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Patent Analysis of

Reagents and methods for breast cancer detection

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10001484

Application Number

US14/660423

Application Date

17 March 2015

Publication Date

19 June 2018

Current Assignee

SANFORD HEALTH

Original Assignee (Applicant)

SANFORD HEALTH

International Classification

C07K4/00,G01N33/574,G01N33/564

Cooperative Classification

G01N33/57415,G01N33/564

Inventor

EGLAND, KRISTI,EVANS, RICK,POTTALA, JAMES

Patent Images

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US10001484 Reagents breast 1 US10001484 Reagents breast 2
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Abstract

The present invention provides compositions including reagents for detecting human autoantibodies against at least two proteins selected from the group consisting of ANGTPL4, DKK1, EPHA2, LAMC2, SPON2, SSR2, GAL1, GFRA1, LRRC15, CD147, CD320, CDH3, LRP10, SPINT2, SUSD2, and CST2, and their use in detecting breast cancer or disease recurrence.

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Claims

1. A composition consisting of between 2 and 25 proteins, or antigenic fragments thereof, wherein the between 2 and 25 proteins or antigenic fragments thereof include at least (i) ANGPTL4 or an ANGPTL4 antigenic fragment, and (ii) DKK1 or a DKK1 antigenic fragment, wherein:(a) the proteins, or antigenic fragments thereof are detectably labeled; and/or(b) the proteins, or antigenic fragments thereof are immobilized on a surface, wherein no additional proteins or antigenic fragments thereof are present on the surface.

2. The composition of claim 1, wherein the between 2 and 25 proteins or antigenic fragments thereof include at least one proteins selected from the group consisting of ANGPTL4, DKK1, EPHA2, GAL1, LAMC2, SPON2, CST2, SPINT2 and SSR2, or antigenic fragments thereof.

3. The composition of claim 1, wherein the between 2 and 25 proteins or antigenic fragments thereof include at least three proteins selected from the group consisting of EPHA2, LAMC2, SPON2, SSR2, GAL1, GFRA1, LRRC15, CD147, CD320, CDH3, LRP10, SPINT2, SUSD2, and CST2, or antigenic fragments thereof.

4. The composition of claim 1, wherein the between 2 and 25 proteins or antigenic fragments include one or both of MUC1 protein and GRN protein, or antigenic fragments thereof.

5. The composition of claim 1, wherein the composition consists of between 2 and 20 proteins, or antigenic fragments thereof.

6. The composition of claim 1, wherein the composition consists of between 4 and 10 proteins, or antigenic fragments thereof.

7. The composition of claim 1, wherein the composition consists of between 5 and 10 proteins, or antigenic fragments thereof.

8. The composition of claim 1, wherein the composition includes proteins, or antigenic fragments thereof selected from one of the following sets: ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, LRRC15, and MUC1; ANGPTL4, DKK1, GAL1, GRANULIN, LRRC15, and MUC1; ANGPTL4, DKK1, GAL1, and LRRC15; ANGPTL4, DKK1, GAL1, GFRA1, and LRRC15; ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, and LRRC15; ANGPTL4, DKK1, GAL1, GRANULIN, and LRRC15; and ANGPTL4, DKK1, GAL1, LRRC15, and MUC1.

9. The composition of claim 1, wherein the composition includes ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15 proteins, or antigenic fragments thereof.

10. The composition of claim 1, wherein the between 2 and 25 proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof.

11. The composition of claim 1, wherein the proteins, or antigenic fragments thereof are detectably labeled.

12. The composition of claim 1, wherein the proteins, or antigenic fragments thereof are immobilized on a surface.

13. The composition of claim 2, wherein the between 2 and 25 proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof.

14. The composition of claim 2, wherein the proteins, or antigenic fragments thereof are detectably labeled.

15. The composition of claim 2, wherein the proteins, or antigenic fragments thereof are immobilized on a surface.

16. The composition of claim 8, wherein the between 2 and 25 proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof.

17. The composition of claim 8, wherein the proteins, or antigenic fragments thereof are detectably labeled.

18. The composition of claim 8, wherein the proteins, or antigenic fragments thereof are immobilized on a surface.

19. The composition of claim 1, wherein the between 2 and 25 proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof, and wherein the proteins, or antigenic fragments thereof are immobilized on a surface.

