agatggtata gcttactaaa cacctatgct atctggtaca gcctgttact cctgggctac 16320aaacctgtac agtatattac tatactgaat accataggaa aatgtaacac agtggtattt 16380gtatatataa atatatctaa acatagaaaa ggtacagtaa aatacggtgt aaaaaatttt 16440aaaaatggta tgcatgtata aagccttacc gtaagtgaag cttgcaagac tagaagttac 16500tctgggtgag ttggtgagtg agtggtgagt gaatgtgaag gcccacgaca ttactacaca 16560ctaccgtaga cttgatgaac actgcaccct taggctacac tagatttttt aaaaagtaaa 16620gtaattgcgc tacaatgtta tgacagctat gatgtcacta aatgatgaga gtttgtcagc 16680tccattaatc ttacgggatc actgttgtat atgtagtcca cagttgacca gaacattgtt 16740atgtagcaca taactaatgt aaatattgca aatcacatct gactactgag cttttgcttc 16800aaagtagaca cttaaagata agaatttgcc atcctttcag ttggtctcag tgcctaggtc 16860taggtatgtt acattttgtt gtgttgagtg tgattctgtt attcagacat gtaatgttca 16920tacaacatgg cctctaatac ccagcctgta gtgcccaacc aaagaaacag cagtttgatc 16980cggtgctgga agagaaactg ctgtttgggc ttgctgagca attgtattgg tctccaatgt 17040ggcctgcaga tatggcaaca agaaactgtc atgtattctc tttgatatac acacgtgtgc 17100accccacatg taagccttca cccttcttaa ttctatgtat attttatata ttgtgtatat 17160agttctattg ttttagattg ctatatgtgc attatatctt tgtacaacac acaattttgt 17220acagtgtggt atacaaaaac cacatacatt atactttatg gatgacttga gatttttcaa 17280gaaaaatgtc gattctgtac aatcatagac tctgtttgtt tgcacatact catttctaga 17340acatggcacc ccagaaatac ctctttcagt tactagctac ttagtaccaa cttttcaggc 17400tcacaaaaca gtagtttgtt caatgttaca tagtaggcaa attactatta tggttctgat 17460ctagaattat aagcaaacgt gctgtttcat tgcatcagac cttcccttct tcctcggtgt 17520aagagagatc aagtcttctt ggctcagtag caatgtcttc tgtccagcta gcaagaatgg 17580cacaggaggc tcagggtaca cttctcatta caaggcctta agggaagtgc tgcttctcca 17640cacagagcag ataggacaaa aattgggtgt ttgcacaaag ggagcaatga ttccataagg 17700aggtgaaagt aacacataga tttgtcacct tatttacatt ttctatcttc ttgtctctag 17760gcaaaggggg taacagaaac ctgcttagtg tttttataaa gtaaattctg cctagtatca 17820ttgaatttcc tttagcacat ccatagacat acaacagaac agataggaga aagaaacata 17880aataacagga agtgctgggc gtggtggctc acgcctgtaa ttccagcact ttgggaggcc 17940gaggcgggca gatcacttga ggtcaggagt ttaagaccac cctggccaac atggcaaaac 18000cccgtctcta ccaaaaatac aaaaaattag ctgggcgtgg tggcgcgtgc ctgtaatccc 18060agctatcggg aggcttaggc agcagaatcg cttaaaccca ggagttggag gttgcggtga 18120gctgagatca cgccactgca ctccagcctg ggtgacagaa tgagactctg tctcaaaaag 18180tgataataat aaagagataa cagaaagtcc agtggtcaca ggaccagatg ttttgagatg 18240cttgtaattc caaatgttcc atttccatac tgttctaata tgactcaagt aattcatctg 18300gggaatgaaa cagatttttc aaaagcgcct tcagaatttg ggctgagaaa atatcactat 18360atgtatattt atattttttt aaaaagctga gtgattaaac attatttcct gtttattcaa 18420tataccctat aattgacaat gtggtagacc ctgggtatat aatggtaaac aacacaaaca 18480tactctgtga ttgtacggtc aacgttttcc ttgaaaaatc tctttaatta ctcataaagt 18540acaatgtata tagtttttat atatcacctt gtacagaaat atacatttta tctctgtata 18600aaacatacag aaatctgaaa catgatgaag ttatatatgc aatgtatgca atacatgtat 18660ttaaaagtgt gaatttatgt gtatatatat atgtatcaaa gagaatacac atgcacagat 18720gatagtttgt tgtggccata ttaagccatc tttgcatttt aaggataatt tctacttgta 18780gaaattatct acaagtagat aaacacatta tctaaacaca tgttgtagag tcacactctg 18840ccacacccat ctaatttttc atatttttgg tagagacagg gtttcgccat gttgcccagg 18900ctggtcttga actcctgggc tcgagcagtc tgcctacctc agtctcccaa agtgttggga 18960ttacatgcct aagttagact taatatcata cccggcctgg tatgatagga tttcattcct 19020aggaaaacca agagtcaact gagaaattgt tagaatgaat aagaattcta gagtaaatag 19080atgtaaggca aatatttaaa aatcaatagt tcttccttat gatagcagta attgtttaga 19140aaacgggaaa aaaacccatt tagaatagta acagaaaata taaaattata aatgagccaa 19200tgtgaagaaa cctaaaaatc tacttggatg ttggaggtta cagggaaagg aatatcatgt 19260ttctgaatgg gaagtccagc aattacagat accagttttc ctcattaaat tacaggttta 19320gcctaattct cttctttaaa aaaaaaaaag tgagaaatta gcaaggtaat ttttaatttt 19380gtctgagaaa ctataaacag ataaaagtaa ggtatctttt gagtggaaaa tgaaggacag 19440agcgctaacc ctgtagctta ttagctatca aaatatacaa tacaattaga gtaaatgaaa 19500gagttgaata ctggcataca cattgatagt aaaattggta aaagggaata gagatcccag 19560aaacactcta aatatgataa agaagtaatc agaaattagg agaaaaagga aaactaagat 19620ttgtttttga gagtattaaa tacttaggaa aatagacttt tgaatcttta tttcatctta 19680cataccaaaa taaagtctag gtaagttgaa gagttaattg gaaaagagta aacttatgcc 19740tagtgtcatt ttaaaaagac atttatgtga atggggaaat actttccaaa gcttcaagaa 19800ttgtaagtac tcaaaaggag gaacaatcat tcgttcgact ctcaaataag attggggcat 19860ggcaatgtgc cccctctagt ccaagctact caggagactg aggcaggaag actgcttgac 19920ccccggagtt taaatgcagc ctgctagcaa gactccctat ctttaaaaaa acaaaaaaag 19980aaaaaaatct ctttctctca cctcctctcc ccccaacccc catacaagtg aacaaattaa 20040aagatgtgca gaccaggatt agagttctgc aaaaattcaa tagatgtttg tctttaaatt 20100tttactacaa atactttcaa aagcaccatg acccatatgg atgaatgagc aaagaatttg 20160agaagttggg ctcatttact aaagaggaaa tgcaggtaca agcataaggg acatgttttt 20220ccctgacagg taattgaaga agtgaaattt taatatgtag tgattttttt ttttttactt 20280tttgagttag cagaagtatt tttctaatga tgtttttgct ggtgagggtg ccatgcaatg 20340tgcttgtcca tttatttcca ttgacagtct gaattggttt aaccctttcg aaaagcacgt 20400gaacaatttg catgccttta aaatcaacat acagtttgac ccaataattc tctttgcagc 20460attagatata ataatttaca acagcttatg tatttaacaa ctaaactgtt taacatctcc 20520cgtggaaata ggttgtcata gaaaatgttt atgaagagtt tacaataatt tgcggaaatg 20580tttttgtagt gaaaaaagat acaaaattat gtatactata tgagtacaag tatacaaaac 20640tatagaaaac aaagaaataa accaaaaata tccagacaaa actgtaagag aatacataaa 20700agatgctaat atggtgtctt aggtacgttt tcttttggtt tcatcattca tctgtgcacc 20760gctatatttc cgatacttac agagcacaat cccaggccca taatagacct ttcagtcagt 20820gcttaagtga ataaatttct gtaataaaca tgggattcat ttacattgaa agaaatatga 20880atacactttc catccaagtt aagaaaagga aaaatgtggg aagcactcta gaaaggaaac 20940tgattaatac cttacaatta gattttgttt tcagaatgtt ttcacatctc tattttatgc 21000gatccttttc tgttctaatt taataatatc attacttacc taagttagaa atttgtgcta 21060accggctggg cgtggtgcct catgcctgta atcccagcac tttgggaggc caaggcgggt 21120ggataacctg aggtcaggag ttcaagacca agcctgacca acatggcaaa accccgtctc 21180tactaaaaaa tataaaatta gccaggcatg gtggcgcata cctgtaattc cagctactca 21240gaaggctgag gcagaagaat cacttgaacc caggaggcgg aggtagcagt gagccaagat 21300tgcgccattg tactccagcc tgggcaacaa gagtgaaact ctgtcttaaa aaaaaaaaga 21360aaaagaaaaa agaaatttct gctaaccttg attctatccc ttttgacctg ccaaatccaa 21420ctgattacca aatcttgggt attctttctt agaaatgtct ctggatttta gccccatctt 21480ttctcataat cactgcctta gtttattcct ttctcatttc actttactgt ccctacctgt 21540ccttctacct ttctactctc ctcttttact ttccacactg ctgtaaaaat tgtcaggtaa 21600atagcagaat tttgtgtgtg tgctattata aataggctgt atggaagaga ctggggggag 21660attaacactg ctttcacact gctattacaa taatcaccct aaaatacaaa attgatctgc 21720ttaaagctta gtaattcctt atgacctata ttgatgcttc tcaaacttgg gttgtataag 21780atcacccagg agacttgata atgtgcaaat tatccagacc ctagaaattc tggttgagtg 21840gatccatagt actgggaacc tgaattttat ttttatttta attttttgaa acaagagtct 21900cactgtgaca cccaggctgg agtgcagtgg ctgatcataa ctcactgcag ccttgaactc 21960ctgggcttaa gtagtcctcc cacctccacc tcccaaagcc ttaagattgc aggcatgagc 22020cactgtgttg agcctggaac ctgaatttta ataaggcttc cctctcatga tccttacgca 22080tatgatccca ggacaagatg ttgaagaaca gaccttggga taacatgcag tctcattaat 22140ttactgtaat tctcacaatt tggcatcagc cttcttttct gtgctcattt tctacatttt 22200tttctcatgg acaccttaca gtctagtcac tctgaactgc tacatgctca cctaggttat 22260tgtggacccc tgttcttgtg ctcatgctgt tctgtggttc acagcctttt ttgctatggc 22320ttctcctact cagtaacatg ttgatcttgg tacactatct tcagcttata tgtaatctga 22380gtaaagtatt cccagactca ctctcctaga caaagtctat ctctccctct tctgtatttc 22440catagcctct attacaacac acatatacta aattacactt cttttcatac cttcctcact 22500agaccatgaa cctcttgaag tcagggatgc catattttag atcttttatt taatcttaga 22560gtaaaagcct ggcacaaaat aattgcatgc taaatatttg tcaaatcatt aacaatccaa 22620taaagaaggc aaggagtaat tagtcccatt ttcagtgagg aaaatgatat cttgagaaat 22680taagtatttg cttttaatta cccagcttgt aagcgtaggc tcaagactta cacataagaa 22740atctggcttc ttttctggtt ttggtcttta cagaaaagat agagaataac tgtaaaatta 22800ccccttccat ttttacccac acggctcaca tgttcattta aattaccccc tccattttct 22860acccataaag ctcacatatt agtttatgtt ttatcatttt atgggtaaag gtgttttaat 22920aaacttttac atcatattca cactttataa aaataaaata atgcctattc ctctagggta 22980tgaacagatt gctggatatt aagccatttg tctctcactt tcacatctaa ccttctttac 23040tctgctttgt aatgcttatt tatttctctt gcagaaagca tgttatagtg ccttatggga 23100tgtctacaaa tatttgttta atcaaattga gctaatattt atgtaggaaa cattttattc 23160tgcaacaatt aaggcaaaca gatatatgtg tgtaagtatt cacatgggtt tccactgtag 23220ttttgaaagc tcagtttcca aagtttagat gtgtttagcc ttactgtaga cttcactctt 23280aaggtgttga tggaaatcaa caaccagctg caaaattcag atccagtatg ccataggata 23340catgtagcca cggtttaaac caaatgttag caatgaaata tttagtagca actgttcttt 23400taaaagaaac attttagata attagagtcg tcttgtaata tcttataccc agctgtcagc 23460tttttacttt ttctgccaat atttctggaa ctgaaaccaa gaaaatggaa attagggcac 23520cacactttga aatgtattaa acacttgaaa aactgttata tcttatagga tttccaaaaa 23580gagttcttca gtgcctcaga agatttttat aaaaagcaat tatctacaat aaccctcccc 23640agtattaagc tgaaatatac acatacctta gtaaataata ggagcatttg ggaagtggaa 23700gaattgcctc ttaatagcta ctggacttga agattattaa atttgagcat tctaattcca 23760tttttgaaag tttctgatat tgtttcttaa ttttgttacc atgtttaatc agtatctcac 23820tagcctttca ttcatttctt tagcttctgt tactatcttt ctacttttaa gatactcata 23880tcttcatttt ctgccacatc aactttcagt aatattcctg attcctcact atgttagaca 23940gtgataatag tgtctcattc ttcctttcaa gtctttccca cccttctaca aaatgttata 24000tataattact ttttaaattg tcaagtattg ataacatttg cattctgttc tataaccata 24060tttaaatcct ctttcttttt aactatagat agattgtaat agttgaacct agtactattt 24120acattaatat tatataaaaa tgtttcactt tacaaccaag tagagtgcta cagttggatt 24180tctttctctg aatctgtgtc cagcatcatg aactacgcaa tgaatattct ttgcattcag 24240atcatatggt ttatcttttc atatattcca catactgctc aaaattgtgc tcttttgctt 24300catttttagc atgatgttat acattttttg ttttccccta aagtttccaa ttacttttct 24360tttttctatt gtgaaaacca taaaaacggt attcagcgtg ccattagtgc tgctggactc 24420ctcagttctt catctgttgc ttcaaacctg ttgtattgtt tttcctgaat actttttact 24480ctggaattca gatttcatct tcaagtttgg gcatattact ttctgtaatt aaagtttatc 24540tcaggactgc tttttcttaa tgatttgttt acgttatttg accactggtt cagttccacc 24600atttctagca tgtggtctct tcctctttct tgacctccac ctttggttgg ctagagtaat 24660taatcaatta agtgccttag aattggttca ggggaagtaa aactttggaa aactaattgt 24720ctgaaaagac ttttattttt tctttacaca tgatagtttg ctgagtatag aattcctggt 24780caaattgttt tccagcattt taattactgc attgtttttt acttgttatt gatgagaata 24840gcattgccag tcagataatt tttcttttgc agattacttg tttttccatc tctgaaagct 24900tttaagcttt tatctctatc ttttattgtc tagtatagtt ctttacattt attctgccca 24960ctggtccctt ttaatctaaa catgtaagtc tctccagctc ccagatgttt ttcttttata 25020tctttaattt cctttcttcc atttttctct tacctcagaa cttgtgcaat aaggaagtat 25080tcagttgttg aacctctttg atttattgta tatctttttc acccttgttt cattatgtct 25140gtctttttgc tctatgttct agcaattttt taaattttgc aaatataggt gaaatgttat 25200atccttatga ttttattttt atattgtgtt tgttattcta ttaagattca tttcaatata 25260aggtataaat tggaaaccca ctcttttttc ccaaatgtct agccgtttcc aggtggtctc 25320ttccctattg atttgaaatg atacctttat catgtaacac attctgtgtc tattttttta 25380atttctgggt tttttttcct tttgtctggc tctttatgag ccgtatctta ccattgtgat 25440tattgtagtt tcataatttt tttttctaga gaagagaagg tttggttaaa cttttgagat 25500gtttatttat tttcccacgt caacaatcat ggactatcat gttatgttta tgagtggctg 25560agtcatatct cctgaggaaa acatagcacc attgagtgct acagcatcct atagagaact 25620tcagtttttg gtcatactgt taagacctct tggtttcaca gccccacaag ccctctttga 25680agtgacctat ccagtagggc tagacctttc tctccccctg ccttctcccc tcccctaatt 25740tttctggtct cctgcaattt ttttttaaac attagaatca gcatgcttaa ttctacacac 25800aaacacaaaa atcctattgg cattttattg ggattacatc atatgtgtag attatctaag 25860gaagaattga cattttggga attggttgtc ttcctatccg agaatgtgga atgtttttcc 25920atgtcttcaa tcttttgtgt tacttgggag cattttaaag ttttcttcgt ttattttaaa 25980gcagttaaaa ttcatttttt ttcagttcac gtgactaatc aattctacca ggaccgtcta 26040ttaaataatc tgtcctaatt aaggttctgg ggtttttccc tatacaaaat tgcagcaact 26100attgtactta agtttgtttc tgaactcagt tctgttctct ggatctgcat gggccaatac 26160tattctattt ttataactaa ggatttaata cctacttagg ctagcccctt ctcattactc 26220ttctttcaaa cattcagtat attaaaagct gttataatac ttcttactct gttttagagg 26280tggacataga cattacacac atacaaaaaa actgcaagcc aatctcactt aaagtattag 26340ttatcttaaa taaaatatta gcagattcag tagtttgtta aaacacacac acataccata 26400gccaaatggc atttattctg gaaatacaaa ggtcatttta atcttaggaa atctgtaaca 26460gacattaatt ctctctcttt ccctgcctcc cttcctccct ccctcttcct ttctccagca 26520cagacatgca catgcgcgtg cacacgcgcg cgcgcacaca cacacacaca cacacacgac 26580cttagtaaaa tagggataga aagctattct ttgacgtact aagtaataga tataccaaac 26640tagaagcttc agtcaggctt aataatgaaa gactcgcatt gtcattcata ttacgaataa 26700ttcaagaaac aatcaatagc tatttttttg ccttgctttt gtaagggaaa aaagtaatat 26760aaaggagaag acatattcgc aaattttaat ttcctgtatc agtttctcct gaccactcac 26820tcacattctc cccaaagtga agccaatgtt ggtaacggag tgaattctag tgggggacat 26880tggaggggat atagaaatac gtttgttcat atatgctttt taacacagat ttttaattgt 26940attagtataa aagtgtgata aacatgcttt cattttaaat atcttctgaa cctataaatt 27000atattcccct tttaaatttt tgtttaattt ttatcatcaa aataaatgta tcttccagta 27060aggacagtgt gaggaggttg gatgatttcc ttcttcagaa aacaggcatc aaaatactgg 27120agtaactgtg ttaatgtcag ccaaaataga ttgtaagaaa aggagtatta ttagagataa 27180agaggaacat ttcaaaatta tgaagtgaca aatatggcag gaaaatataa aaattctaaa 27240tgtatatgca cttaagagag gcacaaaatg catgaaacaa agattgatag aactgagaga 27300tgaaataggc aattcaatat ttagaagagt tggagatttt aatactcttc tctcaacagt 27360tgataggata agtaaacaga aaacaggata cagaagacag gaataaagca atcagccaac 27420tcaatctaat tgacatctgc agaataccca acaacagtag aaaacacatt attctaaagt 27480gcattggtaa cattagccag ggtgaatagt atactggccc acaaaaatga aatgttttaa 27540taaatatgga agaattgaaa ttatatggta tgttttgcaa ctacaaagga attaaaaatc 27600atggagagaa ataaggaaga aataagtaaa atccccaaat atgtgcaaat gaatactatt 27660tctgagtaac ccatgggtga aaaaagaatt cactgggcaa aatagaaaat atttttaact 27720gagtgaaaat gaacacaaaa tgtaagaaaa tatttggtat gtagctctca ggcagtgcta 27780agaaagaaat gtttggcttt aaatgcttat gccagaaagg aagaaaggtt aaaaagtcaa 27840ttatctaagc tactacctta agaagctaga aaagaaagag taaagtgtac caaagtaagt 27900agaagaaagg gagtaataaa gatcagaaca gaaatcaagg aaatcaaaag ctggttcttt 27960aaagagttca gtaaatcttt agctaaaatg aacaagaagg gatgtaaatt agaaatactg 28020agaatgaaag aatgggtatt actacagata ctataaatac taaaaagata aaggaatatt 28080atgaacaagt ttacaacaac caatttggca acttagatga aatgaacaaa ttctttgaaa 28140gatactaatt accaaaactg actccaaaag aagtagaaaa tctaaataga cctatataga 28200gtaaataaat tgaattagta gtttaaaact tccaataaag aaaatctggg ctttaatggc 28260atagctagta attctgtcag acaccaaagg aagaaaaaaa tacaagtccc aaccacattt 28320tcagaaaagc aggagggagg agagcatcac aattcatttt atgaggacag tattactaga 28380taccaaagcc aacacaaagg catcccaaga aaagaaaact agcagccagt attcctcata 28440aatgtagaca caaaaatcct tagcaaaata ttagcatgtt taattagcaa tattatattt 28500aaaaggataa tactataatc atggtcaagt aggatttatc ccagcaatgc aaggctcact 28560taacttagaa atatcaattc tgtgtaatat accatactaa taagatatat ttgaaaaaaa 28620agtgatcatc tcaatagatg cagaaaagta tttgacaaaa ttcaatacaa attataataa 28680aaactcttag caagctagga attgaaggca acttcctcaa cctgataaag aatgtctgtg 28740aaaaaaaaaa tcatacttca tggtgaatga ttttatagtt ttccacttaa gatgaagaac 28800aaggcaagga tgtttgctct tactgctttt attcagcatt atcctggagg tcctagctaa 28860tgcagtaagg caagaaaaag aaatttaaag gatatttgga atggaaagga agaagtaaaa 28920ccaaatagat ggcatttata cagaaagttc taaagaatct accaaaaaga aaaaaaaaaa 28980atactagaac taacaagtga cttaggcaag gctgtagagt aaaagataac aaacaggaat 29040taattatatt tctaaacact agcaatgaac catccaaaaa tgaaattaaa aaaccattta 29100cagtagtatt gcatagctaa atacttagta ataagaatag atacacaaac ctgtacactg 29160aacactacaa aatattgttg atggaagtta aagaagaact aagttaattg agaggtacac 29220tatattcatg aattggtaca ctatgttcag tgcagtccca gtcaaaatct tgtcagattt 29280tctcatagaa attgacaggc tggttttaaa atttatatgg aagtgcaaaa aacataacca 29340cacataaaaa ctataaaatc tgaacactta caagtacctg atttgtactt gctataaagc 29400tacaataatc aaaatagtac agtattagca taaggataga catatagatg gatgaaagag 29460atttgaaaat ctagaaagag taattcttaa tatagtaata tttttcaatt aatttcttat 29520tttggacaat tggtatatcc tatagatctt tttcccgtga tttaatgaaa tgtaacattt 29580ataataattt tctttttatt gaacattttt gctttgtata ttatatgtta ccttatttgt 29640taataaaaac acataaataa acttggttag gtattattta tgtggtaact gattttcaac 29700aaaggtgcca aggtaattca atgggaaaag aatagtcttt tcaaaaaaaa gaaaggccgg 29760gcgcggtagc tcacgcctgt aatcccagca ctttgggagg ctgaggcggg ttgatcacga 29820ggtcaggaga tcgagaccat cctggctaac acacagtgaa accccgtctc tgctaaaaat 29880acaaaaaaaa aaaaaaaaaa aaaaaactta gccaggtgtg gtggcgggcg cctgtagtcc 29940cagctactcg ggaggctgag gcaggggaat ggcaggaatc cgggaggcgg agcttgcagt 30000gagcctatat cgtgccactg cactccagcc tgggtgacag agggagactc catctcaaaa 30060aaaaaagtgc tagaataaca aaaaatttat taaaaatgga

