Patent application title: PREDICTING TUMOR RESPONSE TO ANTI-ERBB3 ANTIBODIES
Inventors:
IPC8 Class: AC12Q16881FI
USPC Class:
1 1
Class name:
Publication date: 2018-07-12
Patent application number: 20180195120
Abstract:
A diagnostic method for predicting quantitatively whether a human tumor
will be sensitive or resistant to treatment with an ERBB3 inhibitor, e.g,
an anti-ERBB3 antibody, is disclosed. The method is based on measurement
of NRG1 expression at the RNA level, or at the protein level, in a tissue
sample from the tumor.Claims:
1. A method of treating a tumor with an anti-ERBB3 antibody, the method
comprising administering the anti-ERBB3 antibody to a subject with a
tumor likely to be sensitive to the antibody, wherein the subject is
identified by: (a) measuring NRG1 gene expression in a tissue sample from
the tumor, thereby determining an NRG1 score, wherein the expression of
one or more genes is measured in addition to NRG1 and the one or more
genes does not include ErbB1, ErbB2, ErbB3, or betacellulin (BTC); and
(b) determining that the NRG1 score is equal to or above a threshold
score defined by a threshold determination analysis, indicating that the
tumor is likely to be responsive to treatment with an anti-ERBB3
antibody.
2. The method of claim 1, wherein the anti-ERBB3 antibody inhibits or prevents binding of NRG1 to ERBB3.
3. The method of claim 1, wherein the anti-ERBB3 antibody inhibits or prevents dimerization of ERBB3 without inhibiting binding of NRG1 to ERBB3.
4. The method of claim 2, wherein the anti-ERBB3 antibody is selected from the group consisting of: (a) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 3; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 6; (b) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 13; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 16; (c) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 42; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 16; (d) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 47, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 49; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 50, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 51, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 52; and (e) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 57, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 42; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 16; (f) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; (g) an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 10; (h) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; (i) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 44; (j) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 54; and (k) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.
5. The method of claim 3, wherein the anti-ERBB3 antibody is selected from the group consisting of: (a) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 23; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 25, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 26; (b) (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the s amino acid sequence of SEQ ID NO: 31, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 33; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 35; (c) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 28; and (d) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 37.
6. The method of claim 2, wherein the anti-ERBB3 antibody comprises (i) an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 3; and (ii) an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 6.
7. The method of claim 2, wherein the anti-ERBB3 antibody comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
8. The method of claim 2, wherein the anti-ERBB3 antibody comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 10.
9. The method of claim 1, wherein the step of measuring NRG1 gene expression is performed by measuring the level of NRG1 protein.
10. The method of claim 1, wherein the step of measuring NRG1 gene expression is performed by measuring the level of mRNA encoding NRG1 protein.
11. The method of claim 1, wherein the threshold determination analysis comprises a receiver operator characteristic curve analysis.
12. The method of claim 1, wherein the tumor is a solid tumor.
13. The method of claim 1, wherein the expression of one or more other genes measured in addition to NRG1 serves as a control or standard for data normalization.
14. The method of claim 1, wherein the expression of one or more other genes measured in addition to NRG1 is used to classify the tumor as likely to be responsive to treatment with an anti-ERBB3 antibody.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent application Ser. No. 14/349,916 filed Apr. 4, 2014, which is a U.S. national stage entry under 35 U.S.C. .sctn. 371 of International Patent Application No. PCT/US2012/058871, filed Oct. 5, 2012, which claims the benefit of and priority to U.S. Provisional Patent Application No. 61/636,183, filed Apr. 20, 2012 and U.S. Provisional Patent Application No. 61/544,206, filed Oct. 6, 2011; the entire contents of each application are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The field of the invention is molecular biology, oncology, and clinical diagnostics.
BACKGROUND
[0003] Most cancer drugs are effective in some patients, but not in others. This results from genetic variation among tumors, and can be observed even among tumors within the same patient. Variable patient response is particularly pronounced with respect to targeted therapeutics. Therefore, the full potential of targeted therapies cannot be realized without suitable tests for determining which patients will benefit from which drugs. According to the National Institutes of Health (NIH), the term "biomarker" is defined as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic or pathogenic processes or pharmacological response to a therapeutic intervention." (Biomarkers Definitions Working Group, 2001, Clin. Pharmacol. Ther. 69:89-95)
[0004] The development of improved diagnostics based on the discovery of biomarkers has the potential to accelerate new drug development by identifying, in advance, those patients most likely to show a clinical response to a given drug. This would significantly reduce the size, length and cost of clinical trials. Technologies such as genomics, proteomics and molecular imaging currently enable rapid, sensitive and reliable detection of specific gene mutations, expression levels of particular genes, and other molecular biomarkers. In spite of the availability of various technologies for molecular characterization of tumors, the clinical utilization of cancer biomarkers remains largely unrealized because few cancer biomarkers have been discovered. For example, a recent review article states:
[0005] There is a critical need for expedited development of biomarkers and their use to improve diagnosis and treatment of cancer. (Cho, 2007, Molecular Cancer 6:25) Another recent review article on cancer biomarkers contains the following comments:
[0006] The challenge is discovering cancer biomarkers. Although there have been clinical successes in targeting molecularly defined subsets of several tumor types--such as chronic myeloid leukemia, gastrointestinal stromal tumor, lung cancer and glioblastoma multiforme--using molecularly targeted agents, the ability to apply such successes in a broader context is severely limited by the lack of an efficient strategy to evaluate targeted agents in patients. The problem mainly lies in the inability to select patients with molecularly defined cancers for clinical trials to evaluate these exciting new drugs. The solution requires biomarkers that reliably identify those patients who are most likely to benefit from a particular agent. (Sawyers, 2008, Nature 452:548-552, at 548) Comments such as the foregoing illustrate the recognition of a need for the discovery of clinically useful biomarkers and diagnostic methods based on such biomarkers.
[0007] There are three distinct types of cancer biomarkers: (1) prognostic biomarkers, (2) predictive biomarkers, and (3) pharmacodynamic (PD) biomarkers. A prognostic biomarker is used to classify a cancer, e.g., a solid tumor, according to aggressiveness, i.e., rate of growth and/or metastasis, and refractiveness to treatment. This is sometimes called distinguishing "good outcome" tumors from "poor outcome" tumors. A predictive biomarker is used to assess the probability that a particular patient will benefit from treatment with a particular drug. For example, patients with breast cancer in which the ERBB2 (HER2) gene is amplified are likely to benefit from treatment with trastuzumab (HERCEPTIN.RTM.), whereas patients without ERBB2 gene amplification are unlikely to benefit from treatment with trastuzumab. A PD biomarker is an indication of the effect(s) of a drug on its molecular target while the patient is taking the drug. Accordingly, PD biomarkers often are used to guide dosage level and dosing frequency, during the early stages of clinical development of a new drug. For a discussion of cancer biomarkers, see, e.g., Sawyers, 2008, Nature 452:548-552.
[0008] Tumors driven by EGFR or HER2 often respond to treatment with inhibitors of EGFR or HER2, but these tumors invariably develop resistance to these inhibitors. At least one mechanism of acquired resistance to anti-EGFR or anti-HER2 treatment is activation of ERBB3 (also known as HER3) signaling. See, e.g., Engelman et al., 2006, Clin. Cancer Res. 12:4372; Ritter et al., 2007, Clin. Cancer Res. 13:4909; Sergina et al., 2007, Nature 445:437. NRG1-induced activation of HER2-ERBB3 heterodimers also has been associated with resistance to EGFR inhibitors (Zhou et al., 2006, Cancer Cell 10:39). Thus, ERBB3 plays an important role in development of drug resistance, as well as being involved in tumor initiation and maintenance, through its heterodimerization with EGFR and HER2. Consequently, there has been interest in development of ERBB3 inhibitors, especially anti-ERBB3 antibodies, since ERBB3 lacks kinase activity.
[0009] As with other types of targeted therapy, some, but not all, tumors respond to anti-ERBB3 therapy. Therefore, there is a need for diagnostic methods based on predictive biomarkers that can be used to identify patients with tumors that are likely (or unlikely) to respond to treatment with an ERBB3 inhibitor such as an anti-ERBB3 antibody.
SUMMARY
[0010] The invention is based, in part, on the discovery that neuregulin-1 (NRG1) expression in a tissue sample from a mammalian tumor (e.g., a human tumor) correlates with sensitivity of the tumor to treatment with an ERBB3 inhibitor, e.g., an anti-ERBB3 antibody. Surprisingly, it has been discovered that the correlation is strong enough that measurement of NRG1 expression alone is sufficient for useful classification of a tumor as sensitive or resistant to treatment with an ERBB3 inhibitor. Accordingly, the invention provides a method of identifying a tumor that is sensitive to treatment with an ERBB3 inhibitor. The method includes: (a) measuring NRG1 gene expression in a tissue sample from the tumor, thereby determining an NRG1 score; and (b) comparing the NRG1 score against a threshold score defined by a threshold determination analysis. An NRG1 score equal to or above the threshold score indicates that the tumor is likely to be sensitive to treatment with an ERBB3 inhibitor, e.g., an anti-ERBB3 antibody. Alternatively, an NRG1 score below the threshold indicates that the tumor is likely to be resistant to treatment with an ERBB3 inhibitor, e.g., an anti-ERBB3 antibody. In certain embodiments, the method is not based on the expression of any other gene than NRG1.
[0011] Measurement of NRG1 expression can be at the protein level, e.g., by immunohistochemistry (IHC) involving a chromophore or fluorophore conjugated to an anti-NRG1 antibody. Alternatively, measurement of NRG1 expression can be at the RNA level, e.g., by measuring the level of mRNA encoding NRG1, e.g., by quantitative PCR or microarray. The threshold determination analysis can include a receiver operator characteristic curve analysis. Methods of the invention are useful for testing various types of tumors, e.g., solid tumors, including, e.g., breast tumors, lung tumors, kidney tumors, colorectal tumors, head and neck tumors, esophageal tumors, ovarian tumors, and pancreatic tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic diagram showing the CDR.sub.H1, CDR.sub.H2, and CDR.sub.H3 sequences (Kabat definition) of the immunoglobulin heavy chain variable regions sequences for the anti-ERBB3 antibodies denoted as AV-203, 04D01, 09D03, 11G01, 12A07, 18H02 and 22A02 (which correspond to the boxed regions in FIG. 2).