20. The composition of claim 2, wherein the between 2 and 25 proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof, and wherein the proteins, or antigenic fragments thereof are immobilized on a surface.

21. The composition of claim 8, wherein the between 2 and 25 proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof, and wherein the proteins, or antigenic fragments thereof are immobilized on a surface.

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Claim Tree

  • 1
    1. A composition consisting of between 2 and 25 proteins, or antigenic fragments thereof, wherein the between 2 and 25 proteins or antigenic fragments thereof include at least (i) ANGPTL4 or an ANGPTL4 antigenic fragment, and (ii) DKK1 or a DKK1 antigenic fragment, wherein:
    • (a) the proteins, or antigenic fragments thereof are detectably labeled; and/or
    • (b) the proteins, or antigenic fragments thereof are immobilized on a surface, wherein no additional proteins or antigenic fragments thereof are present on the surface.
    • 2. The composition of claim 1, wherein
      • the between 2 and 25 proteins or antigenic fragments thereof include at least one proteins selected from the group consisting of
    • 3. The composition of claim 1, wherein
      • the between 2 and 25 proteins or antigenic fragments thereof include at least three proteins selected from the group consisting of
    • 4. The composition of claim 1, wherein
      • the between 2 and 25 proteins or antigenic fragments include one or both of MUC1 protein and GRN protein, or antigenic fragments thereof.
    • 5. The composition of claim 1, wherein
      • the composition consists of between 2 and 20 proteins, or antigenic fragments thereof.
    • 6. The composition of claim 1, wherein
      • the composition consists of between 4 and 10 proteins, or antigenic fragments thereof.
    • 7. The composition of claim 1, wherein
      • the composition consists of between 5 and 10 proteins, or antigenic fragments thereof.
    • 8. The composition of claim 1, wherein
      • the composition includes proteins, or antigenic fragments thereof selected from one of the following sets: ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, LRRC15, and MUC1; ANGPTL4, DKK1, GAL1, GRANULIN, LRRC15, and MUC1; ANGPTL4, DKK1, GAL1, and LRRC15; ANGPTL4, DKK1, GAL1, GFRA1, and LRRC15; ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, and LRRC15; ANGPTL4, DKK1, GAL1, GRANULIN, and LRRC15; and ANGPTL4, DKK1, GAL1, LRRC15, and MUC1.
    • 9. The composition of claim 1, wherein
      • the composition includes ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15 proteins, or antigenic fragments thereof.
    • 10. The composition of claim 1, wherein
      • the between 2 and 25 proteins, or antigenic fragments thereof comprise
    • 11. The composition of claim 1, wherein
      • the proteins, or antigenic fragments thereof are detectably labeled.
    • 12. The composition of claim 1, wherein
      • the proteins, or antigenic fragments thereof are immobilized on a surface.
    • 19. The composition of claim 1, wherein
      • the between 2 and 25 proteins, or antigenic fragments thereof comprise
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Description