ttgatggttc atagacctaa 30120acttcagttc ttaccccagt aattacctac gtggtgtatt gacaacaaag gaagaaaact 30180tgtagaggtg ggttttggca gtgttttttt gtttttgttt agaggtggag tttcactctt 30240gttgcccagg ctagagtgca atggcacaat ctcggcacac tgcaacctcc gcctccctgg 30300ttcaagtgat tcttctgcct cagcctccca agtagctggg attacaggcc cctgccacca 30360cacctggcta attttttgta tttttagtag agacagggtt tcgctatatt ggccaggctg 30420gtctcaaact cctgacctca ggtgatccac ccgcctcggc ctcccgaagt gctgggatta 30480caggcgtgac ccattgcgcc cggcccggaa gtgttttaaa acttggttgt agtggtggtt 30540tttacaacta tacattcacc aagactcatc aaactgaatt ttaatttata cctcaggtaa 30600agctaaaata aagtaataca tgcatattat tttcaaatta caactgaaag gttaataaaa 30660acatcaggcc gagcatggtg gctcatgcct gtaatcccag cactttggga ggctgaggtg 30720ggcgggtcat gaggtcagga gttcgagacc agcctggcca acacagcgaa accctgtctc 30780tactaaaaat acaaaaaaat tagctgggca tggtggcaag cacctgtaat cccagctctc 30840gggaggctaa ggcaggagaa tagcttgaac ctgggaggcg gaggttgcag tgagccgaga 30900ttgtgccact gcactctcta gcctgagcag cagagctaga ctccatctca gaaacaaaca 30960aaacatcagt tcattactgt atctcacttc ccccccattc cctctcacaa ctttgtcttg 31020tctcatgtca tgcttcagga aaccactttc aactatttaa gctactactt ctggtatttg 31080ccaccttata ataatggggt atattgctct gtcttggtcc accaatattt gacattattc 31140attagttttc tcttataatt ttaatgctca tatcttcctt tgccttatct catcatccta 31200tatttaatca ttactggtta aatccacatt tagttatttc attattcaat gaccatatag 31260taataaagca aatgctacat ggggttatat tttttctgtt acaactttct tgttgttttt 31320taattaattg cttcatctca gaaatctgct tagttaccat tgtgtctgaa tttgtctttc 31380catgggcttc aatccatctt tgcaataact ctcacatgtg ataattagtt tggttatttt 31440cctataatat ccttcataga gccttcagta tttctgattt aatcttgatt gcttgctagg 31500tctgtttcac agctgttatc tcaagactct tttttgtctt catcctggaa attccctaca 31560cctgcttcct gtgttgaagc attgattcct cctatgcccc ataccttcat ggttgactac 31620ctcattctat aggagcttat cctctcgtgg catcttgaag agagataact gggaggtaat 31680ttttttgaga ttttacatac ctgaatatgt gttattctac tcctacaatt atgtgatatt 31740ttaagtgagt gtagaatgct agattgaaaa tcatttttac ccagatattg ctctggtatc 31800ttccagcttt caattttgct agagttctaa gtgccactct gtttcctgat tttttattta 31860ttcttttttc tctctgtctg gaagtgatta atctcttctc ttcattttat ggtatggtac 31920cttggtgtgc atcgtttatc tgttgtgctg tgtactccgt aagtccctgc tgcttgcaga 31980cttacattct tctcggaagt atccttgcct tttttggtag ttccctctcc tttgttttca 32040ttattctctc tggaactcct actagccaga tactacactt cctagattgt ttttctaaat 32100ttttaccatt ccctcctctt actcatccct ttctttttat tctgccttct ggaaaattgc 32160cttaacttta acttcaaaca tttgttgatt tttttttttt ttcagttctt attctctggt 32220cttattggtt cctctgcata aggatgcagt cttttatctt tacagtttct ttgacatttt 32280cctctgtccc atcattctct gtttctcacc acttctgttt cattttggtc tcaatcttta 32340atattggaga cattcctcaa atgcatgaag atcctcagtg gtcatttata tttaaaagat 32400gtgaaaagct gaccgaaagc tctgggtgtg aagtcagagc tcttgtctat tggtaaacta 32460tgctgtagga aatcttcata tcacaatttt tctttaggtt agttttgttt tctctagttt 32520tgaatccttt ccagaggaaa tctatagtct tctctgtggg ggctgcttat gttttagaga 32580aatactgaag aaagggactt tgggtctctt tagagttgca cataatcttc tggttttagt 32640catatctact tctgtatcta ttgaagtcca aagcatcttg agtttagttt ttccagaaat 32700tatgccttct gatttctgca tgattgggaa gtcactgagt acaccaactg gagttggaga 32760cctggaattc cagtagttcc aggaacctcc tagtcctgaa cttaatgggg tttcatgaga 32820attgactggc ttctttaggc acttaaattt aacattcctc acctctggtg aggtttttgt 32880ttgttgtttg ttttttctcc ctgtctttgt atattatggt ttagggttac agttttccag 32940cttcatcaaa aaaagagttt ttctcattct ccttgtttag aaatgattgg tgacacaaag 33000gtctgtactc tgatatttta agatccgaag tgtattctac ccctttttta taaactattt 33060tgcagctgtt ccttcacgac tttgctacat cttggagatc ttttcatgtc agtacctaca 33120agtctatatc attgatatta tcagctatat agtactccat aaaatgcttt attataattt 33180atgtaatcat cttcctattg atgaagattt tggtggtttc tagtttttgc tttcatgaat 33240attgttgcaa tgaatattgt ttggcttact tttgggtgaa tttttacaaa gacatccata 33300taataagttc ccagtagtgg ggttgcctaa tctaagaata agtgtattta aaatttgaat 33360catattgtca gactgtctcc cagaaagttt acattcctac caacaatctg agagagctgt 33420ctttgcagtt actaggtttc atcaaacttg tttttttcag tatggtaggt ttaaaaatgg 33480ggatacattt ttgtttttat ttgcattttt aaatattttc ttaggttagt tggctactta 33540aatttctttt tctgaaaact ttgtatttat agccttttaa aattcttatt gacttgcctg 33600aactatttgt aaattacaga aattagccct ttgtcgtatg tgttgcaggt gcttttccag 33660tttgccagtg gtcattcatt ttggtttatg gtacttttga tagaccagaa tctttgactt 33720ttatttagtg agatttatcc atctttttct gtgtggcatc ttggtattat agcattaata 33780ttctcttttc tttttttttt ttttttttga gatggagtct cactctgtcg cccaggctgg 33840cgtgcagtgg cctggtctcc actcactgca acctccgcct cctcgtttca agtgattctc 33900ctgcctcagc ctcctgagta gctgggatta caggcatctg ccaccacacc cagctaattt 33960ttgaattttt agtagagatg gggtttcatc atgttggtca ggctggtctc aaactcctga 34020tctcaggtga tccgcccgcc ttggcctccc aaagtgctgg aattacaggc atgagccacc 34080acgcccaggc tatagttttt ctttatctga acaataacta attataaagt atagtggaag 34140agaaaaaccc attcagaaaa ttaacaaaaa gatcacttat ctagaaataa acttaaacca 34200tagcagagct tacatgaaga aatcttttaa aactgagcaa ggatttgttg gacaggacac 34260agcactaatc tcaaaagaaa atttgatgaa taggtcttat tcatagtttt tgctcatcaa 34320aagacagcat taagaaaatg aatagacaag ccacagaatg agttaaaaaa acagaaattc 34380ccagcattta caagggatta aagatgtgta tcagaatata taaagaacct ctgaaggcca 34440ggtgtggtgg tttacacctt taatcccagt gctctgggag gccaaggtgg gaggatcatt 34500taaagctagg agttcaagac cagcctggta acatagtgaa acaagattct gtctttacaa 34560aaaaaaaaaa aaaaaagtgt ttgggttttt ttttgtgttt tttttttttt gaagttagct 34620aggcatggag gcatgtgcct atagtcccag ctattcagga gcttgaggct gtagtaagct 34680atgatcatgc cactgcactc cagcctgggt gaccaagtgc gaccttgtct ctaaagatca 34740aaaaaaaaaa aaaaaaaact cagaaattaa catttaaaaa attatgaact acttaatata 34800aaaatgggca aaagactcga gcagacattt cacagaagat atacaaatgg ccaataagca 34860catgaaaaga tttccaccat cattattagt catcagtaaa tgcaaattaa aaccatgata 34920agataccact acaaattcaa accatcatga gatacccact agaatagctg aaataaaagt 34980gactgatact actgaatagt tatgatatag accaactgga attctaactt attgctggtg 35040ggaatataag aactatttct tataaagtta aacataacat ttacctaatg tgtcaggagg 35100ttcccagaat catccctagg ttccatggtt cgctaggaca actcacagga ctcagcatga 35160tgtcgtactc atggctctat cacttactac aatgaaaaga cacaaagcac agtcagcaaa 35220gggaaaaggt gaatggggca aaatccaggg gagaccaggt acaagcttcc aacaatcctc 35280ttccgtagag tctcacaaga cacacttaat tcccccagca aagagctgtg acaatacttg 35340tgaaatctaa ccagccaggg aagttcgcta cagattcagt gcccagggct tttaccgggg 35400tctgatcata ccctctgctc agcgcacacc caaattccag actcccaaag gaaagcaggt 35460attaagcata aaccatattg ttatcataaa tgctttagac acatggagcc agtcttatca 35520gatgggaatc agggtaggaa tcctcctaaa atccaagatc caagttccca gacaccaagc 35580cagccttgca aacaggcttt tttaaggata agcataagaa tattaactct tttctgcaag 35640tttacaacct agcattttgc taagtataac aaaaattgat gtaggaattt tgatagccga 35700tgctggaaac agcccacata ttcaacaact ggaaattgta taaacaagct gtagtatatt 35760ttggaatact acttaaaaag aactaaccac taatacatta aaacattgat gaatcccaac 35820ctgttgcact cttctcattc tgtacctgac agtatgttaa ccaaaagaag ccaaatacaa 35880gagtatacac tgtgattcaa tttatgtaaa gttaagactc agcaaaacta atctataatt 35940acagaaacca aaatgtgatt gcctgggggc agagagagaa attaattgga aaagagcatg 36000acagaatgtt ctaggataat agaaatgtat attatatatc ttgttttgtg cagtggttac 36060gtgggtgtaa gaagttgtca aaagtcatca aacttaatgc ttaagatgtg cagagttcgt 36120tgtatgttaa ctatagttca atttgaaaaa ttaactactg agaaacattg aaatccaatg 36180acattcctat ccaaatacat atttggatag gaaaaacatc ttgtaaagat gtcagttctc 36240cctgttctca aaataacaag aagttttgcc ttgatttttg tttttgaatt tagcaaaata 36300ttcaaaagtt catctggaat tatcagccaa gtttaataag actgtgaagg gactttacag 36360aagctacttg ggctgaattt aaaggatgag tatgattaaa atggcaataa atgtgaatat 36420gaggtaaaag attatggagc atatttacaa aggcaaaaaa ctgtaaaggg aaaagattga 36480tctagtaaga ttagagcttg gagtgtggga aaaataagag aagagcacag ttgtaatgtt 36540tgaggcttga gtcaaggctg tattgcatag ttactagaga tccacaatat tgtcttaatt 36600gagtaatggt gtaattagat atatgttttt agatagccaa ttctagtgac actgtagata 36660atgaagtggg tggggagaaa ttggatactt tgtgctttca gggcctcttg tcttagttca 36720tactgtctac ttaacctgtt gcactcttct cattctgtac ctgtcagaaa tcaggcccac 36780tttctcctaa aattaccacg ttctgccttg tagattgtta gataataagg tcttttcaac 36840ctcatttata tacttcattt atatacttgt atgagtgttt ttctcctcct ttcctttcca 36900tcacccatcc ctttgcttca gctgctagtg tcttaataga aggtaattgc ttaatgaatg 36960cttgttgaat tagagacaga tcagtcctgc atatgagaac tcttttaggg tctaatagtg 37020atagagaaga ttgaaaaaaa aaggtgatga atttgagggt aatttaaggg gtggaagtga 37080tgtcatgcaa gagaggagtt gaaagcacca aatgttttaa ccttggagat tgagaaagtg 37140gtgatgtcat gaagaatgta ttccacattt gaataatgag cagagtaaga ggtaccaaca 37200aatgaaacaa ttaaaggagg ccataaaaac aacagagatc atttcaacat tctattgagg 37260aaatttgggg ggacacactt taattaaata ataacatcag tatttcttta cctttgtaac 37320ttttaattaa tgaagatgtt ttaagatgaa atataaaatg tgaacctgag aactgtttaa 37380catttaacat tatggatata catatccatg tttagaatgt tctctgtgta ttaatgcatg 37440attttgtcat aaatacccca ctaggaagag aaaggtcgga gagagtgtgt gtgtctctat 37500gtgtgtgtat ttgtgtgttt tgtcctggca aaaagacaaa ctgagagaac agaagaacaa 37560aagagcagta ctgaaaatag ctagattaaa ggttgttttt ctgtcaccac acaccagata 37620tggagagtag taaaagcaga cagatggaca gttctgttag ttttggagtg tgtgaataga 37680gaaaagaaac attctgttcc ttttattcag aaaaggaatt tctagactaa attactggta 37740tcagcaataa gtttcatatt ctgaccaaag aaacattatt ttttttttac cctagggata 37800atgactcatg ctgttctcag aattttgctt ttgtggaaac taaagtgcag tcataaatac 37860ggcactgtta tttagatctt agtgctaatg atactattgt gagtagacta gtgatgggta 37920agaacactag tattataaac ccagttgtaa caaagaaacc aagcacatct taattagctg 37980attctagatg ggaaagtaga ttttttaaac aaattaacta attgttttaa ttctgtgaat 38040tatgcaatag aaataggacc cataatttta tttttttaat acttcattat tttgaggggt 38100ttatgtcaga agttaaattt ctaacttcca atattgacag tggagataat taaacatatt 38160ttctctgcct tttttcctta tgttcctcaa aattagtata agtctactaa atcatagtca 38220catacaccat aaagcaccta tcaaaagagt atcagctagg tgaagttcaa agattatatt 38280ctagttttat tttaaccttt aaagtcttgt aatttaagaa atagaggttt gaatgtcttc 38340tttatatacc atagtactag atgctctcac tctgaatttt taccatgtca ggatactcca 38400tcacatattt atctctgtaa ttataattat tgggcacata cacaaaagag ttgtccttta 38460cggtatctct gtccaaccta aaaactttcc catttctctt cactttaatc tcttcagtcg 38520ttccctttcc tgaattacct ctttactccc tagtgacggc ttaaagtttt agatcttaaa 38580gttccctttt tcaaactttc ttacgtcttc ctctgtgcct ttattttaat gtcagcagaa 38640gttagctcat gaatcataac atgtgaagca gttgtttgga ataattgaca gcttttaatg 38700acttatgtta aatagccagt ttaaaagttt ttgtcagttg ataagcttta taaagaagca 38760ttttaatgta ttaaatcatt gtaagtaatt tattgtttaa ctgcatgcct tgggtggtta 38820actaatgtca tacagagcat cagtgaataa gagtggcagt attggctggg tgctatgact 38880catgcctcta atcccagcac tttgaaagtc agaggcagaa agatcaccag agcccaggag 38940ttcaagatca gcctgggcaa catggcgaga ccccatctct gcattaaaaa aaatttagct 39000gggcatggta gcacacacag gtagtcctag ttcctagcta ctcgtgaagc tgaggcagga 39060gaattgcttg agcctggggt tggggctgta gtgagccgtg ttcgtagcac tgtactccag 39120cctggataac tgagagagac cctgtctcaa aaaaaaaggg taatattcta ctcactgatc 39180atgtatgttg ctgcttttac acagttatac ccatttcagt ttgtcattct cattgcttgc 39240tttttaaggt atagtaatta taatttccaa gatacttggt acattgaact gtacataagt 39300ataaataaat agtaaatgct tgaaagggaa aataaagacc acttactaat atttctctcc 39360caccattgaa gtattttggc tgtattgtat tatcgaaaag atggattttt tagaaatctg 39420tgcttacatg acttcacatt gttgttgctt tcattcatag aacttgtctt cacatcttag 39480ctaggtgaca cttgtgctga aattaagcta ccctctctct gattgcactt ctgggagatc 39540tgagtttctt tgcttgccaa agttattact tcaagctttc ctgttgtgaa atagcccttt 39600tgccagagat caaatactga aggatgataa atcactatta gtcaataatt gcttgcaatg 39660gacattttca ttattactaa cataatacta ggatattttg ttattgccag aaaatggggc 39720agatggcaac caattacatc aggatataaa gttattgttt ggtctttatc atggaaaaag 39780cattgttatt taaatcaaat ttttactgta tatcatgtct ttcatccaat gtcgtaacat 39840cccagataga cttggggtgt attcacttaa aaaaatacaa tatcttacgt tcattagagt 39900atagttggaa gaaaatgtat tatttttgca agaagctgtt tatggattta tctgattaat 39960ttatgtagag ccaggaaaaa gttgttttga ccaataaatt tggcatttga aatgagcagg 40020ttaagaatag acattaagtg atcaccttaa gtgcaataaa atttcagtgc actatacatt 40080ctgtgcttta tatctatttg taagaatacc aataatatat caatatggta taagaggaaa 40140gaataagaga ttactataat agctcctaag aaaggaaatc agtccaagga cataggctga 40200ggttgtgatg tggacaaaaa gataatagca aggatagata aataactaga caaaaatgtc 40260agggacctaa gtatttatat ggcaaacaag gatactcagt acatctagag ccagactttt 40320tccagtgtct aatccttgtt gtatattaaa tactttggga actaaaatat gttttatgac 40380actgtgttaa ctgatgcata attatgtaat ggaagccatt agggcctgtg tcttggcagg 40440aattaaacta aaagcaaggt ccttctagta tttttggtag aagattattg gctagtgcct 40500attttcacag cacatactac taaaattagg aggacgacag aagacgagca ttgcccctcc 40560ctgtgcaaaa ttgatagaca cattcaagaa gtattccata tttttaagct aatcttttta 40620tttgattcac ttaccagcag gctgttttcc tgctgcaaag caaattggtg gagtccattt 40680acaccttctc cttagcctat aagtattggc agaatcacca ccacgatcac agcagcacag 40740tccagacagt gtcatttgat tttgaaaata ttgtccttgc aacagccagc taggacaatg 40800acatactaat atctgctttg cttattattt gtgcctggga gtatgtaatc atagtcaaca 40860gacaccatag ctacagtttt atgacacttg gtctcaggaa atagagaact ctacagaaga 40920cagtagacca tgatgaagct ttatcattag tccctggttc tctcccctcc agatcacttt 40980ggccttctga cagacagtac agataaatct gttaagtttg gaatgttgaa ttggtcaaaa 41040atgaaaataa tagtccaaag caaaaagacc ttctgtgaaa catatgtgga tgcaattagg 41100tgaatatatt gtatctgctg gtgtccccaa agttgatagc tttatttatt ggaccagcct 41160agactgaaaa tcttctgttt aggcattttg gtggggattt tttcccctct gtggataccg 41220actgttatat ttaaggaaga tagaacaatt ggttctacca agaaacctgt agctaaccca 41280agtttcactt tatgatggca gcactttaaa agtttggtat gaaagactca tctcttcttc 41340attctcaaga caatagggct aatcagtgag atgcaggcaa gttttaattt atctaaatac 41400taaagagtat cactagaagg gatagtactt gaatacaagt ccctatggtg actaaattat 41460gtcctcaccc catgatataa ggaaataagc tagccgttga tttttgatga agaaagactg 41520tgggaacagt gtgtagcaac tgttttccaa gaagttatcc actctcaccc atagtagtgg 41580aaagacttga aagaaagttg ttgaggtgtt cagagccaca gaaaggccta ttaagtcatt 41640gagctatact agaaagaaac ataaaaacaa aagagcagaa gccatttgat aaacttcagg 41700ggggagaagt tctaatccct gatgacttac agaaacatct tgccttcagc ttcttacaga 41760tctttaaaag aatcaggtca agtgatttga aagacatatt gctaatatta cctttttcta 41820gattctgtag aaataaacca tttttcctct gctctcacac tacaataatc aacacagaag 41880acttctgtaa ctgctggtcg ccaagaagta tatggggatt tctccccacc aacaatcagt 41940cagttgattc tgccgcagat tcttcaatag acagcagttg agtatcctgt aattcaattc 42000agttctgaca ctatctgcct agagatagca tcagatccca tagattgagg gctcagtcac 42060aagactgcca tccatttcca atggcagtca taagcttcag gttgttttac ttgtgcttct 42120gactgactgg ctataaatca gcgttcccat gaccccctcc ttgggtttga ttaatttgat 42180agagcagctc atagaactca gggaaataca tttctcggtt tattataaag aatactacaa 42240aggatataga tgaagaagat gcatagggca gggcttgtgg gaaggggtgt ggagcttctg 42300tgccctctgt ggacacacca ccttccagga acctccactt gttcagctat ccagaaggtc 42360ctcgaactca gtcctttttg gatttttatg gaaacttcat taagtagcat gattgattaa 42420ataattggcc attggtgatc agcttaacct tcagcccctc tctcctctga ggaggtgggt 42480agggggtgga ggtaggggta ggactaaaag ccccagttct ttaatcttgc cttggttttt 42540ctggtgacca gccctcatcc tgaagcaacc taggggctgc cagccaccag tcaactcatt 42600agcatacaga aagaccctta ccactttgga gattccaagg aatttaggag ctgtgtgcca 42660ggagagagga cagagactaa atatgtattt cacaatatca catagatgtt cttaatttaa 42720ttaagatgtt aatctagata ttcttaaact cttaatgaac tcaacaagct gagccctgga 42780taatgtaccc ttttgtttct ttctttagct cacaagaagc caggaaacaa ctattttata 42840agtcagccaa gtccagaatg gtgttagctt cattatggca agaaagagtg aagttacttt 42900tcgtgaagcc ctgggctttt tgaagaaaaa gagaggagag gtggttttaa cattgacttt 42960aagttgaaaa gtagagccct tttatacttt tttctttaat aaatccatca atcaagaatg 43020gaacaatgtg tggttagcat catctttgaa gttccaagtc acaaagggat tttcttcctg 43080aagtatgtgg gatgtgagtc atttgtaaat gggaattacg aaggacaatg agccctattt 43140tttttggtta tcatcaatat aggtttggtt ataaacttgt tgtaaagagt taaaccatca 43200ggaccttttc tcttagagtt ccacaggagc tgaaatgttt gcttagtagg aaatcgcagt 43260aacatttcaa gaacccttcc gtattttgca tttcctttat attagaccat gattccttgc 43320taagttggtc tcttcattga tctcattggt taaattttca ttgaggcaac caggtgcata 43380gacttctatt ttcactgtct tcagtatata cagttggtcc tgaaatggat atacagctgg 43440caaacttaaa taccttagga ttgtttctct ctttatgcaa aagatgctat tttttagtca 43500ttttataagg gaagtgtttt ttttttccta atttaaaaaa ttaccctcta tatgataacc 43560aaatttcctt taagtgttgc ttcagtgaac caaatagtgt tttactaagt gtaggagttc 43620tagctagata atatctacca tctctttaca acaagcctga aacagtaagt tctgtttccc 43680tttctggaca catgcattta gataacattg atccagtaga taagagaaag ggaggccggg 43740tgcggtggct cacacctgta atcccagcac tttgggaggc cgaggtgggc ggatcacgag 43800gtcaggagat cgagaccatc ctggctaaca cggtgaaacc ccgtctctac taaaaataca 43860aaaaaattag ctgggcgtgg tggcgggcgc ctgtagtccc agctactcag gaggctgagg 43920caggagaatt gcttgaaccc gggaggtgga ggttgcagtg agccgagatc gcgccactgc 43980actccagcct gggcaacaga gcaagactcc gcctcaaaaa aaaaaaaaaa aaaaagagag 44040aaacggatcc tttattgaat ttctgtgttc caagcactat gctgagaact ttacatatac 44100catctcattt aattcttttg tttgtttttc tttgggatgg agtcttgctc tgtcgcccag 44160ggtggagtgc agtggcacca tctcggctca ctgcaacctc cacctcccgg gttcaagcta 44220ttctcctgcc tcagtcgccc gagtagctgg gactacaggc gcccgccacc atgcctggct 44280aattttttgt atttttagta gagatggggt ttcaccatgt tggccaggct ggtctcgatc 44340tcctgacctt gtgatccgcc cgcctcggcc tcccaaaatg ctgggattat aggcatgagc 44400caccatgccc gaccctcatt taattcttat aacattactg tgagatagta actattttta 44460ttcacctttt gcaaataagg aaactgcctt caaatgagcc tacaaagaag acttaagaat 44520tggagagttc tccctagggt cagtggaatt caggatgatg gtgcactgat ctcaactaaa 44580acgaaactac tggttggacc atttcttctc aggactagat tgtgttaata cacattcttc 44640tctttgctac tatatgttaa ataatctgga gatttccata atgtttccag gttccttggt 44700tactcatttg ctacaggtgt tgctaaagat gtgtaaaagc tcaagtcact agttaggctc 44760tctttgtggt ttgattagct tttgtccttt tctttggcca ggagacactc taattctgtc 44820cacctgtctt accagtgcat gccattggtc acaaggtggg ttggttaata taaaggaata 44880aatgaattgg agaccaagta ttgtactact ggaaaaacaa aggcaaatgt tagtatattt 44940ttttccattt ggaatagtag tcattctttt agttctgtat tttgaaatca ttatttacca 45000atacagtctt cttgatgtga aaatgcaaac agagttttca gtgttacctg atttagggtc 45060actggaactg agagtgatgg tttacttctg actgcctgct gctgctttta tgtgaaactg 45120tgataatctg ccttgactcc cttcctccta ggaaagaaag