[0013] FIG. 2 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin heavy chain variable region for the anti-ERBB3 antibodies denoted as AV-203, 04D01, 09D03, 11G01, 12A07, 18H02 and 22A02. The amino acid sequences for each antibody are aligned against one another, and Complementary Determining Sequences (CDR) (Kabat definition), CDR.sub.H1, CDR.sub.H2, and CDR.sub.H3, are identified in boxes. The unboxed sequences represent framework (FR) sequences.
[0014] FIG. 3 is a schematic diagram showing the CDR.sub.L1, CDR.sub.L2, and CDR.sub.L3 sequences (Kabat definition) of the immunoglobulin light chain variable regions sequences for the anti-ERBB3 antibodies denoted as AV-203, 04D01, 09D03, 11G01, 12A07, 18H02 and 22A02 (which correspond to the boxed regions in FIG. 4).
[0015] FIG. 4 is a schematic diagram showing the amino acid sequence of the complete immunoglobulin light chain variable region for the anti-ERBB3 antibodies denoted as AV-203, 04D01, 09D03, 11G01, 12A07, 18H02 and 22A02. The amino acid sequences for each antibody are aligned against one another, and Complementary Determining Sequences (CDR) (Kabat definition), CDR.sub.L1, CDR.sub.L2, and CDR.sub.L3, are identified in boxes. The unboxed sequences represent framework (FR) sequences.
[0016] FIG. 5 provides the amino acid sequence defining the (A) full length AV-203 immunoglobulin heavy chain and (B) the full length AV-203 immunoglobulin light chain.
[0017] FIG. 6 provides the amino acid sequence defining the (A) full length 04D01 immunoglobulin heavy chain and (B) the full length 04D01 immunoglobulin light chain.
[0018] FIG. 7 provides the amino acid sequence defining the (A) full length 09D03 immunoglobulin heavy chain and (B) the full length 09D03 immunoglobulin light chain.
[0019] FIG. 8 provides the amino acid sequence defining the (A) full length 11G01 immunoglobulin heavy chain and (B) the full length 11G01 immunoglobulin light chain.
[0020] FIG. 9 provides the amino acid sequence defining the (A) full length 12A07 immunoglobulin heavy chain and (B) the full length 12A07 immunoglobulin light chain.
[0021] FIG. 10 provides the amino acid sequence defining the (A) full length 18H02 immunoglobulin heavy chain and (B) the full length 18H02 immunoglobulin light chain.
[0022] FIG. 11 provides the amino acid sequence defining the (A) full length 22A02 immunoglobulin heavy chain and (B) the full length 22A02 immunoglobulin light chain.
[0023] FIG. 12 is a scatter plot with a linear regression trend line showing the relationship between the in vivo efficacy of AV-203 in 25 xenograft models (expressed as percentage tumor growth inhibition (TGI)) and NRG1 RNA expression, as measured by quantitative RT-PCR, represented by the Ct value. The 25 data points are represented by (.box-solid.). The linear regression is shown by a solid line, and the 95% confidence interval by dotted lines. Among 25 tumors tested, the Rho value was -0.601, with p=0.0015 (Spearman correlation).
[0024] FIG. 13 is a scatter plot with a linear regression trend line showing the relationship between the in vivo efficacy of AV-203 in 25 xenograft models (expressed as percentage TGI) and ERBB3 RNA expression, as measured by quantitative RT-PCR, represented by the Ct value. The 25 data points are represented by (.box-solid.). The linear regression is shown by a solid line, and the 95% confidence interval by dotted lines.
[0025] FIG. 14 is a Receiver operator characteristic (ROC) curve based on the data in FIG. 13, to determine the optimum threshold PGS score. This ROC curve indicates that the optimum threshold is Ct=22.9, which yields a false positive rate of 0.13, and a false negative rate of 0.2.
[0026] FIG. 15 is a box graph summarizing AV-203 in vivo efficacy in xenograft models separated by high NRG1-expressing tumors (Ct=<22.9) and low NRG1-expressing tumors (Ct>22.9).
[0027] FIG. 16 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against LU-10 human primary lung tumor xenografts dosed at 20 mg/kg in NCR nude mice.
[0028] FIG. 17 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.), human IgG control (.box-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against LU-58 human primary lung tumor xenografts dosed at 20 mg/kg in NCR nude mice.
[0029] FIG. 18 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.), human IgG control (.box-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against LU-08 human primary lung tumor xenografts dosed at 20 mg/kg in NCR nude mice.
[0030] FIG. 19 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.), human IgG control (.box-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against LU-44 human primary lung tumor xenografts dosed at 20 mg/kg in NCR nude mice.
[0031] FIG. 20 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against CAL-27 human head and neck cancer xenografts dosed at 20 mg/kg in CB17-SCID mice.
[0032] FIG. 21 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.), human IgG control (.box-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against KYSE-150 human esophageal cancer xenografts dosed at 20 mg/kg in NCR nude mice
[0033] FIG. 22 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.), human IgG control (.circle-solid.) and the anti-ERBB3 antibody AV-203 (.circle-solid.) against H520 human non-small cell lung carcinoma xenografts dosed at 20 mg/kg in NCR nude mice.
[0034] FIG. 23 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.), human IgG control (.box-solid.) and the anti-ERBB3 antibody 11G01 (.quadrature.) against BxPC3 pancreatic tumor xenografts dosed at 20 mg/kg in CB.17 SCID mice.
[0035] FIG. 24 is a graph summarizing efficacy data for PBS vehicle control (.diamond-solid.) and the anti-ERBB3 antibody 11G01 (.circle-solid.) against DU145 prostate tumor xenografts dosed at 20 mg/kg in CB.17 SCID mice.
[0036] FIG. 25 is a graph summarizing efficacy data for PBS vehicle control (.box-solid.), human IgG control (---) and the anti-ERBB3 antibody 11G01 (.circle-solid.) against H322 lung tumor xenografts dosed at 20 mg/kg in NCR nude mice.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0037] As used herein, "AV-203" means the humanized anti-human ERBB3 monoclonal antibody whose full-length heavy chain amino acid sequence is SEQ ID NO: 9, and whose full-length light chain amino acid sequence is SEQ ID NO: 10.
[0038] As used herein, "ERBB3" (also known as HERS) means the human protein encoded by the gene identified by Entrez Gene ID No. 2065, and allelic variants thereof.
[0039] As used herein, "ERBB3 inhibitor" means a molecule (small molecule or macromolecule, e.g., an antibody or antigen binding fragment thereof) that binds to ERBB3 and inhibits, neutralizes, prevents or eliminates the biological activity of ERBB3 in a tumor cell.
[0040] As used herein, "NRG1" (also known as neuregulin-1, heregulin, HRG and HRG1) means the human protein encoded by the gene identified by Entrez Gene ID No. 3084, and allelic variants thereof.
[0041] As used herein, "optimum threshold score" means the threshold score at which the classifier gives the most desirable balance between the cost of false negative calls and false positive calls.
[0042] As used herein, "receiver operating characteristic" (ROC) curve means a plot of false positive rate (sensitivity) versus true positive rate (specificity) for a binary classifier system. In construction of an ROC curve, the following definitions apply:
[0043] False negative rate: FNR=1-TPR
[0044] True positive rate: TPR=true positive/(true positive+false negative)
[0045] False positive rate: FPR=false positive/(false positive+true negative)
[0046] As used herein, "response" or "responding" to treatment means, with regard to a treated tumor, that the tumor displays: (a) slowing of growth, (b) cessation of growth, or (c) regression.
[0047] As used herein, a "NRG1 score" is a numerical value representing the level of NRG1 expression in a tumor. The NRG1 score can be based on NRG1 gene expression at the RNA level or at the protein level. For example, an NRG1 score could be expressed as a (1) Ct value from a qRT-PCR assay, or (2) staining intensity in an IHC assay. Ct value and NRG1 expression are inversely related. Therefore, a lower Ct value translates to a higher NRG1 score. The NRG1 score can be interpreted with respect to a threshold score, which can be empirically determined in a threshold determination analysis, e.g., using ROC curve analysis.
[0048] As used herein, "threshold determination analysis" means analysis of a dataset representing a given tumor type, e.g., human renal cell carcinoma, to determine a threshold score for that particular tumor type. The dataset representing a given tumor type includes, for each tumor from a population of such tumors: (a) actual tumor response data (response and non-response to an ERBB3 inhibitor such as an anti-ERBB3 antibody), and (b) NRG1 expression level.
[0049] As used herein, "threshold score" means a score above which a tumor is classified as being sensitive to treatment with an ERBB3 inhibitor.
ERBB3 Antibodies
[0050] The methods disclosed herein can be used for predicting tumor response to treatment with an ERBB3 inhibitor such as an anti-ERBB3 antibody, or antigen-binding fragment of an anti-ERBB3 antibody. In some embodiments, a tumor is classified as sensitive or resistant to an ERBB3 antibody (or antigen binding fragment thereof) that inhibits or prevents NRG1 (e.g., NRG1-.beta.1) from binding to ERBB3, thereby indirectly inhibiting or preventing ligand-induced dimerization of ERBB3 (e.g., anti-ERBB3 antibodies AV-203, 04D01, 12A07, 18H02 and 22A02). In other embodiments, a tumor is classified as sensitive or resistant to an antibody (or antigen-binding fragment thereof) that inhibits or prevents ERBB3 dimerization, without preventing NRG1 binding to ERBB3 (e.g., anti-ERBB3 antibody 09D03 and 11G01).
[0051] In exemplary embodiments, the ERBB3 inhibitor is one of the following antibodies: AV-203, 04D01, 12A07, 18H02, 22A02, 11G01, and 09D03.
[0052] Anti-ERBB3 antibody AV-203 (originally designated as antibody 24C05) comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 3 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 6 as shown in FIG. 3. In an exemplary embodiment, antibody AV-203 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 as shown in FIG. 2, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 as shown in FIG. 4. In another exemplary embodiment, antibody AV-203 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 9 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 10, as shown in FIG. 5.
[0053] Anti-ERBB3 antibody 04D01 comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 13 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the s amino acid sequence of SEQ ID NO: 14, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 16 as shown in FIG. 3. In an exemplary embodiment, antibody 04D01 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 as shown in FIG. 2, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 18 as shown in FIG. 4. In another exemplary embodiment, antibody 04D01 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 19 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 20, as shown in FIG. 6.
[0054] Anti-ERBB3 antibody 09D03 comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 22, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 23 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 25, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 26 as shown in FIG. 3. In an exemplary embodiment, antibody 09D03 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27 as shown in FIG. 2, and immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 28 as shown in FIG. 4. In another exemplary embodiment, antibody 09D03 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 29 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 30, as shown in FIG. 7.