BACKGROUND

For patients with breast cancer (BCa), early and personalized diagnosis is crucial for optimizing treatments leading to long-term survival. Although mammography is the most widely used method to detect BCa, approximately 20% of screening mammograms result in a false negative diagnosis largely due to high breast density. Additionally, 1 in 10 women who get a mammogram will need additional imaging. Yet, the overwhelming majority of these women will not have BCa, as only 2 to 4 of every 1,000 screening mammograms leads to a cancer diagnosis. Therefore, there is an urgent clinical need to develop a novel, minimally invasive diagnostic strategy for the early diagnosis and monitoring of BCa.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides compositions consisting of between 2 and 25 antibody detection markers, wherein the composition includes reagents for detecting human autoantibodies against at least two proteins selected from the group consisting of human ANGPTL4, DKK1, EPHA2, LAMC2, SPON2, SSR2, GAL1, GFRA1, LRRC15, CD147, CD320, CDH3, LRP10, SPINT2, SUSD2, and CST2. In one embodiment, the composition includes reagents for detecting human autoantibodies against at least two proteins selected from the group consisting of human ANGPTL4, DKK1, EPHA2, GAL1, LAMC2, SPON2, CST2, SPINT2 and SSR2. In a further embodiment, the composition includes reagents for detecting human autoantibodies against at least 5 proteins in the recited group. In another embodiment, the composition further includes reagents for detecting human autoantibodies against one or both of MUC1 and GRN. In various embodiments, the composition consists of between 2 and 20, 4 and 10, and 5-10 antibody detection markers. In various further embodiments, the composition includes reagents for detecting human autoantibodies against one of the following marker sets:

  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, and LRRC15;
  • ANGPTL4, DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GFRA1, GRANULIN, LRRC1, and 5 MUC1;
  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GRANULIN, and LRRC15;
  • ANGPTL4, DKK1, GAL1, LRRC15, and MUC1;
  • DKK1, GAL1, and LRRC15;
  • ANGPTL4, GAL1, LRRC15, and MUC1;
  • GAL1, GFRA1, LRRC15, and MUC1;
  • GAL1, GFRA1, and LRRC15;
  • ANGPTL4, GAL1, and LRRC15;
  • DKK1, GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, and LRRC15;
  • GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, and GFRA1;
  • DKK1, GAL1, and GFRA1; and
  • GAL1, GFRA1, and MUC1.

In another embodiment, the reagents for detecting human autoantibodies comprise the at least two proteins, or antigenic fragments thereof. In a further embodiment, the at least two proteins, or antigenic fragments thereof comprise native extracellular domains and/or native secreted proteins or antigenic fragments thereof. In a still further embodiment, the reagents are detectably labeled. In another embodiment, reagents are immobilized on a surface.

In another aspect, the invention provides methods for detecting breast cancer or disease recurrence, comprising contacting a bodily fluid sample from a subject at risk of having breast cancer or breast cancer recurrence with one or more reagents for detecting autoantibodies against one or more of human ANGPTL4, DKK1, EPHA2, LAMC2, SPON2, SSR2, GAL1, GFRA1, LRRC15, CD147, CD320, CDH3, LRP10, SPINT2, SUSD2, and CST2, wherein the presence of autoantibodies against the one or more proteins correlates with a likelihood of the subject having breast cancer or breast cancer recurrence. In another embodiment, the reagents comprise reagents for detecting autoantibodies against one or more of human ANGPTL4, DKK1, EPHA2, GAL1, LAMC2, SPON2, CST2, SPINT2 and SSR2. In various further embodiments, the reagents comprise reagents for detecting autoantibodies two or more, or five or more of the recited proteins. In another embodiment the reagents comprise reagents for detecting human autoantibodies against one of the following marker sets:

  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, and LRRC15;
  • ANGPTL4, DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GFRA1, GRANULIN, LRRC1, and 5 MUC1;
  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GRANULIN, and LRRC15;
  • ANGPTL4, DKK1, GAL1, LRRC15, and MUC1;
  • DKK1, GAL1, and LRRC15;
  • ANGPTL4, GAL1, LRRC15, and MUC1;
  • GAL1, GFRA1, LRRC15, and MUC1;
  • GAL1, GFRA1, and LRRC15;
  • ANGPTL4, GAL1, and LRRC15;
  • DKK1, GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, and LRRC15;
  • GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, and GFRA1;
  • DKK1, GAL1, and GFRA1; and
  • GAL1, GFRA1, and MUC1.