aaataaaata attaaataag 45180aaaaatattt agtagaaaac tctttatcac tgtggaatca tttgtatcct attaaatggt 45240atattatgaa atatatatcg ttttccacat taagtacatt tgccttttct cataatccta 45300tctcaccttc acctttatta atgtttagtc acattttgag cttatatttt aagaaacttt 45360aatttgtcct tttgcatgct gaaatttgaa tttttaaaat tcaagtatgc tgggcacagt 45420ggctcacacc tgtaatccca ggactttggg agaatgaggt gggcagatca cttgaggtca 45480ggagttcaag accagcctgg ccaacatggc aaaaccccat ctctactaaa aatacaaaaa 45540agttagctgg gcatggtagc acatgcttgt agtcccagct actcgggagg ctgaggcagg 45600agaattgctc gaacctcgta aacagaggtt gcagtgagcc gaaatcatgc cactgcactc 45660cagcctgcgt gacacagcaa gactccatct caaaaaaaaa gtaaaaataa aaaaataaaa 45720ttcaaattga aaaagttcat tttcacgatt aaattcatat ttacagtgct gccaaactta 45780aataccttag gattgtttct ctcttcatgt aaaagataca taaagtgact acacatttca 45840gtatctggta tggtttctct ttataaaata caaaatttaa agttcatgct ttatctaata 45900atctaagatc caagtattaa tctatacttc attaatggtt ctttaccaca tttgattctg 45960ctattcaaac tggattcttt taacccaacc ttggtcacat ttactgataa tcagaagtcc 46020acattgctat aaatactact cacctaagcc ttgattattt gtcattaaaa aaaaaatgtg 46080ttagcacaga tcctctctaa acccattcat gggcctcata ttaggaaacc cttgtcttat 46140ataatgcatt aaatctctga aatatatttt tataacatgt ttcttagcac tgtaaatatg 46200aatttgataa tgaaaatgaa ttgcagttcc tcactaaagg aatttaaatt ttttgcatac 46260tagtgatgag gacttgattt tccaccctga agttgtaaag gtctttcagt ctctctaaaa 46320gtgtgagtta aatctgtttt ctcacggcca gcttttaaag gtcagtgaag gacggcactt 46380tgaaagaaat ggataatatg catccttcca gagctagtat atattacagc tgtccctttg 46440gactcactcc agaaacagct cagtgactct tagaggaaga gaattttaga ggccctaact 46500ggggcccaga aaattgtact gaaggatact tgcttcctgt gcatctccac tatagtaaaa 46560tttgtttaaa attacgcttc ttagccctca aatccaaatg gtctcttgag tgcttttgat 46620ttttctttaa ccatccgttt cctactattt ctactgctac ctcgctttgt tcaggccctt 46680gttacatttt cactagagta atttcaacaa tttagttttt ctacctgctc ttctatgctc 46740taggattata gtgcataaaa aaaagtgcac tatatatata gtgcaccccc cccaccaccc 46800ccagcataaa agaccctatc ctgaaaaagt tcatagccta ataggaggtc aaacatgact 46860acagataatt ttaatgagta tgttgaggta cagttataga aatacatata aaaggctgtg 46920gtaacattta aaaagggagt aactgactct agaaatattt aaagtaactt tcaactgcag 46980taataagacc attgaattat taacccagat ttgaatcatg ttgtttctgt agcaaatgta 47040gagggaagtt ttgttctcct gttccaaatc agactaaaga ttcatttggt aatttaaaaa 47100tcagaaaagc tttttttttc tctctctctc tcgttaatgg gttgtagggg ggtgaaagaa 47160ccgtctctgt ctcctctgga ggctcagtat ttgagtccaa gaaataaact tgacagtaga 47220cagattaaca ggagaaaagg catgtaaatg tatcgtatgc atatgcatag gggtcctaca 47280aagtatgaga ctcagataag ggccagacgg ctgaagctta aatagtactt ttagctacag 47340aaacaaatat gggcttaagg ctcctggagg gtgaggggaa gaactgtact gtgaacgaaa 47400attgtcttgc tctgcggata aagtctctca gatagtagcc ctcagaagaa taggtgaaaa 47460gtctgtatgg gcctggtgtc ttgggtgtgc ggacctctag tctcctctcc tgtgatagga 47520gttaatgtaa attccagaga tggggttcac aattccattc cttctggagg aacttccgtc 47580ttgataaggg aaacttcaga gaaagccctt tcttcacttg aggagagaag atgaagagac 47640agaggtacag gggaatgtca gaccttggtt ctgatgctgc ttttttagtt caaagaactc 47700atcatgtcaa agtgtcatac tttggggtat agtttcctga cccctaacat gagcaaataa 47760agagatgtaa ctgagttagc cacaaaacta tttttttctg ttggctctat ccaaaaatga 47820gagtggaaaa tgttctgtga acatatatct aaaagtatgg gtaaaacttg ttttacaaac 47880tgttggtcat tcagcaaaca tttaaacagc ttctatactt gtggaacata aataactaga 47940aaatacaagc tgcttatttt agcaaattgt ggctgtttgc attgctcagg ctgccagttg 48000tgccaattgt gctgaggtat gcttcaaaat aactttcttt cttattttta atgacttttt 48060aatgcaggta caatatcgat gttttaaaaa cataagtatt tggtgttttg ttttgttttg 48120ttttgttttt tgagatggag tttcgctctt gttgcccagg ctagagtgca atggcgcagt 48180ctcggctcac tgcaacctct gcctcccagg ttcaaacaat tctcttgcct cagcctccca 48240agtagctggg attacaggct cccgccacca gggcccagct aatttttgta tttttagtag 48300agacggggtt tcaccatgtt agccaggctg gtctcaaact cttgacctca ggtgatctgc 48360cctcctcagc ctcccaaagt gctaggatta caggcatgag ccaccacgcc tggcctaaaa 48420acataagtat tttaaacata atacactttt gaaattgaaa cctgaactga aatatataaa 48480atatactttc tgcatatttt aaagtagtta agccagaaaa catcttaggc tgtatttata 48540gagactggta atccatagtc aagtaatagt tttctgtact ctgccttgga aatactgatt 48600ccagaatatt gtagtagatt ttcattgcca cattttgaga tgaatattag caaaagggca 48660tatccaatga tggcaaaaat gagatggtaa actatcttgc ctgaattatg gttgaaggaa 48720ttggtggaaa aagacccagg ggaaattcta atagctttca agtatgtgat gagatagaga 48780aagtggaact gagggaatga acaagataat ttaggagtaa ctatgtttag ggataacttc 48840agaattactc cacaaaaagt tccctgtcag gcagtaagct cccagcaatg aaagtaccca 48900cttaaatcac ttctgagata ctttcctatt tcaagatttt aacttaaggg cttaaagaac 48960ttaggttgac actgtatagg actgtagttt gcagaggcag ggtttgggat aacttcttag 49020ccactgggca cttatgcggt tgataccagg taattctaat acattacagg gatcagcaaa 49080ctttttttct gcaagatgcc agatagtaat attttaggct ttgcaggcca tatggtctct 49140atagcatctg ctcaactctg ttgttgcagt gcaaaagtat tgttgacatt atgtaaacac 49200agtttgttac ctgtgttcca ataaaacttt atttacaaaa gcaagtggtg agttggattt 49260ggcctgtggg tcatgctttg acgacccttg ccttaactaa tgaaatctag aaatagagtt 49320ctaatgtaaa tatagaaagg ctttcaatct agggattaaa aatcgaactg tcaagatgtc 49380attgtcttct tggtctgaaa gagtacacat caagtgttca aaatagtgaa ttgcccaatg 49440aattaagaaa ccttccagta taaaagtcaa atgacaatgg agaatctgat actgtttttt 49500actctttatt tctgctactt aacgttgttc ttaaaggcgt cgttacttct gctgggtagt 49560actgtctact cagttggact gctgatgcag acccagtcca taccaacaag ttaacacagg 49620ttcaggcctt ctgagaacca aaagattttt aagtacttcc ttcctaaata cagtaacttt 49680taacagtaat tttaacacca gaagcaagaa gtaaacaaaa atctctgatg atcgaggcat 49740ctgttgtcat atattttgtg ctcctaaggt catacagcag agcctcttgt attatctttt 49800ggtctgttgg cctttagttt atagtttgta aaatggtggt cacatgtcaa tgctgactag 49860aagaaggaga tgctactgta atcaggcagg ttggccttcg ctgagaagtg accattgatt 49920gctagaaaac aaagaaatgg gaggcattca gcaacctggg gttgttggtt ttaggaccag 49980aaatgtaccc ccaagtctcc accaaatttc tcaataatcc tgaagagttt tgctgtttgt 50040gttttaatat aacacagtaa ttggtgcttg taatgaggac tataattcat caaaaaggta 50100aattgtgaat tgtgcacacg cacacacact cttctaagca aatgggactg ctctattttg 50160aacagtcttc aaattgaaat aacttatttc gatatgtaga aaattaggat aagcaaaacc 50220aatttattta ctcttaagag ttccagatag tagtgatttg tcagtatgca tatgaatctt 50280tagtcctggc attctgaact caaaacctca atagagtggt ttcttgaccc ttaaagtgtt 50340ttatttttag taaatacatt tttaattctg tgacctcaaa cctccaagct acattatgat 50400ttcataattt tatagtgatt tatcccccca ttgctttaaa tttaataatc ccatcctctt 50460tattggtatt taattttttc tgtagtctaa aggccctttt atttaaaatg ttatatattc 50520tgatttctta attttaggga tttttctcta gacaattgag aatatgtaat ttttacactt 50580taatgttcat ttgattataa cctacacttg aaaagcaatt tttaaagaaa tctgagagaa 50640tttaaaaatc attttaaaag catcatgtca tttttagctc aaaatacttc agagaattta 50700ttttatttta ttttttttgg agacagggtc ttgctctgtt gcccaggctg gaatgcagtg 50760gtgccatcac ggcttgctgc agccttgacc tcccaggcta aaatgatcct cccacctcag 50820cctcctgagt acctgggacc ataggcatgt gccaccacac ctagctaatt tgtttgtttt 50880tttgtagaga tggggtcttg agcccaggct ggtctcgaac tcctcagctc aagggatcct 50940gctgcctcag cctcccaaag tgctgggatt acaggtgtga gccacaaccc ccagcccaga 51000gtctcctttt tactaacggt gtacaacata aaattcaaac tctaacatgg ttttacatct 51060ggcccctacc taccttttgc ctctccttct ttgtcacata ctccacctgt tcccatgcac 51120tttagactca agtaatgaaa aacagcttgt attgctagga acaggccgca ttctttcatg 51180gtctgacttt atacacactc tcttgtggca acctagaaca cctgtctata ggctctctta 51240catcaggcca taccctttct gtgaaatttt taatctgtaa tattctactg aaactttacc 51300tctctgaagc cttccctaat ctcctcaggc ttttccattg ttcatattct gtcttgtaac 51360agttatcaca ttgttaaagt cgttccccag taaactatga gacccttaac tgtggaaggg 51420aaggcatctt tttaaccttt gtttttgtgc catacttagc ctagagcctg acattaagca 51480ggtacttctc aaaaaaaaaa aaaaaaaaca cagggtagag acagataaca agggattcct 51540gacacctacg ctgtagatag ctataacatt tcaataggaa tcttgggaat ctggggaact 51600ggaagcagct gggaaagcac cggcatctga gccactcagt actcttccag gcttgtgtgt 51660gagccttgcc accttcaggt attagcactt gaaatctaac ttctttatga agctccttat 51720ttacttgcct tctcggtgaa aaaaaaaaac aacaacaaaa accgtttcct tccccatctg 51780gtgccagcac tgtataattc ctaataagct tgaaaagata atgttggcaa tactttacaa 51840gtttattgct aatgtttaac atttattagg tgcttgctgt gtgctgatct ctgtgcttac 51900atatttttca tatatcactt catctatctt cacaacagtc cagtgaagta gatgccatta 51960ttatcccaat ttcacaaatg ggaaaaatga gattcattga gataaattaa actacccaag 52020gccatacaac tatagactaa gggggccaaa atttgaatct agtgtagatt caaattagag 52080ctcattctct tattcactac tggggaatat tgcctggcca taactgaagt ccaaataaca 52140gcatggttat aatatgggct ttaaaataat ctagacctgg attcaaaatc catttattgg 52200aggcaagtga cttcacttct gatcttcagt tttctcatat gtaaaatgat aatatctacc 52260tcaaaagtgt tactgcccac ccatgtacat ggtacatact aaaaaagcaa atgttagttc 52320catttgccac ttctacactt tatgtgaacc ctgggggtaa tggtttgtac agaataaggc 52380ctggcatgag aagacaaaaa ccatctgaca aataaaaaag ctgtcattct gtctaccaga 52440atgtcaacca gtgcctgcct tccaggatca acaagtgttt tattaccata atcacattta 52500ttctcttaat gaaaagggtg tgtcttgagt atttaatagt taattaccca taaatatact 52560acagtagaat aaggaataaa accagtgatc tgtctactat agtaactagg tttctttctt 52620ggtaatactt agggaacata tgagggttcc ctgtgggttg ttatttatta tataaaatag 52680ttgctctggt agttccataa tagagacctg tcagtgccat tcagcagaga aaaatcataa 52740acacagctgc tgtttttgtg tggcttgtgg ttttggtttt gttttcattt gaactttttt 52800atgcaggcat gtcatcattt aaaagtagtc atttggcgtt ttcccttgca ttcttcagat 52860tttttgtagg cttaaaaaaa tttaagtcac ttctataatt gttcatacag attgatccat 52920ctggtctttt tactagccaa tgaaattttt gacatgtagg aaaaaggaca attattttaa 52980attctataat ttcagtggat ctcttttata agtttgaaga aaaatagttt caactatcaa 53040aaaatatttt tcttatattc ttcattgttt cctgttaatt tctgtatttt cgtgggggct 53100aaaaaggcta ataatttctg ctattcctac tcttcacatc ctttgtgtgt tataactctg 53160aagcatacca taattatttg tttgcaacca agaattggta cagcataaat gtctaatcaa 53220attaggccct cttgctctct ttaagtcaca ctggctatga ggtggaggtg gattataggg 53280ggcaggcatc aattcaggga gaccatttag gcctttgcag tggtccagac aagaaatatt 53340gatggccggc ccaaggtact aagagtggta tttgaagaat gtagacagaa taaagagaga 53400attaacgaag cttgttgatg ggttagatgt tgattggtgt gagatggaga gaggagtcag 53460ggatggcagg tttcctctct tctttcttgt tgtgtttgtt caactcttgc ttatcttttt 53520tttttctttg ttttttgatg gagtctcgct ctgtcgccca gactggagtg cagtggagcg 53580atctcggctc actgcaacct ctgcctccca ggtttaagtg attctcctgc ttcagcctcc 53640tgagtaggtg ggattacagg tgcatgccac cacgcccagc taacttttgt atttttagta 53700gagacggggt ttcaccgtgt tggtcaggct ggtctcaaac tcctgacctc gtgatctgcc 53760cacctcagcc tcccaaaatg ctaggattac aggtgtgagc caccatgccc agccacttat 53820ctttaaagga ttaagtttat gtttcctact atgggaaacc atcccacccc aaacttgatg 53880accgcattat gtgcttttat agaacctggc acttctccag gatagcattt attctgtttt 53940gtaagtgtga atgtaattac cctacacaca gcatacacat aatcttcata ttctttgcct 54000tgtcttgtga aggcaagggc catgtctatc ttattcgtca ttagattccc acatccaaca 54060tagtcctggg gacagcacca atgcactttt ggtgcataag caaatagtgt catttatagc 54120tcttacctac aatatctgat agactaatca aatatagtag gttatctggg cctttttgat 54180tcatgtctct agcttaactt tcattttttt cttatttggt atctctcact ttgccttttg 54240atatactctt acagtttcgc tcactgagta aaagaaaata taaacagcaa gaagtaaact 54300tgtgttttat ggattttgat aacatcttct aaaagacccc ccaagattgt tgatgtctaa 54360aaaaattaag ggccttcaac tcataataat acttaatagt tcttaaaata ttacaaactg 54420attggaacat tgcactaaca gagagagtca tggatcttat ttatgtgtag attcatttca 54480gtcagaagta ttaggactgg tttatttgag aaagtaaata gatcattaca aattcatcgt 54540catacttcaa ggaaaattca ttgatgacac tttctaatat tgtgactata tacctaaatc 54600atactttaag aacagaatgt ctaagatatc acctagataa gaatcaatac aaaaagaaaa 54660aggaaaatta caaagattga tttaagaaag acaaaagact aagccataaa atttctcatt 54720attacaggtt gagggcaggg agtgaaaaag gaaaaggatt tgacttctct aggcatggtt 54780ttacttattt taaaataaga gcagtggacc catgatctga aagattgttt ctggcctaaa 54840ttctgttttt ccgtaagata gaagagaatg ataaaggtga tatgcaatgg gaattttggg 54900ttgaattgca acttactttt cattcttccc atagcctcat ctctgtcttg atgaaacctt 54960agagaatcca ctcaaatata gtgttttctt aaatgacttt attaaattta attaaataca 55020aatggttttt ctaatctctg ctgaaataaa ttctttgtag cctaatttag agcaacagtc 55080tagattagaa tacttaagat gaattattac tacttagcct aatcaagtca aactatggaa 55140aatgagtttt tacattttag cagtgttata aatgtattct tctgtagttg tgtctctttt 55200aacatatgtt ttccttcttt gatttaggtt tctgctttgg gacaaccata catctaattc 55260cttaaagtag ttttatatgt aaaacttgca aagaatcaga acaatgcctc cacgaccatc 55320atcaggtgaa ctgtggggca tccacttgat gcccccaaga atcctagtag aatgtttact 55380accaaatgga atgatagtga ctttagaatg cctccgtgag gctacattaa taaccataaa 55440gcatgaacta tttaaagaag caagaaaata ccccctccat caacttcttc aagatgaatc 55500ttcttacatt ttcgtaagtg ttactcaaga agcagaaagg gaagaatttt ttgatgaaac 55560aagacgactt tgtgaccttc ggctttttca acccttttta aaagtaattg aaccagtagg 55620caaccgtgaa gaaaagatcc tcaatcgaga aattggtatg atacaatatc ctattctaaa 55680atgcaaataa ccataaagct taactgttgt ccctttctaa aatatttctg tctaaaccaa 55740taccttcgta atcttaaata gctttctaaa taaaaatcat aaatctaaag tatgttttac 55800tatcgaacta tggaactatt tttaacacct tgatattatt ccataaggtt ttatttaaga 55860aatgtcattt gtgggatgac ttagatttgt tatatctcag tgttgttatt cttttaaaaa 55920tgattgatag gaatgtttgc tgcctttgct ctaaattgct gaatatatta ttttttatat 55980attaaaaata ttcaggactg tcaactttta atatatatgc attcatcaaa aatttgtttt 56040aacctagcgg tacttttttt actttattgt gatcttccaa atctacagag ttccctgttt 56100gcaaaaaaaa catgttcatg ctgtgtatgt aatagaatgt tatattcttt atgtaatttt 56160attaaaggtt ttgctatcgg catgccagtg tgtgaatttg atatggttaa agatccagaa 56220gtacaggact tccgaagaaa tattctgaac gtttgtaaag aagctgtgga tcttagggac 56280ctcaattcac ctcatagtag agcaatgtat gtctatcctc caaatgtaga atcttcacca 56340gaattgccaa agcacatata taataaatta gataaaggta agaaaatgac taatctactc 56400taatcattac tatagtgcag tcttctacct gtgtctatat ctttgtatag tctttttttt 56460tttttccagc tagatagtaa gcttcttgaa ggcagggact gcttatactg acaagatata 56520gttgagtgct aaatagaaat tatgtacaat caatatttat tggttgaatt tttgtgtgaa 56580tatgatactg atttcttggt aaattgtcta taaaacggaa gaagtatgag tttgaaattt 56640actatttttt aggattcaga attagagctg attatctttt cataaataaa aactataaat 56700agaaacattt tatgaaattt tgggaaaact ttatacattc ttgttattat atatcataaa 56760tacaccagaa aaaaataagt aattttctta agtattaaat gcagtaattc tgatcatggg 56820tgataatata tagggagatt tttgatattt aatatattag tttctagaat taagggaaca 56880agaaactaaa ctatattgtt ttcaaaatgc atgtcattgt atcagataaa tatctatatt 56940tttcagggta acagtaaaat tatcaatttg agttaactct cacacactat taaatatcaa 57000attctgttag tagaataagt taagacatct tattactatt cgtatttttc aaagtagttt 57060aatgcaacat aagatctttc aaaattcttt tctattctag catatatttt aatgctcttt 57120tcattttccc gaacattctt tgtgaaaaat tttcaacata tagacaactt gaaagaaatg 57180tactgtgaac aaccaaacct ctctaaatag tttgtcttcc cgttttcttc atctttctct 57240tttcttcttt tttggactcc tggcttaatc ttaaaaggag aaaattagac aatgcatttc 57300ctctcccctc gtaattttct tacctttcct ccctcacatt ttctgtcatt ctaattgaac 57360atctttaaac ctccattggt tctttcttta cttcccatag ttaccaaaaa cttccacctt 57420aggcactgtc aaacctttga gaccatcatg aatcactatt cataggggca gtacccatga 57480agtatgtcat acagtttaga atggaaatta gcttggtctc aggtacttgt gacccacata 57540tcacagtttt gtgtgcttgt cataaaacat gagatttggg aatgatctgg cagcccgctc 57600agatataaac attttctgtt tctacctgat atttacctag ttttaattgg gctgattaaa 57660aagcatttct gatatggata aagtaatgat agtgaatact tgttgaaatt tctcccttga 57720aaaatgaaag agagatggtg attgcatcta atgttttcct gttatagggc aaataatagt 57780ggtgatctgg gtaatagttt ctccaaataa tgacaagcag aagtatactc tgaaaatcaa 57840ccatgactgt gtaccagaac aagtaattgc tgaagcaatc aggaaaaaaa ctcgaagtat 57900gttgctatcc tctgaacaac taaaactctg tgttttagaa tatcagggca agtatatttt 57960aaaagtgtgt ggatgtgatg aatacttcct agaaaaatat cctctgagtc agtataaggt 58020gagtaacaag tttcaaaata ttaattttta atttaaaaag taatcacatt gaggatgagt 58080atctgtattt tttttttttt tttgagacgg aatctcactc tcgcccaggc tggagtgcag 58140tggcgcgatc tcggctcact gcaggctctg cctcctgggt tcatgccatt ctcctgcctc 58200agcctcccga gcagctggga ctacaggcgc ccgccaccac acctggctaa ttttttgtat 58260ttttagtaga gacaggtttt gcaccatgtt atctaggata gtcttgatct cctgacctcg 58320tgatccactc tccgtggctt cccaaagtgc tgggattaca gacgtgagcc accacgccca 58380gccaagtatc tatttttagg atatacttct tgataagtaa tattagtaaa tagcgtttgt 58440aagcttattc ttttaattct gtgattaatt cgagaggctg aaaatgttgg cagttactcc 58500agctcccaaa tatagatatt ccatggggtt gttgtttttg ttgttgtttg tttgtttttc 58560gagacagggt ctcgctttgt ctctcaggct ggaatgcagt ggtatgatca tggcttactg 58620cagcatcagc ctcccaggct caagcagtcc tctcacctca gcctcctaag tggctgggac 58680cacatgcgtg tgccaccatg cccagctaat ttttgtttgt ttgtttgttt gtttagagac 58740agagtctcat attgtccagg ttggtctcga attctgggca caaacaatcc tcctgccttg 58800gcctcccaca gtgctgggat tacaggcgtg agccattgcg cccagcctat ttcgtgattt 58860ttatcccctc caaccagtgg ctgtggaatg gaattgaaat agacatttca aaaaatccat 58920gacctactgt taagatatac tattgatact ttcctactct ctttttctgt ttattaaata 58980aatacctata agaagcaact acgtttgtgc caagcatatg tcgagtacta atttacataa 59040ccaagtaaaa cctgggcctg gccttcaggt tgcttatttt ctagtggggc ttatgggtaa 59100gaaaataatg tgtatatagt aacatttata ttaaaaatct cgtcttaact accatttcaa 59160aattcagacc agtatatttt aacatttttt gtaaatatcc ataatgttac tggtctccac 59220tatttgtggt tttttgtttt aattttagca aaactaatgt ttctcaggaa atgtttggac 59280aaaaaagcaa gtgaacagca gcctttggat gggacatcag tatgacttaa tagattgaat 59340gacagaggcg tcctaagtga tattactttt ctgttatcat atacaatgtt ttcataatga 59400gtatcctgtg atccttgttt tttttttttt ttttttgaga cggagtcttg ctctgtcgcc 59460caggctggag tgcagtggca ggatctcggc tcactgcaag ctccgcctcc cgggttcacg 59520ccattctcct gcctcagcct cccaagtagc tgggactaca ggcgcccgcc actacgcccg 59580gctaattttt tgtattttta gtagagacgg ggtttcaccg ttttagccag gatggtctcg 59640atctcctgac ctcgtgatcc gcccgcctcg gcctcccaaa gtgctgggat tacaggcgtg 59700agccaccacg cccggccgtg atccttgttt ttaattcctt tttttgcctc cagttaaggg 59760tagaactaca gtttcaaaag ttgaccttaa tttttttctt tcgtgcaatt tatattcaga 59820agtgtttgat tgatcttgtg cttcaacgta aatcctaaat gttagtattt taaatgttat 59880aggaactact agtaaatgtg gtctataatg tttaattttt tatcaccttt gcagattaat 59940atgtagtcat aatactctga catgttactt ttaaaatgaa aaaccttaca ggaaatggct 60000cgccccctta atctcttaca gtatataaga agctgtataa tgcttgggag gatgcccaat 60060ttgatgttga tggctaaaga aagcctttat tctcaactgc caatggactg ttttacaatg 60120ccatcttatt ccagacgcat ttccacagct acaccatata tgaatggaga aacatctaca 60180aaatcccttt gggttataaa tagtgcactc agaataaaaa