[0055] Anti-ERBB3 antibody 11G01 comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the s amino acid sequence of SEQ ID NO: 31, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 33 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 35 as shown in FIG. 3. In an exemplary embodiment, antibody 11G01 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36 as shown in FIG. 2, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 as shown in FIG. 4. In another exemplary embodiment, antibody 11G01 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 38 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 39, as shown in FIG. 8.
[0056] Anti-ERBB3 antibody 12A07 comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 42 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 16 as shown in FIG. 3. In an exemplary embodiment, antibody 12A07 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43 as shown in FIG. 2, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 as shown in FIG. 4. In another exemplary embodiment, antibody 12A07 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 45 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 46, as shown in FIG. 9.
[0057] Anti-ERBB3 antibody 18H02 comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 47, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 48, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 49 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 50, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 51, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 52 as shown in FIG. 3. In an exemplary embodiment, antibody 18H02 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53 as shown in FIG. 2, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 54 as shown in FIG. 4. In another exemplary embodiment, antibody 18H02 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 55 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 56, as shown in FIG. 10.
[0058] Anti-ERBB3 antibody 22A02 comprises an immunoglobulin heavy chain variable region comprising a CDR.sub.H1 comprising the amino acid sequence of SEQ ID NO: 57, a CDR.sub.H2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDR.sub.H3 comprising the amino acid sequence of SEQ ID NO: 42 as shown in FIG. 1; and an immunoglobulin light chain variable region comprising a CDR.sub.L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR.sub.L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR.sub.L3 comprising the amino acid sequence of SEQ ID NO: 16 as shown in FIG. 3. In an exemplary embodiment, antibody 22A02 comprises an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59 as shown in FIG. 2, and an immunoglobulin light chain variable region comprising the amino acid sequence of SEQ ID NO: 60 as shown in FIG. 4. In another exemplary embodiment, antibody 22A02 comprises an immunoglobulin heavy chain amino acid sequence of SEQ ID NO: 61 and an immunoglobulin light chain amino acid sequence of SEQ ID NO: 62, as shown in FIG. 11.
[0059] It is contemplated that the skilled person would understand that a complete heavy or kappa chain antibody sequences can be created by ligating a variable region as described above to a respective constant region sequence(s) to produce active full length immunoglobulin heavy and light chains. For example, a complete heavy chain comprises a heavy variable sequence followed by the murine or human IgG1 or IgG2b heavy chain constant sequence (which are known in the art) and a complete kappa chain comprises a kappa variable sequence followed by the murine or human kappa light chain constant sequence (which are known in the art). It is further contemplated that CDR1, CDR2, and CDR3 sequences from the immunoglobulin heavy and light chains may be interposed between human or humanized immunoglobulin framework regions.
Tissue Sample
[0060] A tissue sample from a tumor in a human patient (e.g., a tissue sample from a tumor obtained from a human patient, e.g., a human patient being considered for treatment with an ERBB3 inhibitor) can be used as a source of RNA, a source of protein, or a source of thin sections for immunohistochemistry (IHC), so the level of NRG1 in the sample can be determined in practicing the disclosed methods. The tissue sample can be obtained by using conventional tumor biopsy instruments and procedures. Endoscopic biopsy, excisional biopsy, incisional biopsy, fine needle biopsy, punch biopsy, shave biopsy and skin biopsy are examples of recognized medical procedures that can be used by one of skill in the art to obtain tumor samples. The tumor tissue sample should be large enough to provide sufficient RNA, protein, or thin sections for measuring NRG1 and ERBB3 gene expression.
[0061] The tumor tissue sample can be in any form that allows measurement of NRG1 and ERBB3 expression or content. In other words, the tissue sample must be sufficient for RNA extraction, protein extraction, or preparation of thin sections. Accordingly, the tissue sample can be fresh, preserved through suitable cryogenic techniques, or preserved through non-cryogenic techniques. A standard process for handling clinical biopsy specimens is to fix the tissue sample in formalin and then embed it in paraffin. Samples in this form are commonly known as formalin-fixed, paraffin-embedded (FFPE) tissue. Suitable techniques of tissue preparation for subsequent analysis are well-known to those of skill in the art.
NRG1 Gene Expression
[0062] As described herein, determining or measuring the level of NRG1 gene expression in a tissue sample from a tumor can be performed by any suitable method. Several such methods are known in the art. For example, determining NRG1 gene expression can be done by measuring the level or amount of NRG1 protein, or measuring the level or amount of NRG1 RNA, in a sample.
[0063] Neuregulin 1 is produced in numerous isoforms upon expression of the NRG1 gene. Relative abundance of the various isoforms appears to vary, depending on factors such as tissue type and/or developmental stage. The EGF-like domain of Neuregulin 1 is essential for binding to ERBB3. It is found in all of the various NRG1 isoforms as a beta variant (NRG1.beta.) or alpha variant (NRG1.alpha.). Therefore, when determining the level NRG1 gene expression as described herein, the NRG1 assay preferably is designed to detect at least the EGF-like domain of NRG1, in order to detect most, if not all, of the NRG1 isoforms present. Accordingly, in some embodiments, PCR primers are designed to amplify a portion of the EGF-like domain. Similarly, in some embodiments, microarray probes are designed to hybridize with a sequence in the EGF-like domain or a sequence conserved across multiple variants. If an anti-NRG1 antibody is used to detect NRG1 protein, the antibody preferably will recognize the EGF-like domain.
[0064] In some embodiments, classification of a tumor as sensitive or resistant to treatment with an ERBB3 inhibitor is based solely on the expression of NRG1 in a tissue sample from the tumor. In other embodiments, expression of one or more other genes is measured in addition to NRG1 expression, to classify a tumor as sensitive or resistant to treatment with an ERBB3 inhibitor. It is contemplated herein that in embodiments when the expression of one or more other genes is measured in addition to NRG1, the one or more other genes do not include ErbB1, ErbB2, and ErbB3 (e.g., monomers, heterodimers and/or homodimers of any of ErbB1, ErbB2 and ErbB3, and/or phosphorylated ErbB1, ErbB2 and ErbB3 either in monomeric or dimeric form). In other embodiments, the expression of NRG1 is not measured in combination with beta-cellulin (BCT) secretion. It is further contemplated herein that the expression of one or more other genes measured in addition to NRG1 may include genes serving as controls or standards, e.g., for data normalization.
RNA Analysis
[0065] Conventional microarray analysis and quantitative polymerase chain reaction (PCR) are examples of methods for determining the level of NRG1 gene expression at the mRNA level. In some embodiments, RNA is extracted from the cells, tumor or tissue of interest using standard protocols. In other embodiments, RNA analysis is performed using techniques that do not require RNA isolation.
[0066] Methods for rapid and efficient extraction of eukaryotic mRNA, i.e., poly(a) RNA, from tissue samples are well established and known to those of skill in the art. See, e.g., Ausubel et al., 1997, Current Protocols of Molecular Biology, John Wiley & Sons. The tissue sample can be fresh, frozen or fixed paraffin-embedded (FFPE) samples such as clinical study tumor specimens. In general, RNA isolated from fresh or frozen tissue samples tends to be less fragmented than RNA from FFPE samples. FFPE samples of tumor material, however, are more readily available, and FFPE samples are suitable sources of RNA for use in methods of the present invention. For a discussion of FFPE samples as sources of RNA for gene expression profiling by RT-PCR, see, e.g., Clark-Langone et al., 2007, BMC Genomics 8:279. Also see, De Andres et al., 1995, Biotechniques 18:42044; and Baker et al., U.S. Patent Application Publication No. 2005/0095634. The use of commercially available kits with vendor's instructions for RNA extraction and preparation is widespread and common. Commercial vendors of various RNA isolation products and complete kits include Qiagen (Valencia, Calif.), Invitrogen (Carlsbad, Calif.), Ambion (Austin, Tex.) and Exiqon (Woburn, Mass.).
[0067] In general, RNA isolation begins with tissue/cell disruption. During tissue/cell disruption it is desirable to minimize RNA degradation by RNases. One approach to limiting RNase activity during the RNA isolation process is to ensure that a denaturant is in contact with cellular contents as soon as the cells are disrupted. Another common practice is to include one or more proteases in the RNA isolation process. Optionally, fresh tissue samples are immersed in an RNA stabilization solution, at room temperature, as soon as they are collected. The stabilization solution rapidly permeates the cells, stabilizing the RNA for storage at 4.degree. C., for subsequent isolation. One such stabilization solution is available commercially as RNAlater.RTM. (Ambion, Austin, Tex.).
[0068] In some protocols, total RNA is isolated from disrupted tumor material by cesium chloride density gradient centrifugation. In general, mRNA makes up approximately 1% to 5% of total cellular RNA. Immobilized Oligo(dT), e.g., oligo(dT) cellulose, is commonly used to separate mRNA from ribosomal RNA and transfer RNA. If stored after isolation, RNA must be stored under RNase-free conditions. Methods for stable storage of isolated RNA are known in the art. Various commercial products for stable storage of RNA are available.
[0069] Microarray
[0070] The mRNA expression level of NRG1 can be measured using conventional DNA microarray expression profiling technology. A DNA microarray is a collection of specific DNA segments or probes affixed to a solid surface or substrate such as glass, plastic or silicon, with each specific DNA segment occupying a known location in the array. Hybridization with a sample of labeled RNA, usually under stringent hybridization conditions, allows detection and quantitation of RNA molecules corresponding to each probe in the array. After stringent washing to remove non-specifically bound sample material, the microarray is scanned by confocal laser microscopy or other suitable detection method. Modern commercial DNA microarrays, often known as DNA chips, typically contain tens of thousands of probes, and thus can measure expression of tens of thousands of genes simultaneously. Such microarrays can be used in practicing the present invention. Alternatively, custom chips containing as few probes as those needed to measure NRG1, plus necessary controls or standards, e.g., for data normalization, can be used in practicing the disclosed methods.
[0071] To facilitate data normalization, a two-color microarray reader can be used. In a two-color (two-channel) system, samples are labeled with a first fluorophore that emits at a first wavelength, while an RNA or cDNA standard is labeled with a second fluorophore that emits at a different wavelength. For example, Cy3 (570 nm) and Cy5 (670 nm) often are employed together in two-color microarray systems.
[0072] DNA microarray technology is well-developed, commercially available, and widely employed. Therefore, in performing disclosed methods, a person of ordinary skill in the art can use microarray technology to measure expression levels of genes encoding biomarker proteins without undue experimentation. DNA microarray chips, reagents (such as those for RNA or cDNA preparation, RNA or cDNA labeling, hybridization and washing solutions), instruments (such as microarray readers) and protocols are well known in the art and available from various commercial sources. Commercial vendors of microarray systems include Agilent Technologies (Santa Clara, Calif.) and Affymetrix (Santa Clara, Calif.), but other PCR systems can be used.