In a further embodiment, the reagents comprise reagents for detecting human autoantibodies against human ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15. In another embodiment, the one or more reagents comprise the composition of any embodiment or combination of embodiments of the invention. In a further embodiment, the contacting comprises use of ELISA. In another embodiment, the bodily fluid sample comprises a serum sample from the subject. In a further embodiment, the method identifies the subject as likely to have breast cancer or breast cancer recurrence. In a further embodiment, the method further comprises treating the subject with an amount of a therapeutic sufficient to treat the breast cancer or breast cancer recurrence.

    • In a further aspect, the invention provides methods for treating a subject with breast cancer, comprising:
    • (a) testing a bodily fluid sample from a subject at risk of breast cancer, and identifying candidate subjects that:
      • (i) have autoantibodies against at least one of ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15; and/or
      • (ii) do not have autoantibodies against GFRA1, GRN and/or LRRC15; and
    • (b) treating the candidate subjects with an amount of a therapeutic sufficient to treat the breast cancer.

In one embodiment, the contacting comprises use of Longitudinal Assay Screening, wherein all target biomarkers may be detected and quantitated within a single test and dilution. In a further embodiment, the bodily fluid sample comprises a blood sample from the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Antigen conformation affects antibody recognition. A, ELISA analysis using an antigen designed to have native conformation. Wells were coated with anti-rabbit IgG followed by the HER-2-ECD-rFc protein generated in 293T cells. Serial dilutions of anti-HER-2 monoclonal antibodies generated against native HER-2, 3F32 (blue), Herceptin (green) or against denatured HER-2, 3F27 (red) were used in ELISA. Reactions were developed after addition of the appropriate secondary antibody. The O.D. is the absorbance reading for the reaction. B, ELISA analysis using a denatured antigen. Wells were coated with purified His-HER-2-ECD generated in E. coli, and serial dilutions of 3F32 (blue), Herceptin (green) or 3F27 (red) were added. After addition of the secondary antibody, the reactions were developed. C, detection of native HER-2 on SKBR3 cells via flow cytometry. Fluorescence indicates antibody recognition of HER-2 on the surface of SKBR3 cells. D, binding competition assay to demonstrate specificity of conformation-carrying antigen ELISA. Wells were precoated with anti-rabbit IgG followed by HER-2-ECD-rFc. Purified HER-2-Fc (black) or CD30-Fc (purple) chimeric proteins were serially diluted and added to a constant amount of Herceptin before addition to the wells. The reactions were developed after incubation with the secondary antibody.

FIG. 2. ROC curve comparison for classification of breast cancer patients. The autoantibody responses against seven antigens (i.e. ANGPTL4, DKK1, GAL1, GFRA1, GRN, LRRC15 and MUC1) were added to a logistic regression model that included age, BMI, race and current smoking status. The ROC curves were determined for all subjects (top) and by specific subtypes of breast cancer including ER positive, invasive, maximum tumor dimension >1 cm, in situ, lymph node involvement and HER-2 amplification (bottom).

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in their entirety.

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

In a first aspect, the present invention provides compositions consisting of between 2 and 25 antibody detection markers, wherein the composition includes reagents for detecting human autoantibodies against at least two proteins selected from the group consisting of human ANGPTL4, DKK1, EPHA2, LAMC2, SPON2, SSR2, GAL1, GFRA1, LRRC15, CD147, CD320, CDH3, LRP10, SPINT2, SUSD2, and CST2. The inventors have unexpectedly discovered that autoantibodies against the recited proteins provide an indication of whether a subject is suffering from breast cancer (BCa). Thus, the compositions of the invention can be used, for example, in diagnostic assays to discriminate between BCa and healthy patients by the detection of antibodies in a sample from the subject or to detect recurrence of disease in a breast cancer patient after treatment. In one embodiment, the composition includes reagents for detecting human autoantibodies against at least two proteins selected from the group consisting of ANGPTL4, DKK1, EPHA2, GAL1, LAMC2, SPON2, CST2, SPINT2 and SSR2.