ttctttgtgc aacctacgtg 60240aatgtaaata ttcgagacat tgataaggta aagtcaaatg ctgatgctta ttattttata 60300gaaattattt tagataacct ttttcttgca ctatacagta atctgttgac ctgtagtatg 60360ttttcagatg gttaggagaa catccaaatc tccgaatgta aaaatatatc aagaatttta 60420cttgagcttc catctacctt agctattata cagctcacag tcctttgtta ataattctaa 60480tattcacaat tctagctctt aaaatcaaaa gttttacaga attcgtttgg cagaaagacc 60540tgggccaacc ttaagtgagg gtttttataa tctttattaa ccccacttag tataaaattc 60600cggtatctta ttaaagaaat attaatgtct ttatgaggta ctgcttcacc agctaaggaa 60660gtagtattta gtaagtacgt gtaccaattt agctttctaa aatatggaaa aactctgaat 60720tacatacctc ccttaagggg attgtgggcc tatatttatg ttttagtagt ctgatgtctc 60780cattgttatt agtggatgaa ggcagcaact aattttggtg aagactctac atcagtatta 60840acgtgttaca tatgtgaaaa aaaggagaac caagctatat ctgaacaaaa attccgtggt 60900tttatatttg agtctatcga gtgtgtgcat atgtgtatgt tgagtgtata cattagtata 60960tacctacttt tttcttttag atctatgttc gaacaggtat ctaccatgga ggagaaccct 61020tatgtgacaa tgtgaacact caaagagtac cttgttccaa tcccaggtaa ggaagtatat 61080agatttatat ttccaaaggt tatattagtg tttagcagta tgatccataa aagtagtata 61140tttttttaga ccagcctggg caacaaagca agaccccatt tctacaaaaa gtttttctta 61200aaaattagct aggcactggc caggcacggt ggctcatgcc tgtaatccca gcgctttggg 61260aggctgaggc gggtggatca cgaagtcgga gtttgagacc agtctggcca agatggagaa 61320accccatctc tactaaaaat aaaaaaacat tagccgggcg tggtggcggg cacctgtaat 61380cccagctact tgggagactg aggcaggaga atcgcttgaa cctggaaggc tgaggttgca 61440gtgagctgag attgtgccat tgcactccag cctgggcaac agagcgagac tccgtctcaa 61500aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaattagct aggcacagtg gcatgtgcct 61560gtagtcccag ccattaggga ggctgagaca ggagaatagg agaattgctt gatcctggaa 61620gttccaggct gcagtcagct atgatcgtgc cactgcattc cagcattagt gagacctgat 61680ctcaaaatat atatatattt tacatcatta gctttataca agctatatat taataaatta 61740ccagaaatgt acatatagac cagcaagact ttctgtcaaa tgagtatcag tcacaggatt 61800attttattga ttctgtctaa agttttatca gaatagtttc tgattgtctc ttttcattgt 61860atcaaagaga gaacagagta tggttataat tttatgctaa tcattcccca cattagatta 61920aaaacctagc attttattct tttcctgtct ccatcatcat tcacatgaag gagaagtaag 61980agttggcaag caactccata accaaactac cctgaggcag tttccttcaa atcactccca 62040agaatagata tttatggctt tcaaataaag ggtctttggt agcaaattaa tttgttagtg 62100ggagagggag ttatggggaa cattttaaat tattactgcc tggatgtgat gaagaaataa 62160aaagtagtat aatccaatag ctgactatcc tgcatttctt tgcttctctc tatacagtca 62220tttgggaata agagtacccc tgaacgtaat agcagtaata tttcaagaga actcaaattg 62280ttattggact ttatgataaa ataacaatag agctaactag tgactcctcc agaagtaaat 62340ctcataatga atttagactg agttctaaga cattcaagaa aaagttctgg tcttgttgct 62400aaagatggcc acccttttag agcctaggta aggttaaaag agatggtagc cttgtctgcc 62460acagggaagg aagatagttc ctcagcctac ctaaaccact gtaggtcaga aaacgtatca 62520ttcaggtcag aaatgagatt gtgacaacaa acaacttcag gatctttttt tttttttttt 62580tttttttgag ttgaagtctc accctgtggc ccaggctgga gtacagtcaa gtgtaagcga 62640ttctcctgcc tcagcctccc tagtagctgg gactacaggc acatgccact gcgcccagct 62700tattttttgt attttcagta gaggggggtt tcaccatgtt ggccaggctg gtcttgaact 62760tctgacctca ggtgatccca cccaccttgg cctcccaaag tgctgggatt acaggcctga 62820gccatcatgc ccggccagga tctttttaaa ttcttgctaa aacaagagtt tatatcccac 62880tcatactggt atgcctcaca ctgattagtg gggtggttgt agcaggaaaa gaaaaacaac 62940atttgaggaa ataatagcca gaatttttcc aaatttggta aaaactataa acccgtcaat 63000ccaaggagtt caacaagccc caagcaaaga ccccaaaata atactggata gaattatcct 63060agctagctgt ttcaagtacc acaagttaaa ctagctttat aaaagtaaaa ggctgggcgt 63120ggtggctcac gcctataatc ccaacacttt gggaagccaa ggcagatgga tcatttgagc 63180ccaggagttt gagaccaccc tgggctatat ggaaaaacac agtctctacc aaaaatacaa 63240aaaattagcc tgggatggtg gtgtgtgtac ctatagtccc agctactcag gagggtgagg 63300tgggaggatc acctgagccc agggaggttg agactgcagt gagccatgat cgtactactg 63360cagtccagtc tagggggcag agtgagaccc cgactcaaaa aaaaaaaaaa aagtaaaaga 63420taaacttatt taataatata tgattaaaat gttgaaatgt tagtaaaata gctatttaga 63480aaggactata tttgtcttta agcattctgt agtctaaaag gtgaagtatt tatggtagtg 63540tgaaaattta agtaaacttt ttaaactttt ctgtgcttac aaaaacaaaa atcagataaa 63600tgaatatgaa gttattaaaa agatagaaaa gtagatttta aaaattctta ctaaatattt 63660gtttcaaatc tgtgcttctt ttatgctaaa ttagatgctt tatgatttct ttgtaaatgt 63720tacaagtatc tcccaaaatc agagtcaatt tggactgtat acacacacac aaccattttc 63780tagaaataaa gaacttaatt gagcatgtgt atatgtgtgt gtgtctgtgt gttgtgggat 63840ggatggactt tagaagaaaa gagaaacatg ctttattttg ttagcatagg gctgaacaag 63900tagatttgta ggacatagct ctcttagagt atagaacact tcttattaat ttatgagcac 63960ttctaaagat ttaggaataa taaaaagtct ggtacattaa agtaatatat ctttaatttt 64020tcttttcttt tttttttttt tggaggcgga gtcttgctct gtcgcccagg ctggagtgca 64080tggcacaaac tcgactcact gcaacctctg cctcagcctc ctgagtagct ggagttacag 64140gcgcccacca ccacgcccag ctaatcttgg catttttagt agagatgggg ttttgccacg 64200ttggccagac tggcttcgaa ctcctgacct caggtgatcc gtccaccttg gcctcccaaa 64260gtgctgggat tacaggtgtg agccaccatg cccagcctaa ttttttttaa atcattgcgg 64320acttaagtcc cataatattt ttctctatat gtttctacaa ttatatgtgt tactgggcat 64380ggtagtatgc gcctgaaatc ccagcaactc aggaggctaa gggaggctga ggcaagagga 64440tcacttgagc ccaggagttc gaagctataa tgcactttgg tcatgcctgt gactagccac 64500tgcacttcag cctgggcaca tagcaatacc ctatctctaa aaaataataa taataataac 64560atttgttttt tcttttacct ttagaattct tgccacattt ccccatttta aatgtgtctt 64620gtttctataa atagaccata tttttaaaaa tctttatcca tacttgagca aaaatgctag 64680aataaatgga acttatttta ctgatgaagt ttattactta gttggctaat taaaaataga 64740attgttatac tattttattg ttgattaaga attattttat ttataatcaa gattagacca 64800gaatgataag acatttgaaa agtttttaaa attactatct aacatatgag cttctatcat 64860gggacaattg ctattctaat tcctttgctt gtgttatctc atttaatttt atgagatagt 64920tatcaacctt atgagggtag gtactattat tagtcccatt tcatagagga ggcaaaagac 64980aaagcaaggg tcacacactt agtgaaaaag gggaaataag attctactcc agtctgattt 65040ctgagcccat actctttatc cattgttata atgttttttt aaaatacaaa actgtttatt 65100gtatgtggtt ctttacaaat atgttcacca aagacagaca taatttcaca tgtagcaccc 65160agtaaattga gggaatttat cattaaaatt caaataattt tttatcttgt tcactttttg 65220tagcagtgtt ttattcttcc cttatgaatg tgaagtttca ttacctttta taaaataatt 65280attcttcaat tcaaatactt tatgtatcca tcattttatt tacctactga tttatttagt 65340ggattccttt ggataaaact atattatgta taaaagtaac taaatgtaat cataatagga 65400aatcttattt tacctttagt tttccttttg tttgactttt atgagcaaaa ttatatatat 65460aaatatatag aaataataaa tatatattta tattatgtat atagtatatt atataatata 65520tattataaat ataaataagt tttttagtga cgtatgtagc acacatgcat aaaagtatac 65580aaatcataat tgtatagttc agtcacaaag taaataccca tgtcttctgc ttttaacaaa 65640tgcacacatt tctgtggttt tattcctaca attggagctg ggtcacaggg aatacatatg 65700ctcactttta ttagattatg ctttccaaat tggttgtaac aaattatacc ccaatactct 65760tagtgtatga gatttcctat tactgcataa ccttaccacc ccttggcatc atcagtcttt 65820atagtttagc cattcttatg tatagtgata tctccatgag attttaattt tcattttctg 65880tatgactaat aatgactccc ttttcatgtt tattaactat ttgaatatga ccttataaag 65940tgccttttcc aatcaatctc tttcctgttt ttcgtttggt tgatctttgt cttcgtgatt 66000tgtaggagtc atttatatac tttgatgaag acttttcttg atgtattatt tttgctttaa 66060aattttacat aggtggaatg aatggctgaa ttatgatata tacattcctg atcttcctcg 66120tgctgctcga ctttgccttt ccatttgctc tgttaaaggc cgaaagggtg ctaaagaggt 66180aaagtatttc agaaggaaca attatgttta cctttaaaaa ctcctgatta taccgctgat 66240tgaatttttt cacaaattgg atgttatttt atatttaaga aaataataat aaacctattt 66300ttaaaatttt aataaatgta tcatggaaga ataccttggg agagcttcag gaatttatga 66360tgaatatgtt ttgagttctt attgatacca tttttaaaaa tgcaaagtga ctatataaca 66420gggattgcat gcaaatatct catgcttgct ttggttcata ttttctattt ataattaaaa 66480tacatgtaat ttcaaatggg gaaaaaggaa agaatgggct taaaccttga aaaatcaatt 66540tttttttttt agatattccc attattatag agatgattgt tgaattttcc ttttggggaa 66600gaaaagtgtt ttgaaatgtg ttttataatt tagactagtg aatatttttc tttgtttttt 66660aaggaacact gtccattggc atggggaaat ataaacttgt ttgattacac agacactcta 66720gtatctggaa aaatggcttt gaatctttgg ccagtacctc atggattaga agatttgctg 66780aaccctattg gtgttactgg atcaaatcca aataaagtaa ggtttttatt gtcataaatt 66840agatattttt tatggcagtc aaaccttctc tcttatgtat atataatagc ttttcttcca 66900tctcttagga aactccatgc ttagagttgg agtttgactg gttcagcagt gtggtaaagt 66960tcccagatat gtcagtgatt gaagagcatg ccaattggtc tgtatcccga gaagcaggat 67020ttagctattc ccacgcagga ctggtaaggc aaatcactga gtttattaag tatcaattat 67080aatctgtgga tttaggtaga tactttctct atggaaaagg atccatatat tttactggca 67140tagatactat gaactctagg accaatattg caatagaatt aagttctcta tatgctaata 67200tttttactgc acctctgctt ttttaaagcc ctagcttgcc ttcattcatt taacaaatat 67260ttattgaatg ccgactatgt gtaaagcact gggaaaggca ctaggtaata aagtcatgaa 67320ctaaacagac cttcattgag tatgcactct aactgtgaaa acagacatta agcagataaa 67380agcagaaata taagattaca gttgaccctt gaacaacatg gatatgaact acactggtct 67440acttatatgt ggattttttt caataaatat attggaaaat tttttggaga tttgtgacaa 67500tttgaaaaaa acttggagat aaactacatt gtccagaagt atcaaaaaaa ttgaagttag 67560gtatgtcatg aatccataaa atatgtatag atacaagtct attttatcat ttacgaccat 67620aaaatataca caaatctatt ataaaaagtt aaaatttatc aaaacataca cacacaaaca 67680cagaccatac atggtaccac tcatagtcaa gagaaatgca aacatatata aagatgcagt 67740attaaattgt atctgcataa aattaactgt agtacatact gtactactgt aataatttca 67800tagccacctc ctgttgctat tgtagtaagc tcaagcattg catatatcca cttaaaatgc 67860tatgtaggct gggtgcagtg gctcatgcct gtaatcccag cactttggga ggccgaagcg 67920ggtgaatcac ctgaggtcag agttcaaaac cagcctggca aacatggtga aatcccgtct 67980ctactaaaaa tacaaaaagc cgggtgtggt ggcatgtgcc tgtaatccca gctactcggg 68040aggctaagtt aggagaattg cttgaacccg ggaggcagag gttgcagtga gccgaggtca 68100cgccattgca ctccagcctg ggcaacagag caagactccg tctcaaaaaa caaaacaaaa 68160caaaatgcta tgtaatgctg atcatctcta catgagtact tcatcacttc agtaaattgt 68220ttattgcagt gaaaagtgat ctctcacagt tcttggatat ttttcatcat gtttagtgca 68280ataccgtgaa ccctgaataa cactatgagg gctgtaagga gtgcctctag tgatgctaga 68340ggtactctca agaagcagag aaacgtcatg acgttacaag caaaagttga attgcttgat 68400gtgtaccata tattgaggtc tgcagctgca gttgccctta tttcagacag acaactaatc 68460ttgtaaacag acaacataaa ctttgtttgt ttttgttttt ttgagatggc gttttgctct 68520tgttgctcag gctagagtgc aatggcacaa tcttggctca ctgcaacctc tgcctcccgg 68580gttcaagtga tcctcctgcc tcagcctcct gagtagctgg gattacaggc atgcgccacc 68640atgcccagct aatttttgta tttttaatag agacggggtg tctccatgtt ggtcaggctg 68700gtcttgaact cctgacttca tgtgatccac ccgcctcggc ctcccaaagt gctgggatta 68760caggcgtgag ccaccgcgcc cggccaaact tttttttttt aataagagat ggggtaggtg 68820gatcacacct gtaatcctag cattttggga ggccgaggcg ggtggatcac gaggtcagga 68880gttcaagacc agcccggcca atatggtgaa accccgtctc tactaaaaat accaaaatta 68940gccaggcgtt gttgtacatg cctgttagct actcaggagg ctgaggcagg agaatcactt 69000gaacctggga gatggaggtt gcagtgagcc gagattgcgc cgctgcactc cagcctgggc 69060aacagagtga gactccgtct caaaaaaaca aaaaacaaga gagagatggg gtctcactgt 69120gttgcctggg ctggtctcga actcctgagc tcaagtgatc ctccctcccc tacctcccaa 69180agtgctagga ttacaggtgt gagccatcat gcccggccaa aagacaaaat aaatgtatag 69240tatcagtaaa tacagtacag tactgtaaat gaattttctc ttccttatga ttttcttaat 69300aacattttat tttcactgtc ttactctatt gtaagaatat aatgtatagt acatatacca 69360agtaagtgtt aatcaactgt ttatatttct gttaagactt ccaatctaca gtagtcattt 69420gtagttaagt ttttggggag tcaaaattta cacatggatt ttcagttgta caggagatca 69480gttgccccta tcccctatat tgtccaaggg tcagctgtat aaatcacatt aagtgccagg 69540aaggaaaaaa caatagtgag ctaagaataa ctaactgagg atttgtgggg aaaaggcaaa 69600tatatatata ttgggttcag gaaagacctc tctgagatga taaccttgaa gttgagactg 69660aagtacagct atacctctga ggtattgtga gtggtctagt tctagaccac tgtactaaag 69720cagaaatcac aatagagtca caaatgtttt ggtttcccag tgcatataaa agttctgttc 69780acattatact gtcatctgta aagtgtgtaa tagtctgtca tctgtaaagt gtgtgatagc 69840cttacgtcta acacaacaat gtacatactt taaaaatatt gctcaaaaat gctgatgatc 69900atgcaaggct tcagcaagtt gtaatctttt tgctggtgga gggtcttgcc tcagtgttga 69960tggctgctga cttaccaagc tggtggttgc tgaagtaacg ttagcgtggc tacagcagtt 70020tctttaagac gacagtgaag tttgccacat tgattgaccc ttccttatgt gaaagatttc 70080tctgcagcat gtgatgctat ttgatagcat tttgcccaca atagaacttc tttcagattt 70140ggagtcagtc ctctcaaaac ctgacgttac agttaaccag ctgcattagc ccctaacaag 70200agagtcagcc tgaagctttg aagccaggca ttaacttctc ttctctagct gtgaaagtcc 70260tagatggcat cttcttccat tataagactg tttagtttac attgaaaatc tgttgcttag 70320tgtagccacc ttcatcagtt atcttggcta gatcttctgg ataacttgca gcagcttctc 70380catcagcact tgctgcttca cagcttcttt tttaaacctc ttataccaac ctctgctagc 70440ttcaaacttt tcctctgaag catcttcgtc tctctcaata ttcatagaat tgaaaagagt 70500tacggccttg ctctggatta ggctttgact taaggaaaag ttgtggctgg tttgatcttc 70560tatctagacc actaaaactt tctccatttc agcattaagg ctgttttgtt ttcttatact 70620tcattcactg aagtagcact tttaattttc ttcaggaact tttcctttgc attcacaact 70680tagctgtttg gcaccagagg cctagccttg gcctgtatgg cttttgacat gcatttctca 70740ctaaacttaa catttctagc ttttgatgtg aagcaagaga cgtggcactc ttcctgtcac 70800ttgaacactt agaggccatt gtagggttat taattggcct aatttcaata ttgttgtgtc 70860tcagggaata gggagtcctg agaggaggaa gaaagaggag caaaggttgg tcggtgaaga 70920agtcagaaca tgcaacactt atcgaataag ttcaccatct taattggctc atggcacccc 70980caactgcatc agagtaacat caaagatcac tgatctttga tcacagatca tcataatata 71040taataataat gaaaagcttg aaatattata agaattacca aaatgtgaca cagagacaaa 71100aaatgagcca tgcatttgga aaaatagtgc caggagactt cctttacata gggataccac 71160cacaaacctt cagtttgttg gaaatgcaat atctgtgagg tatgataaag caaagcacaa 71220taatacaggg tacacctgca caaatatatt gttagttgag cttggaagca gagaaaacag 71280caggtcttca ccatgttatc ccaggttccc caggaaagag cgtatgtaag atggaattta 71340aatattgatc tagttgtcta atcttggcca tagcttttga accacagtat aattatctag 71400gttcaagaac cattaactct ccctgatttc tcaagggcaa agatgtcaat gccacgagaa 71460gatgtttgtg ttcattggtg tttccaaata tattactttt tctttggctt tgttggctat 71520agataaacca gccaatgaat tttgggccaa gaagtccaaa acacccctat cccattaaca 71580gtaacagcag catcaagagg cagcctggta tattgcactt aggaaattta ttgattcata 71640gctgtgttct cagtaaggcg tttaccacta cttttcggaa aacaaattat cttttctgca 71700tatatatttt tctgcattta aaagatacga gtctcttttt tgagcagttc tctttgtaag 71760aattgatatt tgatgactgt attctttctt aaaagaaatt ttatttcaaa gaaaagaaat 71820ttgcttattt tatagcagaa taaaacttct atttttcagt tacaatttgt atctattgga 71880aacttttgta aatctaagat acgacatgga atcttagttt ttctgtatct taaatatttt 71940actatttcat catcctagaa attacttcat ccatattcat tatttccaac tatgctaaaa 72000aagactaaaa taatatgaaa atataaaaga aggatagaat agcctagaag gacaataaaa 72060tagtgaagta aatcatgatg ttacatcatc cttcagtttt cttattgcat tgcccagtgt 72120cttgcatcat ctttccagaa gtatagaaga agtctgcaga cactgtcttt gtaatttttt 72180tgtttggttg gttggtttct ttcaggcttt cattggacac ttgagaattc agaatttttc 72240acttttcacc cagaatgatg acagatgaaa actgaccaag gaagatattt cacaagcatt 72300agacaaatca taatatgaat gtagatagat agtagcagcc tagagcagag tttcccaacc 72360tcagcactat tgacattttg cgccaaataa ttctttgttg taggagccat cctgtgtgtg 72420caatccagga tgtttagcat ccctgtcctc taccctactg caggccagta gtgccatcct 72480gctaagttat ggctatcaaa aatgtctcca gacattagcc aaggttcctg ggtggtcaag 72540gggacacaaa atcaccctag ttgagaacca ctactctaaa ctctaatcat ggtgaagttt 72600attatataaa tgaaatgtca gatgacaaca cctaaactgt ctctcagtta tcttaagtct 72660tctcaaactc aggataatga tgagtaaaga atatatttct aacaacaaaa aggaaatttg 72720atagtatttc taaagacaaa aaggaaattt gtattcacat tcagttagtc attccaccag 72780aatgacttca tcacacaata ttttgtgaca agaacctgaa cagcctgttt tacagtattc 72840ttttcatctt ttattatatg caccaaaatt tttttttaaa ttttcttgaa cctctaaatc 72900tacgttaaaa atttacctga tacactttct aaatggacaa atgccgaagg tagctgtgta 72960tacaaatgtg actagaagga aaaagatgat atagaaataa aataactcct tgagttgatc 73020attctgattg gcatttatag agtagaaatg ttttgtaatt acagaggaaa aaagatggcc 73080tttccttcaa cagttatgag ccgtcagaat tttcaaaaat actgcatttt gacaatgtag 73140tttctagttt gacaatgata tatttatctt caaaaccagg aaaatgtaga taagaatttg 73200gttttataat atttaaattc ttattaaaat gtctaataaa attgttttcc ccatcacttt 73260attcttctgt aagttatttt atatttaaaa tgtaaacaaa taaaaataag taaataaaca 73320gtagcagctt cttttcctga taaatcgagg attgagtatg tattatctct ttcctggact 73380actggaataa cctctccctc cttccacaga gaagccataa taatctttat gaaatacaaa 73440tcaaatcatg gtattcattc tttaaatagc tatcaataaa aataaaatcc caactttata 73500ccctgttcgc aaattttacg tggtctgaat tcagcttaca tttcttcttt cccttgtcta 73560ttgcccatca ggctcactgg ctttattcct tcacaccaaa ctagttattt ccggggtggg 73620aggaaggctt gcagtgtttt ctccatctgc aatagtcttt cccaaatctt agtgtggata 73680aaggttcctt cttgttactt gaatcacaaa tactatgttc tcagtcattc tctgttacat 73740catccagagt acattatatc aattttccaa tatttttatt tatttgattt cccactataa 73800cagaggctct gttagtgcag ggtcttttac tattttgtaa tcccaacagc aagaacaaaa 73860caaggtacat agtacatatt taataaatac ttgttgaaca aatatgtgcc ggtaatattt 73920cttcatgctg ctgaataagt taacagcata taaacacata caaaccaagt ggcatggatg 73980tctgctttca tttttagcct tttaaaaata tatgtaaccc atcctaaggg gtttatattt 74040gttttgcata atacattaat atgtactcat tattcattac acagttaata tatctatatt 74100tgcagggaat atacattgct tggaattata caaaaaaata ttatttttcg ttttctaata 74160ttcaggatac agtgttttaa tgggggtgtt tcttcattct ttttttctta ctggttttta 74220ctttttaaat ttgaaagctt tgcagggatc ataaggatct gttcaggcaa agaacatgaa 74280agggtttaca tttttatcat tttagtgttt cttattctct atatcaaaaa cattcacaga 74340taagttaaca agatcctcat caggaggaaa agtaaattgt tcactaccat cctctagtat 74400cctaatctgg tcttgttgtt ggctaacttc agcagttact attctgtgac tggtgtaata 74460ttaaccaaat aaattactgg atttgttcta caaatattat gtcttagatt ggttctttcc 74520tgtctctgaa aataaagtct tgcaatgaaa ataaattatt ttacaacagt taattagcaa 74580tgtaaaattt attgaaaatg tatttgcttt ttctgtaaat catctgtgaa tccagagggg 74640aaaaatatga caaagaaagc tatataagat attattttat tttacagagt aacagactag 74700ctagagacaa tgaattaagg gaaaatgaca aagaacagct caaagcaatt tctacacgag 74760atcctctctc tgaaatcact gagcaggaga aagattttct atggagtcac aggtaagtgc 74820taaaatggag attctctgtt tctttttctt tattacagaa aaaataactg aatttggctg 74880atctcagcat gtttttacca tacctattgg aataaataaa gcagaattta catgattttt 74940aaactataaa cattgccttt ttaaaaacaa tggttgtaaa ttgatatttg tggaaaatca 75000tactacattg gtagttggca cattaaatgc tttttcttac tctgaattcc tgatatgact 75060ttctttagga ttgtttaaaa tattctagta gttttaggtc aatttagatg tgatttagtt 75120ggtctagata ttataatttt taggggttcc ctttcatttt tcttttttct tacgtttctt 75180caaatagtat aatgccttat tttcatttat gaagaaatta ccctgctgtt ggtgatacgg 75240gtatatttaa ataaaccagt tgcagtgcat ttctgcagaa