[0073] Quantitative PCR
[0074] The level of mRNA encoding NRG1 can be measured using conventional quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) technology. Advantages of qRT-PCR include sensitivity, flexibility, quantitative accuracy, and ability to discriminate between closely related mRNAs. Guidance concerning the processing of tissue samples for quantitative PCR is available from various sources, including manufacturers and vendors of commercial instruments and reagents for qRT-PCR (e.g., Qiagen (Valencia, Calif.) and Ambion (Austin, Tex.)). Instruments and systems for automated performance of qRT-PCR are commercially available and used routinely in many laboratories. An example of a well-known commercial system is the Applied Biosystems 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, Calif.).
[0075] Once mRNA is isolated, the first step in gene expression measurement by RT-PCR is the reverse transcription of the mRNA template into cDNA, which is then exponentially amplified in a PCR reaction. Two commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription reaction typically is primed with specific primers, random hexamers, or oligo(dT) primers. Suitable primers are commercially available, e.g., GeneAmp.RTM. RNA PCR kit (Perkin Elmer, Waltham, Mass.). The resulting cDNA product can be used as a template in the subsequent polymerase chain reaction.
[0076] The PCR step is carried out using a thermostable DNA-dependent DNA polymerase. The polymerase most commonly used in PCR systems is a Thermus aquaticus (Taq) polymerase. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification, i.e., regions of the cDNAs reverse transcribed from genes encoding proteins of interest. Therefore, when qRT-PCR is employed in the present invention, primers specific to each marker gene are based on the cDNA sequence of the gene. Commercial technologies such as SYBR.RTM. green or TaqMan.RTM. (Applied Biosystems, Foster City, Calif.) can be used in accordance with the vendor's instructions. Messenger RNA levels can be normalized for differences in loading among samples by comparing the levels of housekeeping genes such as beta-actin or GAPDH. The level of mRNA expression can be expressed relative to any single control sample such as mRNA from normal, non-tumor tissue or cells. Alternatively, it can be expressed relative to mRNA from a pool of tumor samples, or tumor cell lines, or from a commercially available set of control mRNA.
[0077] Suitable primer sets for PCR analysis of expression of genes NRG1 or ERBB3 can be designed and synthesized by one of skill in the art, without undue experimentation. Alternatively, PCR primer sets for practicing the present invention can be purchased from commercial sources, e.g., Applied Biosystems. PCR primers preferably are about 17 to 25 nucleotides in length. Primers can be designed to have a particular melting temperature (Tm), using conventional algorithms for Tm estimation. Software for primer design and Tm estimation are available commercially, e.g., Primer Express.TM. (Applied Biosystems), and also are available on the internet, e.g., Primer3 (Massachusetts Institute of Technology). By applying established principles of PCR primer design, a large number of different primers can be used to measure the expression level of any given gene, including NRG1 and ERBB3.
[0078] gNPA.TM.
[0079] In some embodiments, RNA analysis is performed using a technology that does not involve RNA extraction or isolation. One such technology is quantitative nuclease protection assay, which is commercially available under the name gNPA.TM. (High Throughput Genomics, Inc., Tucson, Ariz.). This technology can be advantageous when the tumor tissue samples to be analyzed are in the form of FFPE material. See, e.g., Roberts et al., 2007, Laboratory Investigation 87:979-997.
Protein Analysis
[0080] In other embodiments, NRG1 and ERBB3 gene expression can be detected at the protein level. Examples of methods for measuring the level of NRG1 or ERBB3 gene expression at the protein level include enzyme linked immunosorbent assay (ELISA) and IHC analysis.
[0081] ELISA
[0082] Performing an NRG1 ELISA requires at least one antibody against NRG1, i.e., the detection antibody. NRG1 protein from a sample to be analyzed is immobilized on a solid support such as a polystyrene microtiter plate. This immobilization can be by non-specific binding, i.e., through adsorption to the surface. Alternatively, immobilization can be by specific binding, i.e., through binding of NRG1 from the sample by a capture antibody (anti-NRG1 antibody different from the detection antibody), in a "sandwich" ELISA. After the NRG1 is immobilized, the detection antibody is added, and the detection antibody forms a complex with the bound NRG1. The detection antibody is linked to an enzyme, either directly or indirectly, e.g., through a secondary antibody that specifically recognizes the detection antibody. Typically between each step, the plate, with bound NRG1, is washed with a mild detergent solution. Typical ELISA protocols also include one or more blocking steps, which involve use of a non-specifically-binding protein such as bovine serum albumin to block unwanted non-specific binding of protein reagents to the plate. After a final wash step, the plate is developed by addition of an appropriate enzyme substrate, to produce a visible signal, which indicates the quantity of NRG1 in the sample. The substrate can be, e.g., a chromogenic substrate or a fluorogenic substrate. ELISA methods, reagents and equipment are well-known in the art and commercially available.
[0083] Immunohistochemistry (IHC)
[0084] The presence and level of NRG1 in a tumor tissue sample, or clinical specimen, can be determined (e.g., visualized) by immunohistochemistry (IHC) or immunofluorescence (IF). Because clinical specimens often are preserved as formalin fixed paraffin embedded (FFPE) blocks, IHC and IF are particularly useful for measuring NRG1 protein in clinical specimens. Assaying NRG1 by IHC or IF requires at least one antibody against NRG1. Anti-NRG1 antibodies suitable for IHC and IF are commercially available. For example, suitable antibodies can be purchased from R&D Systems (Minneapolis, Minn.), abcam (Cambridge, Mass.), Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), or Novus Biologicals (Littleton, Colo.). Using standard techniques, the anti-NRG1 antibody can be used to detect the presence of NRG1 protein in thin sections, e.g., 5 micron sections, obtained from tumors, including FFPE sections and frozen tumor sections. Typically, the tumor sections are initially treated in such a way as to retrieve the antigenic structure of proteins that were fixed in the initial process of collecting and preserving the tumor material. Slides are then blocked to prevent non-specific binding by the anti-NRG1 detection antibody. The presence of NRG1 protein is then detected by binding of the anti-NRG1 antibody (primary antibody) to the NRG1 protein. The detection antibody (secondary antibody), which recognizes and binds to the primary antibody, is linked to a detectable enzyme or fluorophore. Typically, the tumor sections are washed and blocked with non-specific protein such as bovine serum albumin between steps. If the detection antibody is linked to a detectable enzyme, the slide is developed using an appropriate enzyme substrate to produce a visible signal. If the detection antibody is linked to a fluorophore, the slide is viewed by using a fluorescence microscope. The samples can be counterstained with hematoxylin.
Data Interpretation
[0085] An NRG1 score for a tumor can be interpreted with respect to a threshold score. An NRG1 score that is equal to or higher than the threshold score can be interpreted as predictive of the tumor being sensitive (responsive) to treatment with an ERBB3 inhibitor, e.g., an ERBB3 antibody. Alternatively, NRG1 scores equal to or lower than the threshold score can be interpreted as predictive of a tumor being resistant (non-responsive) to treatment with an ERBB3 inhibitor.
[0086] An optimum threshold NRG1 score can be determined (or at least approximated) empirically by performing a threshold determination analysis. Preferably, threshold determination analysis includes receiver operator characteristic (ROC) curve analysis. ROC curve analysis is an established statistical technique, the application of which is within ordinary skill in the art. For a discussion of ROC curve analysis, see generally Zweig et al., 1993, "Receiver operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine," Clin. Chem. 39:561-577; and Pepe, 2003, The statistical evaluation of medical tests for classification and prediction, Oxford Press, New York.
[0087] NRG1 scores and the optimum threshold NRG1 score may vary from tumor type to tumor type. Therefore, a threshold determination analysis preferably is performed on one or more datasets representing any given tumor type to be tested using the present invention. The dataset used for threshold determination analysis includes: (a) actual response data (response or non-response), and (b) an NRG1 score for each tumor sample from a group of tumors. Once an NRG1 score threshold is determined with respect to a given tumor type, that threshold can be applied to interpret NRG1 scores from tumors of that tumor type. In certain embodiments, a threshold score is determined by measuring NRG1 expression in tissue samples of tumors obtained from human patients previously treated with an anti-ERBB3 inhibitor and shown to be sensitive to the anti-ERBB3 inhibitor and human patients previously treated with an anti-ERBB3 inhibitor and shown to be resistant to anti-ERBB3 inhibitor.
[0088] The ROC curve analysis can be performed as follows. Any sample with an NRG1 score greater than or equal to the threshold is identified as a responder (sensitive). Alternatively, any sample with an NRG1 score less than the threshold is identified as a non-responder (resistant). For every NRG1 score from a tested set of samples, "responders" and "non-responders" (hypothetical calls) are classified using that score as the threshold. This process enables calculation of TPR (y vector) and FPR (x vector) for each potential threshold, through comparison of hypothetical calls against the actual response data for the data set. Then an ROC curve is constructed by making a dot plot, using the TPR vector, and FPR vector. If the ROC curve is above the diagonal from (0, 0) point to (1.0, 0.5) point, it shows that the NRG1 test result is a better test result than random.
[0089] The ROC curve can be used to identify the best operating point, or optimum threshold. The best operating point is the one that yields the best balance between the cost of false positives weighed against the cost of false negatives. These costs need not be equal. The average expected cost (C) of classification at point x,y in the ROC space is determined by the following formula.
C=(1-p)alpha*x+p*beta(1-y)
[0090] wherein:
[0091] alpha=cost of a false positive,
[0092] beta=cost of missing a positive (false negative), and
[0093] p=proportion of positive cases.
[0094] False positives and false negatives can be weighted differently by assigning different values for alpha and beta. For example, if it is decided to include more patients in the responder group at the cost of treating more patients who are non-responders, one can put more weight on alpha. In this case, it is assumed that the cost of false positive and false negative is the same (alpha equals to beta). Therefore, the average expected cost of classification at point x,y in the ROC space is:
C'=(1-p)*x+p*(1-y).
The smallest C' can be calculated after using all pairs of false positive and false negative (x, y). The optimum score threshold is calculated as the score of the (x, y) at C'.
[0095] In addition to predicting whether a tumor will be sensitive or resistant to treatment with an ERBB3 inhibitor, i.e., binary classification, an NRG1 score provides an approximate, but useful, indication of how likely a tumor is to be sensitive or resistant. In general, the higher the NRG1 score, the more likely a tumor is to be sensitive to an ERBB3 inhibitor, and the lower the NRG1 score, the more likely a tumor is to be resistant to an ERBB3 inhibitor.