In various embodiments, the composition includes reagents for detecting human autoantibodies against at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen proteins in the recited group. In various further embodiments, the composition consists of between 2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-25, 9-24, 9-23, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-11, 12, 13, 14, 15, or 16 antibody detection reagents.

As will be understood by those of skill in the art, the compositions may include additional antibody detection markers and controls as is appropriate for an intended use of the composition. In one non-limiting embodiment, the compositions may further comprise reagents for detecting antibodies against one or both of mucin-1(MUC1), HER-2 (41), IGFBP2, and GRANULIN (GRN).

In further embodiments, the compositions comprise or consist of reagents for detecting human autoantibodies against one of the following marker sets, which are shown in the examples that follow (see Table 5) to provide strong predictive value for diagnosing breast cancer:

  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, and LRRC15;
  • ANGPTL4, DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GFRA1, GRANULIN, LRRC1, and 5 MUC1;
  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GRANULIN, and LRRC15;
  • ANGPTL4, DKK1, GAL1, LRRC15, and MUC1;
  • DKK1, GAL1, and LRRC15;
  • ANGPTL4, GAL1, LRRC15, and MUC1;
  • GAL1, GFRA1, LRRC15, and MUC1;
  • GAL1, GFRA1, and LRRC15;
  • ANGPTL4, GAL1, and LRRC15;
  • DKK1, GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, and LRRC15;
  • GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, and GFRA1;
  • DKK1, GAL1, and GFRA1; and
  • GAL1, GFRA1, and MUC1.

In another embodiment, the compositions comprise or consist of reagents for detecting human autoantibodies against human ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15.

The antibody detection markers may be any suitable reagents that can be used to detect antibodies against the recited proteins, including but not limited to the recited protein, a secreted version of the protein (such as a native secreted form of the protein), or an extracellular domain of the protein. Secreted proteins are more easily delivered from tumor cells to lymph nodes, where interactions of immune cells take place resulting in abundant high-affinity antibodies. Membrane surface proteins are commonly released in a soluble form from tumor cells through metalloproteinase-dependent cleavage. The shed proteins are more easily transferred to the lymph nodes than intracellular protein. Thus, in one embodiment the antibody detection marker is a secreted or membrane portion of the recited protein. Exemplary amino acid sequences of the secreted or membrane portion of the recited human proteins are shown below.