agtccattaa gacataaatt 75300ttgtccagta actacagtag aagtggtgac tctatgattc attcatgttg cataagtagg 75360tgaaaaatat gagctatatg aagagtggta taacatatat tcataatttt tcttaactgt 75420taactaaatg taagtactta taatccattt gcattttcct tttgtgttct ttgccattat 75480aactgtgcct aagtatatat gtaaatatat ttccaactat agtgttaaac actgatgtct 75540tttgaatttt aaaaaagcta gtaatgtaag aagtttggga cttcttaaga agattcatat 75600ggagaagtta gacatgtcaa ccttttgaac agcatgcaag aatgtttatg tttattttgt 75660ttctcccaca cagacactat tgtgtaacta tccccgaaat tctacccaaa ttgcttctgt 75720ctgttaaatg gaattctaga gatgaagtag cccaggtaaa tgtatgtttg agattactag 75780ataactgttg tacaaattgg tatgtcactt aaattgtttt ctctcagaaa gtccacataa 75840ataaatgaaa tagactaata gtaatatagt gtagaaaaaa acacccttaa cattatttcc 75900atagataaaa ctaattagaa ctgtaaattc taaggagatt atttatctaa actaatttta 75960aaatcagaag ttaaggcagt gttttagatg gctcattcac aactatcttt cccctttaaa 76020tatgatttat tgtctttctc atacacagat gtattgcttg gtaaaagatt ggcctccaat 76080caaacctgaa caggctatgg aacttctgga ctgtaattac ccagatccta tggttcgagg 76140ttttgctgtt cggtgcttgg aaaaatattt aacagatgac aaactttctc agtatttaat 76200tcagctagta caggtaaaat aatgtaaaat agtaaataat gtttaattac aataataatt 76260tattctagat ccatacaact tccttttaaa aaacctactg cactaactag ttttatgctt 76320aaaaaaaaaa attattacca gtaatatcca ctttctttct gaaaaaattt tctttagatc 76380ggccatgcag aaactgaccc tgatttgttt ttttggaatc acctaggtcc taaaatatga 76440acaatatttg gataacttgc ttgtgagatt tttactgaag aaagcattga ctaatcaaag 76500gattgggcac tttttctttt ggcatttaaa gtaagtctaa ttattttccc attaaattct 76560taaggtacat attacttgct ttcttaatag atttataaat atgtattact tatatacttt 76620tgtttatgtt tggctggaag agttttccat actaaaagta ttttgtacca gtgatgagct 76680tctcaacttt tgctctttga aatttaaaaa gcagtaaatt caaaactaaa ttttagtcat 76740gaatgagagc ttaaatattt ttaaagattt ttgttctact taagtcaaat tttctaggtc 76800cagatgaata ttgctgtagg tttcactgtg tgtatggatt acaatatccc caaacaaaga 76860aaaaaatgtt ttaccttgaa attcagaaca atgtcaaact cccgtggttc ttactgaaaa 76920acaagctaat taagaataaa aaatgttttg tagaatgtga tatatgtagt actcaaaagt 76980tacaggtcat aaaccatata acttttcata aatttagaag cagatttata tctaatatga 77040tattttaagt gttaaaattt aataatggaa cccagaagtt aagttgaaaa caagaagcat 77100aggcgtgtgt cagaagagtc aaacagcatt cactgagcgc tttgttccct ccctcttcat 77160ttgattattt ttgtgctcaa tttccttttt tcatgttttt atatcttgta ctgagattag 77220tcaatgaaaa ctagttgaaa taaacctaaa aactagatgt ttatttaatc acatattcag 77280gaactacctg aaactcatgg tggttttgtt tctaaattac aggttttgaa taattttatt 77340attagtatga ttgtaacatt tattggattt caaaaatgag tgtttaaatt gtttagcaaa 77400gattatttgt atactgattt aagactatat atatatattt ttaattttgc acgattcttt 77460tagatctgag atgcacaata aaacagttag ccagaggttt ggcctgcttt tggagtccta 77520ttgtcgtgca tgtgggatgt atttgaagca cctgaatagg caagtcgagg caatggaaaa 77580gctcattaac ttaactgaca ttctcaaaca ggagaagaag gatgaaacac aaaaggtgtg 77640tgactctagt ttgtgtttga gactcttttc actgcagtgg ggcagagttg tttagaagcc 77700cagtgtatat acagatcatg gtccttggaa tcaagcagat taggatttgg aaccaagttc 77760cactgcctct cagctgtgta gtgttagaca cgtcatgcag gctctcagga ctcattttct 77820ttgtctgtaa aatggaaata atacctgctt cataaggcca ttgtgagaat taaattacac 77880gagatatgca aagaacctat cacaatcctt ggaacataga aggtggccaa taaatgttag 77940atccctttac tttcccttcc tttctcttat tcaggtccct aagtatttac agtgattatt 78000tccttattct gtcatttatt gtctctcagt aatgaccctg aaaatgagtg gaaagaagtt 78060agtttttaca tttccaagtt taaaatggat ttcgagtcac tcagtaaata tatcacactc 78120tagtcatctg ctgtctagct tagtataact aagagtagga aatacaatgt aaactttttt 78180tttttttttt tttttttttt tttgagacag ggtctggctc ttttgcctgg cctggaatgc 78240agtggtgcaa tttcggctca ctgcagcctt gacctcctgg gttcaagcca tcctcccacc 78300tcagcctcct gagtagctag gactatagga gcatgccacc acttccagct aatttttgta 78360tttttagtag agacagtgtt tcgctttgtt gcccaggctg gtctccaatt cctgggctca 78420agcagtctgc ccacctcggc ctcccaaagt gctggaatta caggcatgag ccactgtgcc 78480tagcccaatg taaacttttt tatgaccttt tcctacaccc tattctttat tcaatccaat 78540ccaccacatc ctaaattcac cacctcttac aattaaaagg aagctcattc ctcacctcta 78600gtaaaaggaa aaaaaaaaga aaaatcgtat attagtgagt ccccaaagga cagagaatgt 78660aagttagcag tactgaatga atttttccag catcttcatt actagtactt gaaggaaaaa 78720aaaaaattaa aacaaagtta ttgtattaat gtaaatgtca tacagtgtga gttgatttta 78780ttgaagtgtg atatataaat tcaaatatag gatggtagaa aataattgta ctctttctga 78840tactggaatt tatgagcaag aagggtctag attgccctgg aagaacaaaa tttattgtat 78900ttatatattg taactgagct gagaaaaaag aaactaaatt ttagatacat ttgtaaatac 78960agatgttcct caacttataa cagggttata tcctgataaa cacatcataa gttgaaaata 79020tcgtacaggc aaaaatgggc attttgtaga tgtgatggga tgcgaaaaca caaaacacag 79080tatccaaaaa atgctggcca cacagtacgc tgtagactat tggtcgttta ccctcatgac 79140tgcatggctg actgggagct gcggctcgct gctgctaccc agcatctcaa gaaaatactg 79200tgttgcatat cactagccca ggaagagata aaagtttaaa atttgatgta tagtttctac 79260tgaatgcatg taggaagaga taaaagttca aaatttgaca tataatttct gaatgcatgt 79320tgctttcaca ccatcacaaa gttgaaaaat agtaagtcaa aaccattgta agttggagac 79380catctgtgta ggcatttctt ctaagctgga gtttttatat cacaaattgg ctatgataca 79440gtggattaat taggaacatg aaattcatta ctcagatcat tattggctta aatgagattt 79500tgatagggaa ctaagattgc agtcatagaa acaagagcag ttactttgag tggctttgtg 79560ttttgttgta acaatgagtg ataaaatata agaagcattg tgctaacatt agagttgtca 79620gtgatagaaa tgcctttaca tgaacaagag ctatctagtc agtgatgatt agattttcta 79680actattcatt tgtatataac aagaagaaaa aaacttattt aaattccagc atgaaaatta 79740atttattata attcctagct cctaaccact ttttccccca ctcacatttt atattttcaa 79800taggtgaaag tttcctaaaa taaaatccca gctattgcgt gataactcag cagtctgggc 79860tctgatttga attgattaaa aagggatatt gttttgtagc cacatactaa aacctgaata 79920tagtatgaga ctccatgtaa caaatgtttt tacaagtcct tttcccaaca atttctttta 79980gcctcactag accactttca cccagcaaag agagtatgaa tgctgtgttt tcttaaaagc 80040ctaaaagtaa ggttattaag ctaatgtaac atggtctcct tagtttttcc tctagaacac 80100agatcagcaa gttttcttaa agggccagat agtaactgtt ttagactttg caggcccatg 80160tggtctctgt cttaactatt caactctgct attctcgtaa aagcagccaa tatgtaatga 80220atgggtgtag ctatattcta atacaactca atttacaaaa actggcagct gacctgccgg 80280ttgactagcc ctgctctaca gccaaaagca ttgccactat caccaagcag atttcatctt 80340cttacaggaa ggggtacaca gtgctgccag tcttgcttct gtctctgagt gttgctgctc 80400tgtgttgtag aaaccctctt aattagaaag cagcagtcta ggatgtagat tcagagactg 80460tacagtactg aggttctcat gtgagaaaga gattagcagt tagttttatc ttttattaag 80520tcagtttctt actgtgacta tccttttttt ttaatcaggt acagatgaag tttttagttg 80580agcaaatgag gcgaccagat ttcatggatg ctctacaggg ctttctgtct cctctaaacc 80640ctgctcatca actaggaaac ctcaggtact ttcttggggg tttcattgat atatttaaat 80700aaataccttt tctggataaa atcttgagaa aagtaaaaat gtctgttata attagaatgt 80760tcaataattt atgcttctct ctctcattct cctaccctca aaataagagt agtatatctt 80820aagttcagta ctgcctttat tcagaatgag tttttactac ttaaataata cagtttaaaa 80880ccttctatgg ccagaatttc tgttaccata ggataagaaa tggaaatgta atatctgtaa 80940aactaatgat atatctctat atatttgttg gaaattcata tgcaattata taacttttaa 81000aacttttagt tttttttata ctctttagga atggattcct aaataaaaat tgaggtgaaa 81060gttgtaaatc tttgtaacac ttcaaaaagc tatattgtat ttatatttta aaataaattt 81120cagggtaaaa taataataaa gcaaaggtac ctagtaaagt ttttaactat tttaaaggct 81180tgaagagtgt cgaattatgt cctctgcaaa aaggccactg tggttgaatt gggagaaccc 81240agacatcatg tcagagttac tgtttcagaa caatgagatc atctttaaaa atggggatgg 81300taaggaagag tattaatgag cttatgatgc atgaatttag ctatcttttt atacacagga 81360tatttatgaa ccatgaaaac tactgaaagc catttaagga atatacacat gtgataaaat 81420atgtaatatt tatcagatgt cttgaccttt gaaatatgca tgtataatca atgaaaagaa 81480aagaagtact aggtttagat cagaagtcct gaaatcagtt ttttgttttt tctttttcct 81540gttccctgcc tccaaccccc ctcccgtgga cctgtgtaga gaagtatttt ttgttgttgt 81600tttggttttt tttttaatca gtttctaatt atcatttgct tagctgtgtg agtatccatt 81660cattccataa atattctatg tgctaggccc tggggtttta gcagtgaaga aaacagaatc 81720cctgccctct tggagcttac agcctaacag agaaaagagg gacattaaat gaataattac 81780aaaaataaaa ttgcaagcat gttgcaaagg aagagtgtag tctgctcagg aaccatataa 81840aagaaggtac ttatatggag gaaaaggcta tggggggagt gaaggaggat catggatggg 81900ttttcaatag gaaatgacat ttcagcagtg aattctgttg atcccaatat tttaatataa 81960acatgtgttc aaatttaagg attcagaaaa ataattacta taaatgcttt aattaaaaat 82020ttttgtgaga tgcatgctca cttatcctac attgttattc aaatatgttt atgcaaagac 82080tttaaaagtg gatttaagaa tcagaatatt aagagccatt tgtagtggtg catgcttgtg 82140atcccagttg ctctggagga ttacttgagc ccaggagttt tagaccagcc tgggcaactt 82200agcaagaccc catctcaaaa aattataaat acattaataa agctatgtaa aggctttgag 82260tggacatata cactcattta taaatttttt gaaaagaaat cagaatattg ctttcctgaa 82320gtttcttttg aagagtaaat atagctgtat ttgtttttca tttgaaaacc atgtgatggc 82380gtgatcccca aatttgcatc tgtggcatta aatggtgata catattattt gaatttcaga 82440tttacggcaa gatatgctaa cacttcaaat tattcgtatt atggaaaata tctggcaaaa 82500tcaaggtctt gatcttcggt aggtaaccag taaggcaacc tgtatgttga aagttatcct 82560gaaaaagtga actattaata attatagaag catatagagg catatgtcta aaaagaaatg 82620tatgcagtaa ttatcagtag ttgattacac tatagtactt tgacatatcc tcctcttact 82680tagaatagct aaattatatt cagctaagta aaaagagcta ataagctaat aaacactctg 82740ctactgcctc tggagtgtca ccattaggaa caagacaaag gagacacaaa agacccctgt 82800tttcattcag aaattaagag tttaaaagaa agtttcataa tgggaatttt ctaattctaa 82860aactgtataa atcctcaaat attggaatca gttttgcagc aaaattatgc aaccagctat 82920cttagagttt ttaccagtct gttggtttct acccaacttt ccacaatatc agtattatca 82980ccaagttttc tgtcatgctt catcttctcc tagtcattgt tgcttctaaa atgttcttcc 83040ttacctgact tgtctttttg caaatccata gtcattcagg caaagcccac tctgtccaaa 83100gtattttggc ctataagtgg acatagagtc aagtctagct gtaacaacag ggtacattta 83160gtaggaattg caggtaacac cagacaaggg cttatttgta tgcctcttat aaattttaag 83220agtagctagt atacaaaaat gtgtcagtga caaatagttg taccctttta ttgcaaagaa 83280gaaagtggac atttcaggtg acctgctaag gcacaaaatt gtcatcaact tcaaatttgt 83340aactcctgat acgtagctta acccctgctg ctttccaaaa atggttaatg ttatatgaat 83400caaatttcat tttttggtat aatttcctgt gtcatgagag atccacatgc cactactacc 83460atgtccccca aaggaataga ctgtaaaaaa gattatagag ctaagaaaga gtaaaggaga 83520taaccgtgta acgctggtta attacattgt ttaattataa agtaatgact ttgcaaaaaa 83580gcatctgaag ttaagatgat tagaaatatt aatggttttt tagtcttgta ttttctgaat 83640attttttaac ttaagtaaat ataagtaaaa attttatttc aagtttaact ttagattaaa 83700attatttaag taaaatcata acagcatttt tatttatact tggcaaaact ataattcaga 83760atcgggttta ttttaaccct gcttcttttt tttaataaca agacttagaa gacaattaaa 83820ttctgtaaac tgtgagcatg ttcaacagtg ataaaaacat aaaataagaa agaatgagca 83880ggagtacaac cttaagatat ttccctattt acgagggcag gtagaagaaa aggttgtgta 83940agagaagtca tcaaaggaag ttggagtact agaggaagtg attgacaatt tgtagggcca 84000gggcttcaag ggggttaaaa gaataaggcc tgtgaaaaag cacttggatt tagtgactca 84060tcattagttt taagagttta ctcactagag tggtaggtgt agaagctagg ctacaaggaa 84120ttaagtaaga aaatgaaaat aagtattgac cgtattagga atttaagaaa taaaggaaag 84180gtggaaaata tggagagtag cttacgagaa gaacagagca aggaaaacca aactttggat 84240acttacgtgt caatctgaat gttaggtgct ttttttcatt gtctctttaa atcctcacat 84300ctgcaccctt gactaagtgt cctatttaat tttctatgtg aataaatgag aggtacccta 84360aaaagcattt gtgccacatc ccaagtatta tggttgtatc aaagggaaac atttgtggga 84420ttatttgagt tgtgagctga actagctgct gtttttatgg aacacgattt ttacttaaat 84480aactagtagg caagctatgg ttattcagac atggatattt ggcagacatt ttcttgaaaa 84540tgaacaatat gtgcctgtct ctttgaagaa aacaactgtg acagtatttg ttgccaatga 84600taaaattcaa acttgcaaca aaaatttcat tagaaattca aaattagaat tttgaaaatt 84660ctaattttca ttatctgccg ctgaacctaa tagagtccct aaacttaaca tctttctgat 84720gagattcttg gtaatattaa caattgtggt tttttagtat ggtataatga aaagtatcaa 84780catttggaac atctacaaat gaccatgcag gatactataa aatcatgtat gggtaagaga 84840tccattaaaa ggcaagaaag acaaattgat tttaatgtaa ctgagtatga aaagtttatt 84900gatacagttt tagattacat tttgcaactt aagaaactta tcttgtcagg ttttagaata 84960gtaccaaaga aaaatatcac aattatctga aaagtctatt ataatactcc ttctttttct 85020acctacctat ctttgtgagg ccagattttc tttttatact ttaattaaaa cattgcatta 85080gattgcatac aaaagcatat atgagaatcc agctgggtac tatgaagcca gacatttaaa 85140aattttttta aatgtgaaag aatttcactc ttctcactaa attttttaaa tgcatttttt 85200gtttcatgaa atatgttact tatattaaca tgtcatagtt tgttgctttt aaatgaatta 85260gtaaatattt aaaattttta ttttccctat ttcaatttct aatatcatta gatgtaattc 85320acataactaa aagctctttg gagtcctcag taatttctac aagtgtaaag gagtcctgag 85380accaaaattg agaacttctg ccctacagtt ttatttcgat gtcaattaaa catctttaaa 85440ttaacacata tttttaaaat cataaatgtt tgtaaaattt taattgccac aataattttc 85500ttaattcttt tctgataaaa atacagtgct ttctcagttg aaggttataa taaatgatga 85560caggtaattt tgaggattat tccaggagta tgtttatcac accataaaaa agaaaattaa 85620aattggggaa aggcagtaaa ggtcatgcat gacaaattta ctaataaaat actcatgttt 85680tagcctgtta aaacatttgc tattttaaaa ttccatcatt taattgtaaa cgtgttactc 85740ctctttcaga atgttacctt atggttgtct gtcaatcggt gactgtgtgg gacttattga 85800ggtggtgcga aattctcaca ctattatgca aattcagtgc aaaggcggct tgaaaggtgc 85860actgcagttc aacagccaca cactacatca gtggctcaaa gacaagaaca aaggagaaat 85920gtgagttgta ttattctttc ttcctatgtt aatctaagtt tttgttagat gagtctgtcg 85980gtgtttgtgt attcctctga gttagaacag agaaaacaat tgtactttct atggaaaaaa 86040atatgctcaa cctttgaaat atttgatgtt aatggattta aatgattata attactttta 86100atttggtaaa atcttaaaca ttcatcttat gtattatcta aaatgtattg ttattgctta 86160ttctttttaa aacaaatgaa tattgcacat tcaaaatttt atttctaatt cattgttaaa 86220atgattagaa aaaaataatt ttaatgacat gctaagtatt ttttcacatg aagaattatg 86280ctttggtcag ggaacatctg gaaatttcct tagaaaccca tgaaaacttc acaatctcaa 86340aatctttgga cataatttcc ttattcgttg tcagtgattg ttttcattgt ttaaatggaa 86400acttgcaccc tgttttcttt tctcaagttg gcctgaatca ctatatttcc atactactca 86460tgaggtgttt attctttgta gatatgatgc agccattgac ctgtttacac gttcatgtgc 86520tggatactgt gtagctacct tcattttggg aattggagat cgtcacaata gtaacatcat 86580ggtgaaagac gatggacaag taatggtttt ctctgtttaa aatgttttgg tgttcttaat 86640ttattcaaga cattttgtat ctgcatatat caaactataa cataatttct tatttttgaa 86700agctgtttca tatagatttt ggacactttt tggatcacaa gaagaaaaaa tttggttata 86760aacgagaacg tgtgccattt gttttgacac aggatttctt aatagtgatt agtaaaggag 86820cccaagaatg cacaaagaca agagaatttg agaggtgagc tcgagcaatt aaaaacacaa 86880aataaagagt tctggctgct ctattagaaa caatcaatat ttttcaagca atttcaaaat 86940aataaatgtt ggctgggtgt ggtggttcat gcctgtaatc ccaactcttt gggaggccga 87000ggctggagga tcacttgagc tcaggaattg aaaccagcct gggtaacata gagagaattc 87060atgtctacaa aaaaatttaa aaagtagcca ggcgtggtgg catgcactgt agtcccagct 87120actcaggagg ctgagatggg aggatcactt gagcccagga ggttgaggtt gcagtgagcc 87180attatcacgc cactgtactc caacctgggc aacagagtga gaccctgtat caaaataaat 87240aaaatgaaat aatagaagtt cttttaccac ttcagcaaaa actatttttt gtttgttttt 87300tattctatct tatttcaata gcttttgggg tacacgtggt ttttgattac atggatgaat 87360taaatagtgg taaagtctga gattttagcg cacctgtaac ccaagtagtg tgctttgccc 87420ccagtatata cttttttatc cctcactgtt cttcccaccg tctcccatct gagtctctgt 87480agtccattat atcactctgt atgcctttgt gtacctatag cttagctccc acttacaagt 87540gagaacatac tgtattttca ggttttccat tcctggcctc cagctccatc ccagttgctg 87600caaaagacat tatttcattc ttttctgtgg tggagtagta ttctgtggtg tatatgtaca 87660ctttctttat ccactcattg gtcagtgggt acttaacgtt ggtttcatat ctttgcaatt 87720gtgaattgtg ctgcagtaaa catacgatat gcatgcagat gcctttttga tataatgact 87780tcttttcctc tgggtagagg gattcctgga ccaaatggta gatctacttt tagttcttta 87840agaaatctcc atactgtttt ccatagaggc tgtactaatt tacattccca ccaggagtgt 87900ataagcattc tcatttcacc acatccatgt caacatctgt tgttttttga ctttttaatt 87960atggccattc ttgcaggagt aaggtggtat ctcattgtgg ttttaatttg catttccctg 88020atgattaatg atgttgagca ttttttcatg tttgttgacc atttatatat cttcttttga 88080gaattgtcta ttcatgtcct tagcccactt tttttttttt tttttttttt ttttttgaga 88140tggagtctca ctcttgttgc ccaggctgga gtacaatggc atgatctcag ctcaccgcaa 88200cctccacctc ctgggttcaa agcaattctc ctgcctcagc ctcccaagta gctgggacta 88260caggcacatg ccaccacaca cggctaattt ttgaattttt agtagagacg gggtttcacc 88320atgttggtca ggctggtctt gaattcctga cctcaggtga tccacccgcc tcagcctccc 88380taagtgctgg gattacaggc ttgagccact gcatcctgct ttaacccact ttttgatggg 88440attctttgtt tcttgctgat ttgagttcct tatagattct ggatattagt cctttgtcag 88500atgcatagtt tgggaatatt ttctcccatt ctacgggttg tctgttaact ttgatgattc 88560tttcttttgt tatgcagagg ctttttagtt taattaggtc ccatttattt tgtttgtttt 88620tgtcgcattt gcttttgggg tcttagtcat ttattatttg cctaggccag tgtttaaaag 88680agtttttcct aagttatctt ctagaatttt tatggtttca ggtcttagat ttaagtcttc 88740gatccatctt gagttgattt ttgtataagg tgagagacag ggatccagtt tcattctttt 88800acatgtggct agctagtttt cccaccacca tttattaaac agaatgtcct ttctccaatt 88860tatgttttta tatgtctaag atcagttcgt tgtaagtttt tggctttatt tctaggttct 88920ccattctgtt ccattggtct gtgtgtctgc ttttgtgtgt atctgctgtt ttggtaatta 88980tagtttcctg gtataatttg aagtccagta atgtgatgcc tccagatttg ttctttttgc 89040ttagtcttgc tttggctatt cagactcttt ttggtcggtt ttatatgaat tttaggattt 89100ttttttctaa ttctgtgaaa aatgatggtg acagtgtttt gtagttttcc ttgaaaagat 89160ctttcacccc cttggttaag tacattccta ggtatttttt tgaagctaaa agggcttgtg 89220ttcttaattt gattctcagc ttggttgttg ttggtgtata gcaatgctgc tgatttgtgt 89280acactgattt tgtaacctga gactttactg aattcattta tcaaatctag gagtcttttt 89340ttttatcttt tttaagacag agtcttgctc tgtcccccag gctagagcgt agtggtgcga 89400tctcagctca ctgcaacctc cacctcctgg gtttaagcaa ttctcctgcc tcagctttcc 89460aagtagctgg gattacaggc atgcaccacc atgctcagct aatttttata ttttttagta 89520gagatggggt ttcactttgt tggccaggct ggtcttgaac tcccaatctc gggtgatcct 89580cctgcctcgg cttcccaaag tgctgggatt atagaaatga gccaccacac ctggctgaaa 89640tctaggaatt ttttggagaa ctcttgggtt ttctaggtat atgattatgt cattggtaaa 89700cagctatagt ttgacttcct cttttccaat ttggatgcca atgtgggctt ccttgtcttg 89760ttctagttct cagggggagt gctttcagct tttccccatt cagtatgata ttggccgtgg 89820gtttgtcaca tgacttttat tattttgagg taagtccctt ctatgcctag tttgttgagt 89880gaaaaactat tttaattttt tttttttgta tttatttgtg tgatgctgtg aaggaaaatg 89940gaaagggaat acaatttaat ttgttgagct aattaaggcc taaaaagaaa gtaatcctta 90000aacttcataa cacattaaag gtttttattt actgagcttt aaatagttgg actccacctc 90060tatattgaca aataatgtat agtgcttaat acgacatttt ttggtcatac actttgagga 90120aagtcagtca accataatca ccttgtttat tcataacttt tttaccacct tatggtatct 90180cattagacta tatcagactt taaagtactt tttacaatct tctataaaat tctgctttgt 90240ctacaacaca gtcctgactc tagcttaagc acaaaaggat ttagtatgaa ccaattcata 90300catttaatac ttattaaact cctgacatgc caggcattgt