Test Kits
[0096] Also disclosed is a diagnostic test kit comprising certain components for performing methods of the invention. A diagnostic test kit enhances convenience, speed and reproducibility in the performance of diagnostic assays. For example, in an exemplary qRT-PCR-based embodiment, a basic diagnostic test kit includes PCR primers for analyzing expression of NRG1. In other embodiments, a more elaborate test kit contains not only PCR primers, but also buffers, reagents and detailed instructions for measuring NRG1 expression levels, using PCR technology. In some embodiments, the kit includes a test protocol and all the consumable components needed for the test, except the RNA sample(s).
[0097] In an exemplary DNA microarray-based embodiment, a test kit includes a micro fluidic card (array) designed for use with a particular instrument. Optionally, the micro fluidic card is a custom made device designed specifically for measurement of NRG1. Such custom micro fluidic cards are commercially available. For example, the TaqMan Array is a 384-well micro fluidic card (array) designed for use with the Applied Biosystems 7900 HT Fast Real Time PCR System (Applied Biosystems, Foster City, Calif.). It is understood that additional probes can optionally be included on a fluidic card to measure the expression of one or more additional genes. Such additional genes may be included to serve as controls or standards, e.g., for data normalization, or may be otherwise informative.
[0098] In some embodiments, the test kit contains materials for determining NRG1 content by IHC. An IHC kit, for example, may contain a primary antibody against NRG1, and a secondary antibody conjugated to a reporter enzyme, e.g., horseradish peroxidase. In some embodiments, the secondary antibody is replaced with a conjugated polymer that specifically recognizes the primary antibody.
EXAMPLES
[0099] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the invention in any way.
Example 1: Xenograft Tumor Response to AV-203
[0100] Evaluation of tumor response to AV-203 was performed as follows. In order to establish xenograft tumor, tumor cells were initially grown in culture at 37.degree. C. in an atmosphere containing 5% CO2, using medium containing 10% fetal bovine serum. Cells were inoculated subcutaneously into the flank of 8-week old female NCR nude or CB.17 SCID mice (Taconic Labs) with 2-10.times.10.sup.6 cells per mouse in 50% matrigel (BD Biosciences, Cat No. 356237). Tumor measurements were taken twice weekly using vernier calipers. Tumor volume was calculated using the formula: width.times.width.times.length/2. When tumors reached approximately 200 mm.sup.3, the mice were randomized into two groups of 10 mice each, PBS vehicle control or AV-203 dosed intraperitoneally (IP) at 20 mg/kg twice weekly. In some studies, a second control group was used, which received human IgG dosed at 20 mg/kg IP twice weekly.
[0101] In total, 25 xenograft tumors were treated with AV-203. Response to AV-203 was varied, ranging from -10% tumor growth inhibition (TGI) to tumor regression. "Tumor regression" means that a tumor is smaller at the end of the evaluation period compared to the size of the tumor at the beginning of the evaluation period prior to treatment. Based on the tumor growth inhibition achieved, responders (defined as those with TGI>60%) and non-responders (defined as those with TGI<60%) were identified. Of the 25 tumors evaluated, 10 were found to be responders (e.g., a hNRG1 Ct value equal to or less than 22.9), and 15 were found to be non-responders (Table 1). These groups enabled the identification of a molecular marker for AV-203 responsiveness.
TABLE-US-00001 TABLE 1 Summary of Data from Xenograft Studies Xenograft Cancer TGI hNRG1 hERBB3 Model Type (%) Ct Ct HCC2429 Lung -10.0 23.8 23.4 H23 Lung -3.8 24.2 24.5 AN3CA Endometrial 0.0 27.6 22.8 LS1034 Colorectal 0.0 32.4 18.6 MFM-223 Breast 0.0 35.5 19.7 MFE-296 Endometrial 5.0 32.3 23.8 H716 Colorectal 11.0 34.0 21.4 H441 Lung 11.1 31.7 20.9 SW1990 Pancreas 29 18.6 21.4 HCC827 Lung 32.0 36.0 21.5 Snu16 Gastric 35.0 31.8 20.5 LOVO Colorectal 49.0 28.6 18.4 H1048 Lung 53.0 26.0 20.8 H1993 Lung 56.6 24.4 22.6 A431 Skin 58.0 21.2 21.6 H322 Lung 62.6 22.4 21.2 CAPAN-1 Pancreas 66.0 24.2 19.2 A498 Kidney 68.3 22.9 21.3 H522 Lung 68.6 17.5 24.2 Calu-3 Lung 69.0 20.8 19.4 A549 Lung 74.0 20.6 20.3 BxPC3 Pancreas 76.5 20.1 19.3 MDA-MB-453 Breast 85.3 30.8 18.5 H358 Lung 89.0 22.2 27.3 HCC95 Lung 95.0 17.7 22.2
Example 2: Relationship Between AV-203 Response and NRG1 Levels
[0102] For the 25 tumors that were evaluated, RNA was prepared from an untreated healthy tumor. Flash frozen tumor samples were pulverized using Covaris CryoPrep.TM. system (Covaris Inc. Model CP-02). Approximately 30 mg of pulverized tumor material was transferred into a 2 mL SafeLock.TM. tube (Eppendorf, Cat. No. 02236652). One mL of TRIzol, (Invitrogen, Cat. No. 15596-026) and one (5 mm) stainless steel shaker bead (Qiagen, Cat. No. 69989) were added to each tube. The tubes were then placed in racks in the Tissue Lyser II.TM. (Qiagen, Cat. No. 85300) for cell lysis. The samples were shaken for two 30-second cycles. The racks then were rotated and shaken again for two more cycles.
[0103] The total RNA (aqueous phase) was extracted from the cell lysate by the addition of 200 .mu.L chloroform to each sample. The samples were shaken vigorously for 15 seconds and centrifuged at 12,000 rpm for 15 minutes at 4.degree. C. The upper supernatants (350 (IL) were transferred to new 2 mL SafeLock.TM. tube and placed in QIAcube.TM. Automated Purification Instrument (Qiagen, Cat. No. 9001292) for automated RNA isolation with RNeasy.TM. Mini QIAcube Kit (Qiagen, Cat. No. 74116). A DNase I treatment step was included in the RNA isolation. The isolated total RNA concentration was measured with NanoDrop.TM. (Thermo Scientific, Model 1000), and the RNA integrity was determined by electrophoresis to verify the position of the 18S band and detection of any RNA degradation. The RNA was aliquoted into two 1.7 mL microtubes (Axygen Cat. No. MCT-175-C) and stored at -80.degree. C.
[0104] Human NRG1, ERBB3 and .beta.-Actin expression levels were determined using quantitative, real-time RT-PCR. Total tumor RNA expression was assayed using QuantiTect.TM. SYBR Green RT PCR Kit (Qiagen Cat. No. 204245) and run on a Applied Biosystems Thermocycler, Model 7900 HT Fast Real-Time PCR System (Applied Biosystems, Cat. No. 4329001). Each RNA tumor sample was assayed in quadruplicate in 20 .mu.L reactions. Each reaction contained 50 ng of total tumor RNA, 10 .mu.L of 2.times. QuantiTect.TM. SYBR Green RT-PCR Master Mix, 0.2 .mu.L QuantiTect RT Mix and forward and reverse gene specific primers (synthesized by Eurofins MWG Operon) at final concentration of 900 nM. The reactions setup using Qiagen BioRobot Rapidplate liquid handling system in a 384-well plate (Applied Biosystems, Cat. No. 4309849) and sealed with MicroAmp Optical Adhesive Film (Applied Biosystems, Cat. No. 4311971). The Real-Time plate was assayed with the following Program: 30 minutes at 50.degree. C., 15 minutes at 95.degree. C., followed by 40 cycles of 15 seconds at 95.degree. C., 30 seconds at 54.degree. C., 30 seconds at 72.degree. C. Cycle threshold (Ct) value averages were then calculated in Microsoft Excel. Ct value is defined as cycle number at the threshold level of log-based fluorescence. A low Ct value reflects a high specific RNA level (i.e., a low Ct value reflects high expression of NRG1).
[0105] AV-203 tumor growth inhibition in these 25 tumors was then plotted against NRG1 expression levels (represented as Ct values) within each tumor. As shown in FIG. 12, a positive correlation was observed between tumor growth inhibition and NRG1 expression. More specifically, increased tumor growth inhibition after treatment with AV-203 correlated with increased NRG1 expression (lower Ct value). This correlation was found to be highly statistically significant (Table 2).
TABLE-US-00002 TABLE 2 Statistical Analysis of NRG1 Expression and AV-203 In Vivo Efficacy Number of XY Pairs 25 Spearman r -0.601 95% confidence interval -0.8093 to -0.2583 P value (two-tailed) 0.0015 P value summary ** Exact or approximate P value? Gaussian Approximation Is the correlation significant? (alpha = 0.05) Yes
Example 3: Relationship Between AV-203 Response and ERBB3 Levels
[0106] As described for NRG1, ERBB3 levels were also determined by quantitative RT-PCR from these 25 tumor models. AV-203 tumor growth inhibition in these 25 tumors was then plotted against ERBB3 expression levels (represented as Ct values) for each tumor. As shown in FIG. 13, tumor growth inhibition did not correlate with increased ERBB3 expression, even though ERBB3 is the target for AV-203.
Example 4: NRG1 Threshold Determination
[0107] Using the Ct values for NRG1 from the AV-203 responding (sensitive) and non-responding (resistant) xenograft tumor models, a receiver operator characteristic (ROC) curve was generated to determine an NRG1 expression threshold useful to predict AV-203 tumor response (FIG. 14). In general, a ROC curve is used to determine if the test result (e.g., an NRG1 biomarker test result) is significantly different from a random event and to determine the optimum threshold score (e.g., an optimum threshold NRG1 score). For example, if the test results are random, a diagonal line would divide the ROC space. In this example, the ROC curve is above the diagonal line indicating that the test is achieving a high degree of separation between responders and non-responders (FIG. 14). As shown in FIG. 14, the optimum threshold is Ct=22.9, which yields a false positive rate of 0.13, and a false negative rate of 0.2. Results of the ROC analysis indicate that AV-203 tumor response can be predicted by high NRG1 expression level, using a cut-off at Ct value 22.9. Using the xenograft tumor models listed in Table 1, the Ct value cut-off of 22.9 (e.g., equal to or less than 22.9) predicted AV-203 response with statistical significance (FIG. 15). As shown in FIG. 15, increased TGI was observed following treatment with AV-203 in tumors with a low Ct value (which indicates high NRG1 expression and a high NRG1 score).
