U.S. patent application number 15/163552 was filed with the patent office on 2016-09-15 for identification of non-responders to her2 inhibitors.
The applicant listed for this patent is Roche Molecular Systems, Inc.. Invention is credited to Victoria Brophy, Neil Jones, Astrid Kiermaier, Jayantha Ratnayake.
Application Number | 20160265067 15/163552 |
Document ID | / |
Family ID | 48573606 |
Filed Date | 2016-09-15 |
United States Patent
Application |
20160265067 |
Kind Code |
A1 |
Brophy; Victoria ; et
al. |
September 15, 2016 |
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.
Inventors: |
Brophy; Victoria; (Martinez,
CA) ; Jones; Neil; (Therwil, CH) ; Kiermaier;
Astrid; (Loerrach, DE) ; Ratnayake; Jayantha;
(Bedford, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Molecular Systems, Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
48573606 |
Appl. No.: |
15/163552 |
Filed: |
May 24, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14363982 |
Jun 9, 2014 |
9376715 |
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PCT/EP2012/074857 |
Dec 7, 2012 |
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15163552 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6827 20130101;
C07K 16/3015 20130101; C12Q 2600/156 20130101; C07K 16/40 20130101;
C12Q 2600/106 20130101; C12Q 1/6886 20130101; C07K 2317/24
20130101; C07K 2317/76 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 16/30 20060101 C07K016/30; C07K 16/40 20060101
C07K016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 2011 |
EP |
11192861.0 |
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.
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,
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6,333,348 B1, WO 01/05425, WO 01/64246, US 2003/0022918, US
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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
* * * * *
References