ANGPTL4
(SEQ ID NO: 1)
KSPRFASWDEMNVLAHGLLQLGQGLREHAERTRSQLSALERRLSACG
SACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVA
QQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEMAQP
VDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKM
TSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSI
TGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQL
GATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNL
NGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAAS
DKK1
(SEQ ID NO: 2)
VSATLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQ
TIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCM
RHAMCCPGNYCKNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYS
RRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKE
GQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTC
QRH
EPHA2
(SEQ ID NO: 3)
KEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVM
SGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFN
LYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEE
RSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPET
IAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCL
CQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCE
CEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSG
GREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDL
EPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRST
TSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDD
LAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNL
LAMC2
(SEQ ID NO: 4)
TSRREVCDCNGKSRQCIFDRELHRQTGNGFRCLNCNDNTDGIHCEKC
KNGFYRHRERDRCLPCNCNSKGSLSARCDNSGRCSCKPGVTGARCDR
CLPGFHMLTDAGCTQDQRLLDSKCDCDPAGIAGPCDAGRCVCKPAVT
GERCDRCRSGYYNLDGGNPEGCTQCFCYGHSASCRSSAEYSVHKITS
TFHQDVDGWKAVQRNGSPAKLQWSQRHQDVFSSAQRLDPVYFVAPAK
FLGNQQVSYGQSLSFDYRVDRGGRHPSAHDVILEGAGLRITAPLMPL
GKTLPCGLTKTYTFRLNEHPSNNWSPQLSYFEYRRLLRNLTALRIRA
TYGEYSTGYIDNVTLISARPVSGAPAPWVEQCICPVGYKGQFCQDCA
SGYKRDSARLGPFGTCIPCNCQGGGACDPDTGDCYSGDENPDIECAD
CPIGFYNDPHDPRSCKPCPCHNGFSCSVMPETEEVVCNNCPPGVTGA
RCELCADGYFGDPFGEHGPVRPCQPCQCNNNVDPSASGNCDRLTGRC
LKCIHNTAGIYCDQCKAGYFGDPLAPNPADKCRACNCNPMGSEPVGC
RSDGTCVCKPGFGGPNCEHGAFSCPACYNQVKIQMDQFMQQLQRMEA
LISKAQGGDGVVPDTELEGRMQQAEQALQDILRDAQISEGASRSLGL
QLAKVRSQENSYQSRLDDLKMTVERVRALGSQYQNRVRDTHRLITQM
QLSLAESEASLGNTNIPASDHYVGPNGFKSLAQEATRLAESHVESAS
NMEQLTRETEDYSKQALSLVRKALHEGVGSGSGSPDGAVVQGLVEKL
EKTKSLAQQLTREATQAEIEADRSYQHSLRLLDSVSRLQGVSDQSFQ
VEEAKRIKQKADSLSSLVTRHMDEFKRTQKNLGNWKEEAQQLLQNGK
SGREKSDQLLSRANLAKSRAQEALSMGNATFYEVESILKNLREFDLQ
VDNRKAEAEEAMKRLSYISQKVSDASDKTQQAERALGSAAADAQRAK
NGAGEALEISSEIEQEIGSLNLEANVTADGALAMEKGLASLKSEMRE
VEGELERKELEFDTNMDAVQMVITEAQKVDTRAKNAGVTIQDTLNTL
DGLLHLMGM
SPON2
(SEQ ID NO: 5)
QPLGGESICSARAPAKYSITFTGKWSQTAFPKQYPLFRPPAQWSSLL
GAAHSSDYSMWRKNQYVSNGLRDFAERGEAWALMKEIEAAGEALQSV
HEVFSAPAVPSGTGQTSAELEVQRRHSLVSFVVRIVPSPDWFVGVDS
LDLCDGDRWREQAALDLYPYDAGTDSGFTFSSPNFATIPQDTVTEIT
SSSPSHPANSFYYPRLKALPPIARVTLLRLRQSPRAFIPPAPVLPSR
DNEIVDSASVPETPLDCEVSLWSSWGLCGGHCGRLGTKSRTRYVRVQ
PANNGSPCPELEEEAECVPDNCV
SSR2
(SEQ ID NO: 6)
EEGARLLASKSLLNRYAVEGRDLTLQYNIYNVGSSAALDVELSDDSF
PPEDFGIVSGMLNVKWDRIAPASNVSHTVVLRPLKAGYFNFTSATIT