Example 5: Primary Human Tumor Model Response
[0108] In order to validate this prediction method for response to AV-203, primary human tumor models were classified as having high or low NRG1 expression, based on a microarray analysis of the primary human tumors. These models were then tested for response to AV-203 treatment.
[0109] Evaluation of human primary tumor response to AV-203 was performed as follows. Primary human tumors were collected from surgical resection. Tumor samples were shipped on wet ice overnight in media containing 10% FBS. Upon arrival, tumor samples were cut into 2 mm.times.2 mm fragments and implanted subcutaneously into five NCR nude mice (Taconic) using a 10 gauge trocar needle. In order to establish xenograft tumor material to be used for efficacy studies, tumors were collected at 500 mm.sup.3 and propagated further into 20 sites. Once these tumors achieved a size of 500 mm.sup.3, they were collected for further propagation, efficacy studies, and molecular characterization. For efficacy studies, tumor fragments were implanted subcutaneously into 8-week old female NCR nude mice. Tumor measurements were taken twice weekly, using vernier calipers. Tumor volume was calculated using the formula: width.times.width.times.length/2. When tumors reached approximately 200 mm.sup.3, the mice were randomized into two groups of 10 mice each, PBS vehicle control, or AV-203 dosed intraperitoneally (IP) at 20 mg/kg twice weekly. In some studies, a second control group was used, which received human IgG dosed at 20 mg/kg twice weekly.
[0110] In total, xenografts of four primary human lung tumors were treated with AV-203. Two were predicted to respond, and two were predicted not to respond, based on NRG1 expression ranking of the microarray data. The two NRG1 high human primary tumors that were predicted to respond to AV-203 treatment did respond, displaying significant tumor growth inhibition. Data from these sensitive tumors are summarized in FIGS. 16 and 17. The two NRG1 low human primary tumors predicted not to respond, did not respond to AV-203 treatment. Data from the resistant tumors are summarized in FIGS. 18 and 19. These data demonstrated that human primary tumor sensitivity to treatment with AV-203 can be predicted on the basis of high NRG1 expression in the tumor.
Example 6: Xenograft Tumor Model Response to Anti-ERBB3 Antibody AV-203
[0111] Two additional xenograft tumors (i.e., a CAL-27 head and neck tumor xenograft and a KYSE 150 esophageal tumor xenograft) were selected for high NRG1 expression based on the Ct value cut-off of 22.9 (e.g., equal to or less than 22.9) outlined in Example 4 and, therefore, were predicted to respond to AV-203. In addition, an H520 non-small cell lung carcinoma (NSCLC) xenograft tumor was selected for low NRG1 expression based on the same Ct value cut-off of 22.9 outlined in Example 4 and, therefore, was predicted not to respond to AV-203. All three tumor models were treated with 20 mg/kg of antibody AV-203. The response to antibody AV-203 ranged from 75.2-79.9% tumor growth inhibition for the sensitive tumors (i.e., CAL-27 and KYSE 150) (TGI, see Table 3 and FIGS. 20-21) and was -8.0% TGI for the resistant tumor (i.e., H520) (see Table 3 and FIG. 22).
TABLE-US-00003 TABLE 3 Summary Table of Xenograft Tumors Treated with Antibody AV-203 Xenograft Cancer TGI hNRG1 Model Type (%) Ct CAL-27 Head andNeck 75.2 20.1 KYSE 150 Esophageal 79.9 19.1 H520 NSCLC -8.0 23.7
[0112] These data demonstrate that the response of solid tumors to treatment with AV-203 can be predicted by measuring NRG1 expression.
Example 7: Xenograft Tumor Model Response to Anti-ERBB3 Antibody 11G01
[0113] To validate the prediction method for response to other anti-ERBB3 antibodies, tumor models expressing high NRG1 levels were treated with an anti-ERBB3 antibody having a different mechanism of action from AV-203. As discussed above, AV-203 inhibits binding of NRG1 to ERBB3, therefore, the following experiment was conducted using an antibody that blocks dimerization of ERBB3 without inhibiting binding of NRG1 to ERBB3, i.e., antibody 11G01. Evaluation of tumor response to antibody 11G01 was performed as described in Example 1.
[0114] Three xenograft tumors (i.e., a BxPC3 pancreas tumor xenograft, a DU145 prostate tumor xenograft and an H322 lung tumor xenograft) were selected for high NRG1 expression based on the Ct value cut-off of 22.9 (e.g., equal to or less than 22.9) outlined in Example 4 and, therefore, were predicted to respond to an anti-ERBB3 antibody. All 3 tumor models were treated with 20 mg/kg of antibody 11G01. Response to antibody 11G01 ranged from 60-72% tumor growth inhibition (TGI, see Table 4 and FIGS. 23-25).
TABLE-US-00004 TABLE 4 Summary Table of Xenograft Tumors Treated with Antibody 11G01 Xenograft Cancer TGI hNRG1 hERBB3 Model Type (%) Ct Ct BxPC3 Pancreas 71.8 20.1 19.3 DU145 Prostate 60.0 22.3 19.6 H322 Lung 61.5 22.4 21.2
[0115] Using the same cut-off of response as described in Examples 1 and 4 (i.e., an hNRG1 Ct value equal to or less than 22.9), all three tumors were considered as responding to antibody 11G01. These data demonstrate that the response of solid tumors to treatment with anti-ERBB3 antibodies including neutralizing antibodies (e.g., AV-203) and dimerization inhibiting antibodies (e.g., antibody 11G01) can be predicted by measuring high hNRG1 expression.
INCORPORATION BY REFERENCE
[0116] The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference for all purposes.
EQUIVALENTS
[0117] The invention can be embodied in other specific forms with departing from the essential characteristics thereof. The foregoing embodiments therefore are to be considered illustrative rather than limiting on the invention described herein. The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
Sequence CWU
1
1
6215PRTArtificial Sequencesource/note="Description of Artificial Sequence
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217PRTArtificial Sequencesource/note="Description of Artificial Sequence
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Asp Ser Val Lys 1 5 10
15 Gly 310PRTArtificial Sequencesource/note="Description of
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Phe Asp Tyr 1 5 10 411PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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10 57PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 5Ala Ala Ser Thr Leu Asp
Ser 1 5 69PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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7119PRTArtificial Sequencesource/note="Description of Artificial
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Gly Leu Val Lys Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asp Tyr 20 25 30
Ala Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Thr Ile Ser
Asp Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Trp Gly Asp Tyr Asp Gly Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val
Thr Val Ser Ser 115 8107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr
20 25 30 Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35
40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ser Tyr Pro Tyr
85 90 95 Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105
9471PRTArtificial Sequencesource/note="Description of Artificial Sequence
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Leu Leu Leu Leu Trp 1 5 10
15 Leu Arg Gly Ala Arg Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly
20 25 30 Leu Val
Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35
40 45 Phe Thr Phe Ser Asp Tyr Ala
Met Ser Trp Ile Arg Gln Ala Pro Gly 50 55
60 Lys Gly Leu Glu Trp Val Ser Thr Ile Ser Asp Gly
Gly Thr Tyr Thr 65 70 75
80 Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
85 90 95 Ala Lys Asn
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100
105 110 Thr Ala Val Tyr Tyr Cys Ala Arg
Glu Trp Gly Asp Tyr Asp Gly Phe 115 120
125 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr 130 135 140
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145
150 155 160 Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165
170 175 Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His 180 185
190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser 195 200 205
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 210
215 220 Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 225 230
235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro 245 250
255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 260 265 270 Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275
280 285 Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295
300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 305 310 315
320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335 Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340
345 350 Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360
365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys 370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385
390 395 400 Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405
410 415 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 420 425
430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450
455 460 Leu Ser Leu Ser
Pro Gly Lys 465 470 10236PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Leu Trp 1 5 10 15
Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30 Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35
40 45 Gln Glu Ile Ser Gly Tyr Leu Ser Trp
Tyr Gln Gln Lys Pro Gly Lys 50 55
60 Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp
Ser Gly Val 65 70 75
80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
85 90 95 Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100
105 110 Tyr Asp Ser Tyr Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile 115 120
125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp 130 135 140
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145
150 155 160 Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165
170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp 180 185
190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr 195 200 205 Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210
215 220 Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 225 230 235
115PRTArtificial Sequencesource/note="Description of Artificial Sequence
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1217PRTArtificial Sequencesource/note="Description of Artificial Sequence
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Gln Asn Phe Lys 1 5 10
15 Gly 1311PRTArtificial Sequencesource/note="Description of
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Ala Met Asp Tyr 1 5 10
1416PRTArtificial Sequencesource/note="Description of Artificial Sequence
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Thr Tyr Leu Glu 1 5 10
15 157PRTArtificial Sequencesource/note="Description of Artificial
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5 169PRTArtificial Sequencesource/note="Description of
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Trp Thr 1 5 17120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Gly Thr 1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His
20 25 30 Trp Leu His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Leu Asp Pro Ser Asp Phe Tyr
Ser Asn Tyr Asn Gln Asn Phe 50 55
60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser
Thr Ala Tyr 65 70 75
80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Leu
Leu Ser Gly Asp Tyr Ala Met Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Ser Val Thr Val Ser Ser
115 120 18112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Leu Gly 1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30 Asn Gly Asn Thr Tyr
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40 45 Pro Lys Ser Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95 Ser Tyr Val Pro
Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105 110 19463PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Thr Gly 1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg
20 25 30 Pro Gly Thr Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35
40 45 Thr Ser His Trp Leu His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu 50 55
60 Glu Trp Ile Gly Val Leu Asp Pro Ser Asp Phe Tyr Ser
Asn Tyr Asn 65 70 75
80 Gln Asn Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser
85 90 95 Thr Ala Tyr Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100
105 110 Tyr Tyr Cys Ala Arg Gly Leu Leu Ser
Gly Asp Tyr Ala Met Asp Tyr 115 120
125 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr
Thr Pro 130 135 140
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser 145
150 155 160 Met Val Thr Leu Gly
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val 165
170 175 Thr Val Thr Trp Asn Ser Gly Ser Leu Ser
Ser Gly Val His Thr Phe 180 185
190 Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val
Thr 195 200 205 Val
Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala 210
215 220 His Pro Ala Ser Ser Thr
Lys Val Asp Lys Lys Ile Val Pro Arg Asp 225 230
235 240 Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
Glu Val Ser Ser Val 245 250