tgtaggtgct ggtaataaag 90360cagtttttaa aaagtcctta ttctcttgaa gtttacattc tagtggggta aagggaatca 90420aaagatgttg gtaagagaag tgagagagga atgctatttt tttatagctt tgtctacgaa 90480agcctctcta attttgtgac atttgagcaa agacctgaag gtattaacat catttgctcc 90540aaactgacca aactgttctt attacttata ggtttcagga gatgtgttac aaggcttatc 90600tagctattcg acagcatgcc aatctcttca taaatctttt ctcaatgatg cttggctctg 90660gaatgccaga actacaatct tttgatgaca ttgcatacat tcgaaagacc ctagccttag 90720ataaaactga gcaagaggct ttggagtatt tcatgaaaca aatgaatgat gcacatcatg 90780gtggctggac aacaaaaatg gattggatct tccacacaat taaacagcat gcattgaact 90840gaaaagataa ctgagaaaat gaaagctcac tctggattcc acactgcact gttaataact 90900ctcagcaggc aaagaccgat tgcataggaa ttgcacaatc catgaacagc attagaattt 90960acagcaagaa cagaaataaa atactatata atttaaataa tgtaaacgca aacagggttt 91020gatagcactt aaactagttc atttcaaaat taagctttag aataatgcgc aatttcatgt 91080tatgccttaa gtccaaaaag gtaaactttg aagattgttt gtatcttttt ttaaaaaaca 91140aaacaaaaca aaaatcccca aaatatatag aaatgatgga gaaggaaaaa gtgatggttt 91200tttttgtctt gcaaatgttc tatgttttga aatgtggaca caacaaaggc tgttattgca 91260ttaggtgtaa gtaaactgga gtttatgtta aattacattg attggaaaag aatgaaaatt 91320tcttattttt ccattgctgt tcaatttata gtttgaagtg ggtttttgac tgcttgttta 91380atgaagaaaa atgcttgggg tggaagggac tcttgagatt tcaccagaga ctttttcttt 91440ttaataaatc aaaccttttg atgatttgag gttttatctg cagttttgga agcagtcaca 91500aatgagacct gttataaggt ggtatttttt tttttcttct ggacagtatt taaaggatct 91560tattcttatt tcccagggaa attctgggct cccacaaagt aaaaaaaaaa aaaaatcata 91620gaaaaagaat gagcaggaat agttcttatt ccagaattgt acagtattca ccttaagttg 91680attttttttc tccttctgca attgaactga atacattttt catgcatgtt ttccagaaaa 91740tagaagtatt aatgttatta aaaagattat tttttttatt aaaggctatt tatattatag 91800aaactatcat taatatatat tctttattta catgatctgt cccatagtca tgcattgttt 91860tgcaccccaa attttttatt gttcatagca gcatggtcag ctttcttctt gatctataga 91920tgaggctcag gcactatccc atttatacca ataaccagtg tataactact taaggaaaac 91980ataaaaactt catcttcttt ccttttattt cttatgtgaa tctcccgtct tccattctct 92040tttataattg agaatgtctc aatcatatga aattagttac cagaattaac acaatttaga 92100ctatcttcct gattccttaa acccctttac tgaagtatac tcatgaataa tactttaaaa 92160tatgggggaa tagaaaccat gaacttttta cctttttaaa ctatttatcc atatctccaa 92220agtagaacat taaaccattt taagatatgt ctcattccca agtagtcaga gctcactctc 92280caactttatt aaatactatt tgagcacagg acacattctt aaacattttg aaaaacatta 92340acccaagatg tagaggctac tgctagtcgt cattctagaa tctgatattt tactctgtat 92400ttgaaatgaa tgattaatgt cctaggaaat tagctttagc agatgtccag gtgccacatc 92460aaaaaagtgc aataattatt gacagttttt tagattaggc atattattgg aaaacaactt 92520tataaagagt gaacattgta tactctagta aaacagcatc actttaaaaa tattcattta 92580tgaaatctgt tacctatagt tgaagtcttg agtagtgaac aagggactct aataccaata 92640ctcttaatat ctggctattt tagatccctt aaagggcata attattggaa atttaggtat 92700ttcactaaag catgtatata atattgccaa caagaaaagt aaatttgaag attaagggaa 92760cttacttctg caaactgtct tgcgatagtt aagcagaatt taaactctgt tttaagcagg 92820aaaccagaaa gattattttg cagttgtaga agatttcata acttattaaa acttattaac 92880attttgtgtt gtttagatat aggcagttga tacatactaa catcccagcc ttttcaatat 92940cagggttaaa ttataggaaa actcagtaaa atggtacaaa tctgaaagtt tgatggtaga 93000aactgaagat ttaacagaga actgtgtttt acccgagtgc caaaaatgct gtgagcctcc 93060ttgcacaaaa tttataccac ttttgcattt ttatctatca gtccagatag ttgtctcccc 93120tccttctccc aggacctctc caccattaaa atgcacaaac cacatggccg atttcaccat 93180ttacatttat 9319021068PRTHomo sapiens 2Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met 1 5 10 15 Pro Pro Arg Ile Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25 30 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu Ile Thr Ile Lys His Glu 35 40 45 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60 Glu Ser Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu Arg Glu 65 70 75 80 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln 85 90 95 Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys Ile 100 105 110 Leu Asn Arg Glu Ile Gly Phe Ala Ile Gly Met Pro Val Cys Glu Phe 115 120 125 Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe Arg Arg Asn Ile Leu 130 135 140 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 Leu Pro Lys His Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val 180 185 190 Val Ile Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200 205 Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala 210 215 220 Ile Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln Leu Lys 225 230 235 240 Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile Leu Lys Val Cys Gly 245 250 255 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gln Tyr Lys Tyr 260 265 270 Ile Arg Ser Cys Ile Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 Ala Lys Glu Ser Leu Tyr Ser Gln Leu Pro Met Asp Cys Phe Thr Met 290 295 300 Pro Ser Tyr Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser Ala Leu Arg Ile 325 330 335 Lys Ile Leu Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp Ile Asp 340 345 350 Lys Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 Asp Asn Val Asn Thr Gln Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 Glu Trp Leu Asn Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 Arg Leu Cys Leu Ser Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 Glu Glu His Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr 420 425 430 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser 450 455 460 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val Ile Glu Glu His Ala 485 490 495 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 Glu Gln Leu Lys Ala Ile Ser Thr Arg Asp Pro Leu Ser Glu Ile Thr 530 535 540 Glu Gln Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 Arg Asp Glu Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 Ile Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 Asp Asp Lys Leu Ser Gln Tyr Leu Ile Gln Leu Val Gln Val Leu Lys 625 630 635 640 Tyr Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 Ala Leu Thr Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 Ser Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680 685 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 Gln Val Glu Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile Leu Lys 705 710 715 720 Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln Met Lys Phe Leu Val 725 730 735 Glu Gln Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gln Gly Phe Leu 740 745 750 Ser Pro Leu Asn Pro Ala His Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 Cys Arg Ile Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 Asn Pro Asp Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile Ile 785 790 795 800 Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile 805 810 815 Ile Arg Ile Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp Leu Arg 820 825 830 Met Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys Val Gly Leu Ile 835 840 845 Glu Val Val Arg Asn Ser His Thr Ile Met Gln Ile Gln Cys Lys Gly 850 855 860 Gly Leu Lys Gly Ala Leu Gln Phe Asn Ser His Thr Leu His Gln Trp 865 870 875 880 Leu Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890 895 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly 900 905 910 Ile Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp Gly Gln 915 920 925 Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln Asp Phe 945 950 955 960 Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Cys Thr Lys Thr Arg Glu 965 970 975 Phe Glu Arg Phe Gln Glu Met Cys Tyr Lys Ala Tyr Leu Ala Ile Arg 980 985 990 Gln His Ala Asn Leu Phe Ile Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 Gly Met Pro Glu Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg 1010 1015 1020 Lys Thr Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030 1035 Phe Met Lys Gln Met Asn Asp Ala His His Gly Gly Trp Thr Thr 1040 1045 1050 Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln His Ala Leu Asn 1055 1060 1065 3125DNAHomo sapiens 3agtaacagac tagctagaga caatgaatta agggaaaatg acaaagaaca gctcaaagca 60atttctacac gagatcctct ctctgaaatc actgagcagg agaaagattt tctatggagt 120cacag 125442PRTHomo sapiens 4Glu His Ala Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser 1 5 10 15 His Ala Gly Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu 20 25 30 Asn Asp Lys Glu Gln Leu Lys Ala Ile Ser 35 40 5107PRTMus musculus 5Asp Thr Val Met Thr Gln Ser His Lys Ile Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 6119PRTMus musculus 6Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys Ala Ser Leu Thr Val Asp Arg Ser Ser Arg Ile Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 7107PRTArtificial SequenceDescription of Artificial Sequence Synthetic humanized 574 variable light chain polypeptide 7Asp Ile 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 Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 8119PRTArtificial SequenceDescription of Artificial Sequence Synthetic humanized 574 variable heavy chain polypeptide 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 9107PRTArtificial SequenceDescription of Artificial Sequence Synthetic consensus framework hum kappa1 variable light chain polypeptide 9Asp Ile 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 Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu 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 Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 10119PRTArtificial SequenceDescription of Artificial Sequence Synthetic consensus framework hum kappa1 variable heavy chain polypeptide 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70