255 Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
260 265 270 Pro Lys
Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu 275
280 285 Val Gln Phe Ser Trp Phe Val
Asp Asp Val Glu Val His Thr Ala Gln 290 295
300 Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg Ser Val Ser 305 310 315
320 Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
325 330 335 Cys Arg Val
Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile 340
345 350 Ser Lys Thr Lys Gly Arg Pro Lys
Ala Pro Gln Val Tyr Thr Ile Pro 355 360
365 Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu
Thr Cys Met 370 375 380
Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn 385
390 395 400 Gly Gln Pro Ala
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr 405
410 415 Asp Gly Ser Tyr Phe Val Tyr Ser Lys
Leu Asn Val Gln Lys Ser Asn 420 425
430 Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu
Gly Leu 435 440 445
His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450
455 460 20238PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Pro Ala 1 5 10 15
Ser Ser Ser Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val
20 25 30 Ser Leu Gly Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35
40 45 Val His Ser Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro 50 55
60 Gly Gln Ser Pro Lys Ser Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser 65 70 75
80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95 Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100
105 110 Phe Gln Gly Ser Tyr Val Pro Trp Thr
Phe Gly Gly Gly Thr Lys Leu 115 120
125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro 130 135 140
Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145
150 155 160 Asn Asn Phe Tyr Pro
Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165
170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys
Asp 195 200 205 Glu
Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210
215 220 Ser Thr Ser Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 225 230
235 217PRTArtificial Sequencesource/note="Description of
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1 5 2216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Ser 1 5 10 15
2310PRTArtificial Sequencesource/note="Description of Artificial Sequence
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5 10 2416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Tyr 1 5 10 15
257PRTArtificial Sequencesource/note="Description of Artificial Sequence
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269PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 26Met Gln His Leu Glu Tyr Pro Phe Thr 1
5 27120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 27Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Arg Pro
Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe
20 25 30 Gly Leu Ser Val Gly
Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35
40 45 Trp Leu Ala His Ile Trp Trp Asp Asp
Asp Lys Tyr Tyr Asn Pro Ala 50 55
60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val 65 70 75
80 Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr
85 90 95 Cys Ala Arg Ile
Gly Ala Asp Ala Leu Pro Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Thr Leu Thr Val Ser Ser
115 120 28112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Pro Gly 1 5 10 15
Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser
20 25 30 Asn Gly Asn Thr Tyr
Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser
Asn Leu Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr
Leu Arg Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His
85 90 95 Leu Glu Tyr Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100
105 110 29475PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
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Ala Tyr 1 5 10 15
Val Leu Ser Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Arg
20 25 30 Pro Ser Gln Thr Leu
Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu 35
40 45 Ser Thr Phe Gly Leu Ser Val Gly Trp
Ile Arg Gln Pro Ser Gly Lys 50 55
60 Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp
Lys Tyr Tyr 65 70 75
80 Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
85 90 95 Asn Gln Val Phe
Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala 100
105 110 Thr Tyr Tyr Cys Ala Arg Ile Gly Ala
Asp Ala Leu Pro Phe Asp Tyr 115 120
125 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr
Thr Pro 130 135 140
Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser 145
150 155 160 Ser Val Thr Ser Gly
Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val 165
170 175 Thr Val Thr Trp Asn Ser Gly Ser Leu Ser
Ser Ser Val His Thr Phe 180 185
190 Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val
Thr 195 200 205 Val
Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala 210
215 220 His Pro Ala Ser Ser Thr
Thr Val Asp Lys Lys Leu Glu Pro Ser Gly 225 230
235 240 Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys
Lys Glu Cys His Lys 245 250
255 Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro
260 265 270 Pro Asn
Ile Lys Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr 275
280 285 Cys Val Val Val Asp Val Ser
Glu Asp Asp Pro Asp Val Gln Ile Ser 290 295
300 Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln
Thr Gln Thr His 305 310 315
320 Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
325 330 335 Gln His Gln
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn 340
345 350 Asn Lys Asp Leu Pro Ser Pro Ile
Glu Arg Thr Ile Ser Lys Ile Lys 355 360
365 Gly Leu Val Arg Ala Pro Gln Val Tyr Thr Leu Pro Pro
Pro Ala Glu 370 375 380
Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe 385
390 395 400 Asn Pro Gly Asp
Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu 405
410 415 Glu Asn Tyr Lys Asp Thr Ala Pro Val
Leu Asp Ser Asp Gly Ser Tyr 420 425
430 Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser Lys Trp Glu
Lys Thr 435 440 445
Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr 450
455 460 Leu Lys Lys Thr Ile
Ser Arg Ser Pro Gly Lys 465 470 475
30239PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 30Met Arg Cys Leu Ala Glu Phe Leu
Gly Leu Leu Val Leu Trp Ile Pro 1 5 10
15 Gly Ala Ile Gly Asp Ile Val Leu Thr Gln Thr Ala Pro
Ser Val Pro 20 25 30
Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser
35 40 45 Leu Leu His Ser
Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg 50
55 60 Pro Gly Gln Ser Pro Gln Leu Leu
Ile Tyr Arg Met Ser Asn Leu Ala 65 70
75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ala Phe 85 90
95 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
100 105 110 Cys Met Gln
His Leu Glu Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys 115
120 125 Leu Glu Ile Lys Arg Ala Asp Ala
Ala Pro Thr Val Ser Ile Phe Pro 130 135
140 Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe 145 150 155
160 Leu Asn Asn Phe Tyr Pro Arg Asp Ile Asn Val Lys Trp Lys Ile Asp
165 170 175 Gly Ser Glu Arg
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp 180
185 190 Ser Lys Asp Ser Thr Tyr Ser Met Ser
Ser Thr Leu Thr Leu Thr Lys 195 200
205 Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr
His Lys 210 215 220
Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225
230 235 315PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 31Asp His Ile Ile His 1 5 3217PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 32Tyr Ile Tyr Pro Arg Asp Gly Tyr Ile Lys Tyr Asn Glu Lys Phe
Lys 1 5 10 15 Gly
338PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 33Gly Tyr Tyr Tyr Ala Met Asp Tyr 1
5 3416PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 34Arg Ser Ser Gln Ser Ile Val
His Ser Ile Gly Asn Thr Tyr Leu Glu 1 5
10 15 359PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 35Phe Gln Gly Ser His Val Pro Phe Thr 1 5
36117PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 36Gln Val Gln Leu Gln Gln
Ser Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5
10 15 Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr
Thr Phe Thr Asp His 20 25
30 Ile Ile His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp
Ile 35 40 45 Gly
Tyr Ile Tyr Pro Arg Asp Gly Tyr Ile Lys Tyr Asn Glu Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu
Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Val Asn Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Phe Cys 85 90
95 Ala Arg Gly Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
100 105 110 Val Thr
Val Ser Ser 115 37112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 37Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser
Leu Gly 1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser
20 25 30 Ile Gly Asn Thr Tyr
Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55
60 Glu Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75
80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95 Ser His Val Pro
Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100
105 110 38460PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 38Met Glu Trp Ser Trp Val Ser Leu Phe Phe Leu Ser Val Thr
Thr Gly 1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Leu Val Lys
20 25 30 Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe 35
40 45 Thr Asp His Ile Ile His Trp Met Lys
Gln Arg Pro Glu Gln Gly Leu 50 55
60 Glu Trp Ile Gly Tyr Ile Tyr Pro Arg Asp Gly Tyr Ile
Lys Tyr Asn 65 70 75
80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95 Thr Ala Tyr Met
Gln Val Asn Ser Leu Thr Ser Glu Asp Ser Ala Val 100
105 110 Tyr Phe Cys Ala Arg Gly Tyr Tyr Tyr
Ala Met Asp Tyr Trp Gly Gln 115 120
125 Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro
Ser Val 130 135 140
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr 145
150 155 160 Leu Gly Cys Leu Val
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr 165
170 175 Trp Asn Ser Gly Ser Leu Ser Ser Gly Val
His Thr Phe Pro Ala Val 180 185
190 Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro
Ser 195 200 205 Ser
Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala 210
215 220 Ser Ser Thr Lys Val Asp
Lys Lys Ile Val Pro Arg Asp Cys Gly Cys 225 230
235 240 Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser
Ser Val Phe Ile Phe 245 250
255 Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val
260 265 270 Thr Cys
Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe 275
280 285 Ser Trp Phe Val Asp Asp Val
Glu Val His Thr Ala Gln Thr Gln Pro 290 295
300 Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val
Ser Glu Leu Pro 305 310 315
320 Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
325 330 335 Asn Ser Ala
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr 340
345 350 Lys Gly Arg Pro Lys Ala Pro Gln
Val Tyr Thr Ile Pro Pro Pro Lys 355 360
365 Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met
Ile Thr Asp 370 375 380
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro 385
390 395 400 Ala Glu Asn Tyr
Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser 405
410 415 Tyr Phe Val Tyr Ser Lys Leu Asn Val
Gln Lys Ser Asn Trp Glu Ala 420 425
430 Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His
Asn His 435 440 445
His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450
455 460 39238PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 39Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile
Pro Ala 1 5 10 15
Ser Arg Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30 Ser Leu Gly Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35
40 45 Val His Ser Ile Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro 50 55
60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser 65 70 75
80 Gly Val Pro Glu Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95 Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100
105 110 Phe Gln Gly Ser His Val Pro Phe Thr
Phe Gly Ser Gly Thr Lys Leu 115 120
125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro 130 135 140
Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145
150 155 160 Asn Asn Phe Tyr Pro
Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165
170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys
Asp 195 200 205 Glu
Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210
215 220 Ser Thr Ser Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 225 230
235 405PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 40Ser Tyr Trp Met His 1
5 4117PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 41Met Ile Asp Pro Ser Asp Val
Tyr Thr Asn Tyr Asn Pro Lys Phe Lys 1 5
10 15 Gly 426PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Asn Tyr Ser Gly Asp Tyr 1 5
43115PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 43Gln Val Gln Leu Leu Gln Pro Gly
Ala Glu Leu Val Arg Pro Gly Thr 1 5 10
15 Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe
Ser Ser Tyr 20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Met Ile Asp
Pro Ser Asp Val Tyr Thr Asn Tyr Asn Pro Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Val
Asp Thr Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Tyr Ser Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110 Val Ser Ser
115 44112PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 44Asp Val Leu Met Thr Gln
Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5
10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25
30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Tyr Cys Phe Gln Gly 85 90
95 Ser Tyr Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
45458PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 45Met Gly Trp Ser Cys Ile Ile Val
Leu Leu Val Ser Thr Ala Thr Cys 1 5 10
15 Val His Ser Gln Val Gln Leu Leu Gln Pro Gly Ala Glu
Leu Val Arg 20 25 30
Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe
35 40 45 Ser Ser Tyr Trp
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50
55 60 Glu Trp Ile Gly Met Ile Asp Pro
Ser Asp Val Tyr Thr Asn Tyr Asn 65 70
75 80 Pro Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp
Thr Ser Ser Ser 85 90
95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110 Tyr Tyr Cys
Ala Arg Asn Tyr Ser Gly Asp Tyr Trp Gly Gln Gly Thr 115
120 125 Thr Leu Thr Val Ser Ser Ala Lys
Thr Thr Pro Pro Ser Val Tyr Pro 130 135
140 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
Thr Leu Gly 145 150 155
160 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
165 170 175 Ser Gly Ser Leu
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180
185 190 Ser Asp Leu Tyr Thr Leu Ser Ser Ser
Val Thr Val Pro Ser Ser Thr 195 200
205 Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala
Ser Ser 210 215 220
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 225
230 235 240 Cys Ile Cys Thr Val
Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 245
250 255 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu
Thr Pro Lys Val Thr Cys 260 265
270 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
Trp 275 280 285 Phe
Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 290
295 300 Glu Gln Phe Asn Ser Thr
Phe Arg Ser Val Ser Glu Leu Pro Ile Met 305 310
315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
Cys Arg Val Asn Ser 325 330
335 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
340 345 350 Arg Pro
Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 355
360 365 Met Ala Lys Asp Lys Val Ser
Leu Thr Cys Met Ile Thr Asp Phe Phe 370 375
380 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly
Gln Pro Ala Glu 385 390 395
400 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
405 410 415 Val Tyr Ser
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 420
425 430 Thr Phe Thr Cys Ser Val Leu His
Glu Gly Leu His Asn His His Thr 435 440
445 Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450
455 46238PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 46Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile
Pro Ala 1 5 10 15
Ser Ser Ser Asp Val Leu Met Thr Gln Ile Pro Leu Ser Leu Pro Val
20 25 30 Ser Leu Gly Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35
40 45 Val His Ser Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro 50 55
60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser 65 70 75
80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95 Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100
105 110 Phe Gln Gly Ser Tyr Val Pro Trp Thr
Phe Gly Gly Gly Thr Lys Leu 115 120
125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro 130 135 140
Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145
150 155 160 Asn Asn Phe Tyr Pro
Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165
170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys
Asp 195 200 205 Glu
Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210
215 220 Ser Thr Ser Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 225 230
235 475PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 47Thr Tyr Gly Met Ser 1
5 4817PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 48Trp Ile Asn Thr Tyr Ser Gly
Val Pro Thr Tyr Ala Asp Asp Phe Lys 1 5
10 15 Gly 4912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49Gly Arg Asp Gly Tyr Gln Val Ala Trp Phe Ala Tyr 1
5 10 5011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Ile Thr Ser Thr Asp Ile Asp Asp Asp Met Asn 1 5
10 517PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 51Glu Gly Asn Thr Leu Arg Pro 1 5
529PRTArtificial Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 52Leu Gln Ser Asp Asn Leu Pro Tyr Thr 1
5 53121PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 53Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro
Gly Glu 1 5 10 15
Ala Val Lys Ile Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Thr Tyr
20 25 30 Gly Met Ser Trp Val
Lys Gln Ala Pro Gly Arg Ala Leu Lys Trp Met 35
40 45 Gly Trp Ile Asn Thr Tyr Ser Gly Val
Pro Thr Tyr Ala Asp Asp Phe 50 55
60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Ser Ser Ala Ser
Thr Ala Tyr 65 70 75
80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95 Ala Arg Gly Arg
Asp Gly Tyr Gln Val Ala Trp Phe Ala Tyr Trp Gly 100
105 110 Gln Gly Thr Leu Val Thr Val Ser Ala
115 120 54107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 54Glu Thr Thr Val Thr Gln Ser Pro Ala Ser Leu Ser Met Ala
Ile Gly 1 5 10 15
Asp Lys Val Thr Ile Arg Cys Ile Thr Ser Thr Asp Ile Asp Asp Asp
20 25 30 Met Asn Trp Phe Gln
Gln Lys Pro Gly Glu Pro Pro Lys Leu Leu Ile 35
40 45 Ser Glu Gly Asn Thr Leu Arg Pro Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Tyr Gly Thr Asp Phe Ile Phe Thr Ile Glu Asn
Met Leu Ser 65 70 75
80 Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser Asp Asn Leu Pro Tyr
85 90 95 Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105
55464PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 55Met Gly Trp Leu Trp Asn Leu Leu
Phe Leu Met Ala Ala Ala Gln Ser 1 5 10
15 Ala Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu
Leu Lys Lys 20 25 30
Pro Gly Glu Ala Val Lys Ile Ser Cys Lys Ser Ser Gly Tyr Thr Phe
35 40 45 Thr Thr Tyr Gly
Met Ser Trp Val Lys Gln Ala Pro Gly Arg Ala Leu 50
55 60 Lys Trp Met Gly Trp Ile Asn Thr
Tyr Ser Gly Val Pro Thr Tyr Ala 65 70
75 80 Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu
Ser Ser Ala Ser 85 90
95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr
100 105 110 Tyr Phe Cys
Ala Arg Gly Arg Asp Gly Tyr Gln Val Ala Trp Phe Ala 115
120 125 Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ala Ala Lys Thr Thr 130 135
140 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala
Gln Thr Asn 145 150 155
160 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro
165 170 175 Val Thr Val Thr
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 180
185 190 Phe Pro Ala Val Leu Gln Ser Asp Leu
Tyr Thr Leu Ser Ser Ser Val 195 200
205 Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys
Asn Val 210 215 220
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 225
230 235 240 Asp Cys Gly Cys Lys
Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser 245
250 255 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
Val Leu Thr Ile Thr Leu 260 265
270 Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp
Pro 275 280 285 Glu
Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala 290
295 300 Gln Thr Gln Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 305 310
315 320 Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu
Asn Gly Lys Glu Phe 325 330
335 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr
340 345 350 Ile Ser
Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 355
360 365 Pro Pro Pro Lys Glu Gln Met
Ala Lys Asp Lys Val Ser Leu Thr Cys 370 375
380 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
Glu Trp Gln Trp 385 390 395
400 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp
405 410 415 Thr Asp Gly
Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 420
425 430 Asn Trp Glu Ala Gly Asn Thr Phe
Thr Cys Ser Val Leu His Glu Gly 435 440
445 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser
Pro Gly Lys 450 455 460
56234PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 56Met Phe Ser Leu Ala Leu Leu Leu
Ser Leu Leu Leu Leu Cys Val Ser 1 5 10
15 Asp Ser Arg Ala Glu Thr Thr Val Thr Gln Ser Pro Ala
Ser Leu Ser 20 25 30
Met Ala Ile Gly Asp Lys Val Thr Ile Arg Cys Ile Thr Ser Thr Asp
35 40 45 Ile Asp Asp Asp
Met Asn Trp Phe Gln Gln Lys Pro Gly Glu Pro Pro 50
55 60 Lys Leu Leu Ile Ser Glu Gly Asn
Thr Leu Arg Pro Gly Val Pro Ser 65 70
75 80 Arg Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe Ile
Phe Thr Ile Glu 85 90
95 Asn Met Leu Ser Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln Ser Asp
100 105 110 Asn Leu Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115
120 125 Ala Asp Ala Ala Pro Thr Val Ser
Ile Phe Pro Pro Ser Ser Glu Gln 130 135
140 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn
Asn Phe Tyr 145 150 155
160 Pro Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
165 170 175 Asn Gly Val Leu
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 180
185 190 Tyr Ser Met Ser Ser Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu Arg 195 200
205 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr
Ser Pro 210 215 220
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230
575PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 57Asn Tyr Trp Met His 1 5
5817PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 58Met Ile Asp Pro Ser Asp Ser Tyr Thr
Asn Tyr Asn Pro Lys Phe Lys 1 5 10
15 Gly 59115PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 59Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5
10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25
30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly
Met Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Pro Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Tyr Ser Gly Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110 Val Ser
Ser 115 60112PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 60Asp Val Leu Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5
10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25
30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro
Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Tyr Cys Phe Gln Gly 85 90
95 Ser Tyr Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
61458PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 61Met Gly Trp Ser Cys Ile Ile Val
Leu Leu Val Ser Thr Ala Thr Gly 1 5 10
15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Arg 20 25 30
Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45 Thr Asn Tyr Trp
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50
55 60 Glu Trp Ile Gly Met Ile Asp Pro
Ser Asp Ser Tyr Thr Asn Tyr Asn 65 70
75 80 Pro Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp
Thr Ser Ser Ser 85 90
95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110 Tyr Tyr Cys
Ala Arg Asn Tyr Ser Gly Asp Tyr Trp Gly Gln Gly Thr 115
120 125 Thr Leu Thr Val Ser Ser Ala Lys
Thr Thr Pro Pro Ser Val Tyr Pro 130 135
140 Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
Thr Leu Gly 145 150 155
160 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
165 170 175 Ser Gly Ser Leu
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 180
185 190 Ser Asp Leu Tyr Thr Leu Ser Ser Ser
Val Thr Val Pro Ser Ser Thr 195 200
205 Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala
Ser Ser 210 215 220
Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro 225
230 235 240 Cys Ile Cys Thr Val
Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro 245
250 255 Lys Pro Lys Asp Val Leu Thr Ile Thr Leu
Thr Pro Lys Val Thr Cys 260 265
270 Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser
Trp 275 280 285 Phe
Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu 290
295 300 Glu Gln Phe Asn Ser Thr
Phe Arg Ser Val Ser Glu Leu Pro Ile Met 305 310
315 320 His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
Cys Arg Val Asn Ser 325 330
335 Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
340 345 350 Arg Pro
Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln 355
360 365 Met Ala Lys Asp Lys Val Ser
Leu Thr Cys Met Ile Thr Asp Phe Phe 370 375
380 Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly
Gln Pro Ala Glu 385 390 395
400 Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
405 410 415 Val Tyr Ser
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn 420
425 430 Thr Phe Thr Cys Ser Val Leu His
Glu Gly Leu His Asn His His Thr 435 440
445 Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450
455 62238PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 62Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile
Pro Ala 1 5 10 15
Ser Ser Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val
20 25 30 Ser Leu Gly Asp Gln
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 35
40 45 Val His Ser Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro 50 55
60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn
Arg Phe Ser 65 70 75
80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95 Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100
105 110 Phe Gln Gly Ser Tyr Val Pro Trp Thr
Phe Gly Gly Gly Thr Lys Leu 115 120
125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe
Pro Pro 130 135 140
Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145
150 155 160 Asn Asn Phe Tyr Pro
Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165
170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser 180 185
190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys
Asp 195 200 205 Glu
Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210
215 220 Ser Thr Ser Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 225 230
235
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