75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 11214PRTArtificial SequenceDescription of Artificial Sequence Synthetic pertuzumab light chain polypeptide 11Asp Ile 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 Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 12448PRTArtificial SequenceDescription of Artificial Sequence Synthetic pertuzumab heavy chain polypeptide 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 13214PRTArtificial SequenceDescription of Artificial Sequence Synthetic trastuzumab light chain polypeptide 13Asp Ile 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 Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 14449PRTArtificial SequenceDescription of Artificial Sequence Synthetic trastuzumab heavy chain polypeptide 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly 15217PRTArtificial SequenceDescription of Artificial Sequence Synthetic variant pertuzumab light chain polypeptide 15Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 1 5 10 15 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val 20 25 30 Ser Ile Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 65 70 75 80 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile 85 90 95 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 16449PRTArtificial SequenceDescription of Artificial Sequence Synthetic variant pertuzumab heavy chain polypeptide 16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 17125DNAHomo sapiens 17agtaacagac tagctagaga caatgaatta agggaaaatg acaaagaaca gctcaaagca 60atttctacac gagatcctct ctctaaaatc actgagcagg agaaagattt tctatggagt 120cacag 125181068PRTHomo sapiens 18Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met 1 5 10 15 Pro Pro Arg Ile Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25 30 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu Ile Thr Ile Lys His Glu 35 40 45 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60 Glu Ser Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu Arg Glu 65 70 75 80 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln 85 90 95 Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys Ile 100 105 110 Leu Asn Arg Glu Ile Gly Phe Ala Ile Gly Met Pro Val Cys Glu Phe 115 120 125 Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe Arg Arg Asn Ile Leu 130 135

140 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 Leu Pro Lys His Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val 180 185 190 Val Ile Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200 205 Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala 210 215 220 Ile Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln Leu Lys 225 230 235 240 Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile Leu Lys Val Cys Gly 245 250 255 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gln Tyr Lys Tyr 260 265 270 Ile Arg Ser Cys Ile Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 Ala Lys Glu Ser Leu Tyr Ser Gln Leu Pro Met Asp Cys Phe Thr Met 290 295 300 Pro Ser Tyr Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser Ala Leu Arg Ile 325 330 335 Lys Ile Leu Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp Ile Asp 340 345 350 Lys Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 Asp Asn Val Asn Thr Gln Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 Glu Trp Leu Asn Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 Arg Leu Cys Leu Ser Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 Glu Glu His Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr 420 425 430 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser 450 455 460 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val Ile Glu Glu His Ala 485 490 495 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 Glu Gln Leu Lys Ala Ile Ser Thr Arg Asp Pro Leu Ser Lys Ile Thr 530 535 540 Glu Gln Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 Arg Asp Glu Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 Ile Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 Asp Asp Lys Leu Ser Gln Tyr Leu Ile Gln Leu Val Gln Val Leu Lys 625 630 635 640 Tyr Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 Ala Leu Thr Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 Ser Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680 685 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 Gln Val Glu Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile Leu Lys 705 710 715 720 Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln Met Lys Phe Leu Val 725 730 735 Glu Gln Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gln Gly Phe Leu 740 745 750 Ser Pro Leu Asn Pro Ala His Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 Cys Arg Ile Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 Asn Pro Asp Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile Ile 785 790 795 800 Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile 805 810 815 Ile Arg Ile Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp Leu Arg 820 825 830 Met Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys Val Gly Leu Ile 835 840 845 Glu Val Val Arg Asn Ser His Thr Ile Met Gln Ile Gln Cys Lys Gly 850 855 860 Gly Leu Lys Gly Ala Leu Gln Phe Asn Ser His Thr Leu His Gln Trp 865 870 875 880 Leu Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890 895 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly 900 905 910 Ile Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp Gly Gln 915 920 925 Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln Asp Phe 945 950 955 960 Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Cys Thr Lys Thr Arg Glu 965 970 975 Phe Glu Arg Phe Gln Glu Met Cys Tyr Lys Ala Tyr Leu Ala Ile Arg 980 985 990 Gln His Ala Asn Leu Phe Ile Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 Gly Met Pro Glu Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg 1010 1015 1020 Lys Thr Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030 1035 Phe Met Lys Gln Met Asn Asp Ala His His Gly Gly Trp Thr Thr 1040 1045 1050 Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln His Ala Leu Asn 1055 1060 1065 19125DNAHomo sapiens 19agtaacagac tagctagaga caatgaatta agggaaaatg acaaagaaca gctcaaagca 60atttctacac gagatcctct ctctgaaatc actaagcagg agaaagattt tctatggagt 120cacag 125201068PRTHomo sapiens 20Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met 1 5 10 15 Pro Pro Arg Ile Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25 30 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu Ile Thr Ile Lys His Glu 35 40 45 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60 Glu Ser Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu Arg Glu 65 70 75 80 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln 85 90 95 Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys Ile 100 105 110 Leu Asn Arg Glu Ile Gly Phe Ala Ile Gly Met Pro Val Cys Glu Phe 115 120 125 Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe Arg Arg Asn Ile Leu 130 135 140 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 Leu Pro Lys His Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val 180 185 190 Val Ile Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200 205 Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala 210 215 220 Ile Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln Leu Lys 225 230 235 240 Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile Leu Lys Val Cys Gly 245 250 255 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gln Tyr Lys Tyr 260 265 270 Ile Arg Ser Cys Ile Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 Ala Lys Glu Ser Leu Tyr Ser Gln Leu Pro Met Asp Cys Phe Thr Met 290 295 300 Pro Ser Tyr Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser Ala Leu Arg Ile 325 330 335 Lys Ile Leu Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp Ile Asp 340 345 350 Lys Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 Asp Asn Val Asn Thr Gln Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 Glu Trp Leu Asn Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 Arg Leu Cys Leu Ser Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 Glu Glu His Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr 420 425 430 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser 450 455 460 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val Ile Glu Glu His Ala 485 490 495 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 Glu Gln Leu Lys Ala Ile Ser Thr Arg Asp Pro Leu Ser Glu Ile Thr 530 535 540 Lys Gln Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 Arg Asp Glu Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 Ile Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 Asp Asp Lys Leu Ser Gln Tyr Leu Ile Gln Leu Val Gln Val Leu Lys 625 630 635 640 Tyr Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 Ala Leu Thr Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 Ser Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680 685 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 Gln Val Glu Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile Leu Lys 705 710 715 720 Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln Met Lys Phe Leu Val 725 730 735 Glu Gln Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gln Gly Phe Leu 740 745 750 Ser Pro Leu Asn Pro Ala His Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 Cys Arg Ile Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 Asn Pro Asp Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile Ile 785 790 795 800 Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile 805 810 815 Ile Arg Ile Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp Leu Arg 820 825 830 Met Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys Val Gly Leu Ile 835 840 845 Glu Val Val Arg Asn Ser His Thr Ile Met Gln Ile Gln Cys Lys Gly 850 855 860 Gly Leu Lys Gly Ala Leu Gln Phe Asn Ser His Thr Leu His Gln Trp 865 870 875 880 Leu Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890 895 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly 900 905 910 Ile Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp Gly Gln 915 920 925 Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln Asp Phe 945 950 955 960 Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Cys Thr Lys Thr Arg Glu 965 970 975 Phe Glu Arg Phe Gln Glu Met Cys Tyr Lys Ala Tyr Leu Ala Ile Arg 980 985 990 Gln His Ala Asn Leu Phe Ile Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 Gly Met Pro Glu Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg 1010 1015 1020 Lys Thr Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030 1035 Phe Met Lys Gln Met Asn Asp Ala His His Gly Gly Trp Thr Thr 1040 1045 1050 Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln His Ala Leu Asn 1055 1060 1065 21125DNAHomo sapiens 21agtaacagac tagctagaga caatgaatta agggaaaatg acaaagaaca gctcaaagca 60atttctacac gagatcctct ctctgaaatc actgcgcagg agaaagattt tctatggagt 120cacag 125221068PRTHomo sapiens 22Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met 1 5 10 15 Pro Pro Arg Ile Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25 30 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu Ile Thr Ile Lys His Glu 35 40 45 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60 Glu Ser Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu Arg Glu 65 70 75 80 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln 85 90 95 Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys Ile 100 105 110 Leu Asn Arg Glu Ile Gly Phe Ala Ile Gly Met Pro Val Cys Glu Phe 115 120 125 Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe Arg Arg Asn Ile Leu 130 135 140 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 Leu Pro Lys His Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val 180 185 190 Val Ile Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200 205 Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala 210 215 220 Ile Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln Leu Lys 225 230

235 240 Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile Leu Lys Val Cys Gly 245 250 255 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gln Tyr Lys Tyr 260 265 270 Ile Arg Ser Cys Ile Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 Ala Lys Glu Ser Leu Tyr Ser Gln Leu Pro Met Asp Cys Phe Thr Met 290 295 300 Pro Ser Tyr Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser Ala Leu Arg Ile 325 330 335 Lys Ile Leu Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp Ile Asp 340 345 350 Lys Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 Asp Asn Val Asn Thr Gln Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 Glu Trp Leu Asn Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 Arg Leu Cys Leu Ser Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 Glu Glu His Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr 420 425 430 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser 450 455 460 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val Ile Glu Glu His Ala 485 490 495 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 Glu Gln Leu Lys Ala Ile Ser Thr Arg Asp Pro Leu Ser Glu Ile Thr 530 535 540 Ala Gln Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 Arg Asp Glu Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 Ile Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 Asp Asp Lys Leu Ser Gln Tyr Leu Ile Gln Leu Val Gln Val Leu Lys 625 630 635 640 Tyr Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 Ala Leu Thr Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 Ser Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680 685 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 Gln Val Glu Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile Leu Lys 705 710 715 720 Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln Met Lys Phe Leu Val 725 730 735 Glu Gln Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gln Gly Phe Leu 740 745 750 Ser Pro Leu Asn Pro Ala His Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 Cys Arg Ile Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 Asn Pro Asp Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile Ile 785 790 795 800 Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile 805 810 815 Ile Arg Ile Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp Leu Arg 820 825 830 Met Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys Val Gly Leu Ile 835 840 845 Glu Val Val Arg Asn Ser His Thr Ile Met Gln Ile Gln Cys Lys Gly 850 855 860 Gly Leu Lys Gly Ala Leu Gln Phe Asn Ser His Thr Leu His Gln Trp 865 870 875 880 Leu Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890 895 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly 900 905 910 Ile Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp Gly Gln 915 920 925 Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln Asp Phe 945 950 955 960 Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Cys Thr Lys Thr Arg Glu 965 970 975 Phe Glu Arg Phe Gln Glu Met Cys Tyr Lys Ala Tyr Leu Ala Ile Arg 980 985 990 Gln His Ala Asn Leu Phe Ile Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 Gly Met Pro Glu Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg 1010 1015 1020 Lys Thr Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030 1035 Phe Met Lys Gln Met Asn Asp Ala His His Gly Gly Trp Thr Thr 1040 1045 1050 Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln His Ala Leu Asn 1055 1060 1065 23125DNAHomo sapiens 23agtaacagac tagctagaga caatgaatta agggaaaatg acaaagaaca gctcaaagca 60atttctacac gagatcctct ctctgaaatc actgggcagg agaaagattt tctatggagt 120cacag 125241068PRTHomo sapiens 24Met Pro Pro Arg Pro Ser Ser Gly Glu Leu Trp Gly Ile His Leu Met 1 5 10 15 Pro Pro Arg Ile Leu Val Glu Cys Leu Leu Pro Asn Gly Met Ile Val 20 25 30 Thr Leu Glu Cys Leu Arg Glu Ala Thr Leu Ile Thr Ile Lys His Glu 35 40 45 Leu Phe Lys Glu Ala Arg Lys Tyr Pro Leu His Gln Leu Leu Gln Asp 50 55 60 Glu Ser Ser Tyr Ile Phe Val Ser Val Thr Gln Glu Ala Glu Arg Glu 65 70 75 80 Glu Phe Phe Asp Glu Thr Arg Arg Leu Cys Asp Leu Arg Leu Phe Gln 85 90 95 Pro Phe Leu Lys Val Ile Glu Pro Val Gly Asn Arg Glu Glu Lys Ile 100 105 110 Leu Asn Arg Glu Ile Gly Phe Ala Ile Gly Met Pro Val Cys Glu Phe 115 120 125 Asp Met Val Lys Asp Pro Glu Val Gln Asp Phe Arg Arg Asn Ile Leu 130 135 140 Asn Val Cys Lys Glu Ala Val Asp Leu Arg Asp Leu Asn Ser Pro His 145 150 155 160 Ser Arg Ala Met Tyr Val Tyr Pro Pro Asn Val Glu Ser Ser Pro Glu 165 170 175 Leu Pro Lys His Ile Tyr Asn Lys Leu Asp Lys Gly Gln Ile Ile Val 180 185 190 Val Ile Trp Val Ile Val Ser Pro Asn Asn Asp Lys Gln Lys Tyr Thr 195 200 205 Leu Lys Ile Asn His Asp Cys Val Pro Glu Gln Val Ile Ala Glu Ala 210 215 220 Ile Arg Lys Lys Thr Arg Ser Met Leu Leu Ser Ser Glu Gln Leu Lys 225 230 235 240 Leu Cys Val Leu Glu Tyr Gln Gly Lys Tyr Ile Leu Lys Val Cys Gly 245 250 255 Cys Asp Glu Tyr Phe Leu Glu Lys Tyr Pro Leu Ser Gln Tyr Lys Tyr 260 265 270 Ile Arg Ser Cys Ile Met Leu Gly Arg Met Pro Asn Leu Met Leu Met 275 280 285 Ala Lys Glu Ser Leu Tyr Ser Gln Leu Pro Met Asp Cys Phe Thr Met 290 295 300 Pro Ser Tyr Ser Arg Arg Ile Ser Thr Ala Thr Pro Tyr Met Asn Gly 305 310 315 320 Glu Thr Ser Thr Lys Ser Leu Trp Val Ile Asn Ser Ala Leu Arg Ile 325 330 335 Lys Ile Leu Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asp Ile Asp 340 345 350 Lys Ile Tyr Val Arg Thr Gly Ile Tyr His Gly Gly Glu Pro Leu Cys 355 360 365 Asp Asn Val Asn Thr Gln Arg Val Pro Cys Ser Asn Pro Arg Trp Asn 370 375 380 Glu Trp Leu Asn Tyr Asp Ile Tyr Ile Pro Asp Leu Pro Arg Ala Ala 385 390 395 400 Arg Leu Cys Leu Ser Ile Cys Ser Val Lys Gly Arg Lys Gly Ala Lys 405 410 415 Glu Glu His Cys Pro Leu Ala Trp Gly Asn Ile Asn Leu Phe Asp Tyr 420 425 430 Thr Asp Thr Leu Val Ser Gly Lys Met Ala Leu Asn Leu Trp Pro Val 435 440 445 Pro His Gly Leu Glu Asp Leu Leu Asn Pro Ile Gly Val Thr Gly Ser 450 455 460 Asn Pro Asn Lys Glu Thr Pro Cys Leu Glu Leu Glu Phe Asp Trp Phe 465 470 475 480 Ser Ser Val Val Lys Phe Pro Asp Met Ser Val Ile Glu Glu His Ala 485 490 495 Asn Trp Ser Val Ser Arg Glu Ala Gly Phe Ser Tyr Ser His Ala Gly 500 505 510 Leu Ser Asn Arg Leu Ala Arg Asp Asn Glu Leu Arg Glu Asn Asp Lys 515 520 525 Glu Gln Leu Lys Ala Ile Ser Thr Arg Asp Pro Leu Ser Glu Ile Thr 530 535 540 Gly Gln Glu Lys Asp Phe Leu Trp Ser His Arg His Tyr Cys Val Thr 545 550 555 560 Ile Pro Glu Ile Leu Pro Lys Leu Leu Leu Ser Val Lys Trp Asn Ser 565 570 575 Arg Asp Glu Val Ala Gln Met Tyr Cys Leu Val Lys Asp Trp Pro Pro 580 585 590 Ile Lys Pro Glu Gln Ala Met Glu Leu Leu Asp Cys Asn Tyr Pro Asp 595 600 605 Pro Met Val Arg Gly Phe Ala Val Arg Cys Leu Glu Lys Tyr Leu Thr 610 615 620 Asp Asp Lys Leu Ser Gln Tyr Leu Ile Gln Leu Val Gln Val Leu Lys 625 630 635 640 Tyr Glu Gln Tyr Leu Asp Asn Leu Leu Val Arg Phe Leu Leu Lys Lys 645 650 655 Ala Leu Thr Asn Gln Arg Ile Gly His Phe Phe Phe Trp His Leu Lys 660 665 670 Ser Glu Met His Asn Lys Thr Val Ser Gln Arg Phe Gly Leu Leu Leu 675 680 685 Glu Ser Tyr Cys Arg Ala Cys Gly Met Tyr Leu Lys His Leu Asn Arg 690 695 700 Gln Val Glu Ala Met Glu Lys Leu Ile Asn Leu Thr Asp Ile Leu Lys 705 710 715 720 Gln Glu Lys Lys Asp Glu Thr Gln Lys Val Gln Met Lys Phe Leu Val 725 730 735 Glu Gln Met Arg Arg Pro Asp Phe Met Asp Ala Leu Gln Gly Phe Leu 740 745 750 Ser Pro Leu Asn Pro Ala His Gln Leu Gly Asn Leu Arg Leu Glu Glu 755 760 765 Cys Arg Ile Met Ser Ser Ala Lys Arg Pro Leu Trp Leu Asn Trp Glu 770 775 780 Asn Pro Asp Ile Met Ser Glu Leu Leu Phe Gln Asn Asn Glu Ile Ile 785 790 795 800 Phe Lys Asn Gly Asp Asp Leu Arg Gln Asp Met Leu Thr Leu Gln Ile 805 810 815 Ile Arg Ile Met Glu Asn Ile Trp Gln Asn Gln Gly Leu Asp Leu Arg 820 825 830 Met Leu Pro Tyr Gly Cys Leu Ser Ile Gly Asp Cys Val Gly Leu Ile 835 840 845 Glu Val Val Arg Asn Ser His Thr Ile Met Gln Ile Gln Cys Lys Gly 850 855 860 Gly Leu Lys Gly Ala Leu Gln Phe Asn Ser His Thr Leu His Gln Trp 865 870 875 880 Leu Lys Asp Lys Asn Lys Gly Glu Ile Tyr Asp Ala Ala Ile Asp Leu 885 890 895 Phe Thr Arg Ser Cys Ala Gly Tyr Cys Val Ala Thr Phe Ile Leu Gly 900 905 910 Ile Gly Asp Arg His Asn Ser Asn Ile Met Val Lys Asp Asp Gly Gln 915 920 925 Leu Phe His Ile Asp Phe Gly His Phe Leu Asp His Lys Lys Lys Lys 930 935 940 Phe Gly Tyr Lys Arg Glu Arg Val Pro Phe Val Leu Thr Gln Asp Phe 945 950 955 960 Leu Ile Val Ile Ser Lys Gly Ala Gln Glu Cys Thr Lys Thr Arg Glu 965 970 975 Phe Glu Arg Phe Gln Glu Met Cys Tyr Lys Ala Tyr Leu Ala Ile Arg 980 985 990 Gln His Ala Asn Leu Phe Ile Asn Leu Phe Ser Met Met Leu Gly Ser 995 1000 1005 Gly Met Pro Glu Leu Gln Ser Phe Asp Asp Ile Ala Tyr Ile Arg 1010 1015 1020 Lys Thr Leu Ala Leu Asp Lys Thr Glu Gln Glu Ala Leu Glu Tyr 1025 1030 1035 Phe Met Lys Gln Met Asn Asp Ala His His Gly Gly Trp Thr Thr 1040 1045 1050 Lys Met Asp Trp Ile Phe His Thr Ile Lys Gln His Ala Leu Asn 1055 1060 1065 2531DNAArtificial SequenceDescription of Artificial Sequence Synthetic 542/545 forward primer PIK3CA-9F13T 25uaaaauuuau ugagaaugua uuugcttttt c 312624DNAArtificial SequenceDescription of Artificial Sequence Synthetic 542/545 reverse primer PIK3CA-9R01 26tccattttag cacttacctg tgac 242730DNAArtificial SequenceDescription of Artificial Sequence Synthetic 542 WT probe 27tttcaagaga ggaucucgug uagaaauugc 302833DNAArtificial SequenceDescription of Artificial Sequence Synthetic 542 542K mutation probe 28attttgagag aggaucucgu guagaaauug cuu 332932DNAArtificial SequenceDescription of Artificial Sequence Synthetic 545 WT probe 29ctgctcagta uuunagagag aggatctcgt gt 323034DNAArtificial SequenceDescription of Artificial Sequence Synthetic 545 545K mutation probe 30aatcactaag aggagaaaga uuuucuaugg aguc 343130DNAArtificial SequenceDescription of Artificial Sequence Synthetic 545 545A mutation probe 31ctgcgcggag aaagauuuuc uauggaguca 303230DNAArtificial SequenceDescription of Artificial Sequence Synthetic 545 545G mutation probe 32cctgcccgtg auuunagaga gaggatctcg 303325DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 420 forward primer PIK3CA-7F03 33uuuuggggaa gaaaaguguu uugaa 253429DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 420 reverse primer PIK3CA-7R04 34gattcaaagc catttttcca gatactaga 293523DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 1047 forward primer PIK3CA-20F01 35gaggctttgg agtatttcat gaa 233621DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 1047 reverse primer PIK3CA-20R01 36ccaatccatt tttgttgtcc a 213737DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 420 WT probe 37caatggacag guuccuuaaa aaacaaagaa aaauauu 373831DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 420 420R mutation probe 38gaacacctcc auuggcaugg ggaaauauaa a 313925DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 1047 WT probe 39tgcacatctg gtggctggac aacaa 254025DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 1047 1047R mutation probe

40gacgtcauca uucauuuguu ucaug 254127DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 1047 1047L mutation probe 41gcacttcatg tggctggaca acaaaaa 274227DNAArtificial SequenceDescription of Artificial Sequence Synthetic codon 1047 1047Y mutation probe 42accatgatat caucauucau uuguuuc 2743195PRTHomo sapiens 43Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg 195 44124PRTHomo sapiens 44Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro Leu Pro Thr 1 5 10 15 Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly Pro Lys His 20 25 30 Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly Ile Cys Glu 35 40 45 Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr Phe Glu Ser 50 55 60 Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser Cys Val Thr 65 70 75 80 Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser Cys Thr Leu 85 90 95 Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp Gly Thr Gln 100 105 110 Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val 115 120 45169PRTHomo sapiens 45Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr 1 5 10 15 Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser 20 25 30 Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 35 40 45 Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu 50 55 60 Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp 65 70 75 80 Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His 85 90 95 Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu 100 105 110 Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His 115 120 125 His Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu 130 135 140 Phe Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu 145 150 155 160 Asp Glu Cys Val Gly Glu Gly Leu Ala 165 46142PRTHomo sapiens 46Cys His Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr 1 5 10 15 Gln Cys Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu 20 25 30 Glu Cys Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg 35 40 45 His Cys Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val 50 55 60 Thr Cys Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr 65 70 75 80 Lys Asp Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro 85 90 95 Asp Leu Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala 100 105 110 Cys Gln Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp 115 120 125 Asp Lys Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr 130 135 140 473724DNAHomo sapiens 47tctccctcgg cgccgccgcc gccgcccgcg gggctgggac ccgatgcggt tagagccgcg 60gagcctggaa gagccccgag cgtttctgct ttgggacaac catacatcta attccttaaa 120gtagttttat atgtaaaact tgcaaagaat cagaacaatg cctccacgac catcatcagg 180tgaactgtgg ggcatccact tgatgccccc aagaatccta gtagaatgtt tactaccaaa 240tggaatgata gtgactttag aatgcctccg tgaggctaca ttaataacca taaagcatga 300actatttaaa gaagcaagaa aataccccct ccatcaactt cttcaagatg aatcttctta 360cattttcgta agtgttactc aagaagcaga aagggaagaa ttttttgatg aaacaagacg 420actttgtgac cttcggcttt ttcaaccctt tttaaaagta attgaaccag taggcaaccg 480tgaagaaaag atcctcaatc gagaaattgg ttttgctatc ggcatgccag tgtgtgaatt 540tgatatggtt aaagatccag aagtacagga cttccgaaga aatattctga acgtttgtaa 600agaagctgtg gatcttaggg acctcaattc acctcatagt agagcaatgt atgtctatcc 660tccaaatgta gaatcttcac cagaattgcc aaagcacata tataataaat tagataaagg 720gcaaataata gtggtgatct gggtaatagt ttctccaaat aatgacaagc agaagtatac 780tctgaaaatc aaccatgact gtgtaccaga acaagtaatt gctgaagcaa tcaggaaaaa 840aactcgaagt atgttgctat cctctgaaca actaaaactc tgtgttttag aatatcaggg 900caagtatatt ttaaaagtgt gtggatgtga tgaatacttc ctagaaaaat atcctctgag 960tcagtataag tatataagaa gctgtataat gcttgggagg atgcccaatt tgatgttgat 1020ggctaaagaa agcctttatt ctcaactgcc aatggactgt tttacaatgc catcttattc 1080cagacgcatt tccacagcta caccatatat gaatggagaa acatctacaa aatccctttg 1140ggttataaat agtgcactca gaataaaaat tctttgtgca acctacgtga atgtaaatat 1200tcgagacatt gataagatct atgttcgaac aggtatctac catggaggag aacccttatg 1260tgacaatgtg aacactcaaa gagtaccttg ttccaatccc aggtggaatg aatggctgaa 1320ttatgatata tacattcctg atcttcctcg tgctgctcga ctttgccttt ccatttgctc 1380tgttaaaggc cgaaagggtg ctaaagagga acactgtcca ttggcatggg gaaatataaa 1440cttgtttgat tacacagaca ctctagtatc tggaaaaatg gctttgaatc tttggccagt 1500acctcatgga ttagaagatt tgctgaaccc tattggtgtt actggatcaa atccaaataa 1560agaaactcca tgcttagagt tggagtttga ctggttcagc agtgtggtaa agttcccaga 1620tatgtcagtg attgaagagc atgccaattg gtctgtatcc cgagaagcag gatttagcta 1680ttcccacgca ggactgagta acagactagc tagagacaat gaattaaggg aaaatgacaa 1740agaacagctc aaagcaattt ctacacgaga tcctctctct gaaatcactg agcaggagaa 1800agattttcta tggagtcaca gacactattg tgtaactatc cccgaaattc tacccaaatt 1860gcttctgtct gttaaatgga attctagaga tgaagtagcc cagatgtatt gcttggtaaa 1920agattggcct ccaatcaaac ctgaacaggc tatggaactt ctggactgta attacccaga 1980tcctatggtt cgaggttttg ctgttcggtg cttggaaaaa tatttaacag atgacaaact 2040ttctcagtat ttaattcagc tagtacaggt cctaaaatat gaacaatatt tggataactt 2100gcttgtgaga tttttactga agaaagcatt gactaatcaa aggattgggc actttttctt 2160ttggcattta aaatctgaga tgcacaataa aacagttagc cagaggtttg gcctgctttt 2220ggagtcctat tgtcgtgcat gtgggatgta tttgaagcac ctgaataggc aagtcgaggc 2280aatggaaaag ctcattaact taactgacat tctcaaacag gagaagaagg atgaaacaca 2340aaaggtacag atgaagtttt tagttgagca aatgaggcga ccagatttca tggatgctct 2400acagggcttt ctgtctcctc taaaccctgc tcatcaacta ggaaacctca ggcttgaaga 2460gtgtcgaatt atgtcctctg caaaaaggcc actgtggttg aattgggaga acccagacat 2520catgtcagag ttactgtttc agaacaatga gatcatcttt aaaaatgggg atgatttacg 2580gcaagatatg ctaacacttc aaattattcg tattatggaa aatatctggc aaaatcaagg 2640tcttgatctt cgaatgttac cttatggttg tctgtcaatc ggtgactgtg tgggacttat 2700tgaggtggtg cgaaattctc acactattat gcaaattcag tgcaaaggcg gcttgaaagg 2760tgcactgcag ttcaacagcc acacactaca tcagtggctc aaagacaaga acaaaggaga 2820aatatatgat gcagccattg acctgtttac acgttcatgt gctggatact gtgtagctac 2880cttcattttg ggaattggag atcgtcacaa tagtaacatc atggtgaaag acgatggaca 2940actgtttcat atagattttg gacacttttt ggatcacaag aagaaaaaat ttggttataa 3000acgagaacgt gtgccatttg ttttgacaca ggatttctta atagtgatta gtaaaggagc 3060ccaagaatgc acaaagacaa gagaatttga gaggtttcag gagatgtgtt acaaggctta 3120tctagctatt cgacagcatg ccaatctctt cataaatctt ttctcaatga tgcttggctc 3180tggaatgcca gaactacaat cttttgatga cattgcatac attcgaaaga ccctagcctt 3240agataaaact gagcaagagg ctttggagta tttcatgaaa caaatgaatg atgcacatca 3300tggtggctgg acaacaaaaa tggattggat cttccacaca attaaacagc atgcattgaa 3360ctgaaaagat aactgagaaa atgaaagctc actctggatt ccacactgca ctgttaataa 3420ctctcagcag gcaaagaccg attgcatagg aattgcacaa tccatgaaca gcattagaat 3480ttacagcaag aacagaaata aaatactata taatttaaat aatgtaaacg caaacagggt 3540ttgatagcac ttaaactagt tcatttcaaa attaagcttt agaataatgc gcaatttcat 3600gttatgcctt aagtccaaaa aggtaaactt tgaagattgt ttgtatcttt ttttaaaaaa 3660caaaacaaaa caaaaatccc caaaatatat agaaatgatg gagaaggaaa aaaaaaaaaa 3720aaaa 3724

Claims

1-47. (canceled)

48. A method for detecting the presence of a mutation in exon 9 of Phosphoinositol-3 kinase (PIK3CA) in a sample from a patient with HER2-positive cancer comprising: i) obtaining a nucleic acid sample from the patient; ii) contacting the nucleic acid sample from the patient with primers that specifically amplify a target sequence in exon 9 of PIK3CA and a probe that specifically hybridizes to a mutation in the target sequence in exon 9 of PIK3CA; iii) carrying out an amplification reaction to generate a target sequence amplification product; and iv) detecting the presence of a mutation in exon 9 of PIK3CA by detecting hybridization of the probe to the amplification product, wherein the presence of a mutation in exon 9 is detected and the presence of a mutation in exon 20 is not detected.

49. The method of claim 48, wherein the HER2-positive cancer is breast cancer.

50. The method of claim 49, wherein the breast cancer is early-stage breast cancer.

51. The method of claim 48, wherein the mutation is one or more of the mutations E542 E545K, E545A and E545G in exon 9 of PIK3CA.

52. The method of claim 48, further comprising treating the patient with a HER2 inhibitor if a mutation in exon 9 of PIK3CA is not detected.

53. The method of claim 52, wherein the HER2-inhibitor is an anti-HER2 antibody.

54. The method of claim 53, wherein the anti-HER2 antibody is pertuzumab.

55. A method of treating a patient with a HER2-positive cancer comprising: i) obtaining a nucleic acid sample from the patient; ii) contacting the nucleic acid sample from the patient with primers that specifically amplify a target sequence in exon 9 of PIK3CA and a probe that specifically hybridizes to a mutation in the target sequence in exon 9 of PIK3CA; iii) carrying out an amplification reaction to generate a target sequence amplification product; iv) detecting the presence of a mutation in exon 9 of PIK3CA by detecting hybridization of the probe to the amplification product; and v) treating the patient with a HER2 inhibitor if a mutation in exon 9 of PIK3CA is not detected.

56. The method of claim 55, wherein the HER2-positive cancer is breast cancer.

57. The method of claim 56, wherein the breast cancer is early-stage breast cancer.

58. The method of claim 55, wherein the mutation is one or more of the mutations E542 E545K, E545A and E545G in exon 9 of PIK3CA.

59. The method of claim 55, wherein the HER2-inhibitor is an anti-HER2 antibody.

60. The method of claim 55, wherein the anti-HER2 antibody is pertuzumab.

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