YLAQEDGPVVIGSTSAPGQGGILAQREFDRRFSPH
GAL1
(SEQ ID NO: 7)
LRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGDANTIVCNSK
DGGAWGTEQREAVFPFQPGSVAEVCITFDQANLTVKLPDGYEFKFPN
RLNLEAINYMAADGDFKIKCVAFD
GFRA1
(SEQ ID NO: 8)
DRLDCVKASDQCLKEQSCSTKYRTLRQCVAGKETNFSLASGLEAKDE
CRSAMEALKQKSLYNCRCKRGMKKEKNCLRIYWSMYQSLQGNDLLED
SPYEPVNSRLSDIFRVVPFISDVFQQVEHIPKGNNCLDAAKACNLDD
ICKKYRSAYITPCTTSVSNDVCNRRKCHKALRQFFDKVPAKHSYGML
FCSCRDIACTERRRQTIVPVCSYEEREKPNCLNLQDSCKTNYICRSR
LADFFTNCQPESRSVSSCLKENYADCLLAYSGLIGTVMTPNYIDSSS
LSVAPWCDCSNSGNDLEECLKFLNFFKDNTCLKNAIQAFGNGSDVTV
WQPAFPVQTTTATTTTALRVKNKPLGPAGSENEIPTHVLPPCANLQA
QKLKSNVSGNTHLCISNGNYEKEGLGASSHITTKSMAAPPSCGLSPL
LVLVVTALSTLLSLTETS
LRRC15
(SEQ ID NO: 9)
YHGCPSECTCSRASQVECTGARIVAVPTPLPWNAMSLQILNTHITEL
NESPFLNISALIALRIEKNELSRITPGAFRNLGSLRYLSLANNKLQV
LPIGLFQGLDSLESLLLSSNQLLQIQPAHFSQCSNLKELQLHGNHLE
YIPDGAFDHLVGLTKLNLGKNSLTHISPRVFQHLGNLQVLRLYENRL
TDIPMGTFDGLVNLQELALQQNQIGLLSPGLFHNNHNLQRLYLSNNH
ISQLPPSVFMQLPQLNRLTLFGNSLKELSPGIFGPMPNLRELWLYDN
HISSLPDNVFSNLRQLQVLILSRNQISFISPGAFNGLTELRELSLHT
NALQDLDGNVFRMLANLQNISLQNNRLRQLPGNIFANVNGLMAIQLQ
NNQLENLPLGIFDHLGKLCELRLYDNPWRCDSDILPLRNWLLLNQPR
LGTDTVPVCFSPANVRGQSLIIINVNVAVPSVHVPEVPSYPETPWYP
DTPSYPDTTSVSSTTELTSPVEDYTDLTTIQVTDDRSVWGMTQAQSG
GRN
(SEQ ID NO: 10)
TRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVD
AHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRS
CFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCC
EDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSS
VMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTV
CDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRL
QSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWME
KAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPE
AVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPR
DIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYT
CNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCC
RDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWD
APLRDPALRQLL
MUC1
(SEQ ID NO: 11)
APKPATVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAFNSSLED
PSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAF
REGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA
GVPG
CD147
(SEQ ID NO: 12)
AAGTVFTTVEDLGSKILLTCSLNDSATEVTGHRWLKGGVVLKEDALP
GQKTEFKVDSDDQWGEYSCVFLPEPMGTANIQLHGPPRVKAVKSSEH
INEGETAMLVCKSESVPPVTDWAWYKITDSEDKALMNGSESRFFVSS
CD320
(SEQ ID NO: 13)
AGPSSGSCPPTKFQCRTSGLCVPLTWRCDRDLDCSDGSDEEECRIEP
CTQKGQCPPPPGLPCPCTGVSDCSGGTDKKLRNCSRLACLAGELRCT
LSDDCIPLTWRCDGHPDCPDSSDELGCGTNEILPEGDATTMGPPVTL
ESVTSLRNATTMGPPVTLESVPSVGNATSSSAGDQSGSPTAYG
CDH3
(SEQ ID NO: 14)
EPCRAVFREAEVTLEAGGAEQEPGQALGKVFMGCPGQEPALFSTDND
DFTVRNGETVQERRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPE
NGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETG
WLLLNKPLDREEIAKYELFGHAVSENGASVEDPMNISIIVTDQNDHK
PKFTQDTFRGSVLEGVLPGTSVMQMTATDEDDAIYTYNGVVAYSIHS
QEPKDPHDLMFTIHRSTGTISVISSGLDREKVPEYTLTIQATDMDGD
GSTTTAVAVVEILDANDNAPMFDPQKYEAHVPENAVGHEVQRLTVTD
LDAPNSPAWRATYLIMGGDDGDHFTITTHPESNQGILTTRKGLDFEA
KNQHTLYVEVTNEAPFVLKLPTSTATIVVHVEDVNEAPVFVPPSKVV
EVQEGIPTGEPVCVYTAEDPDKENQKISYRILRDPAGWLAMDPDSGQ
VTAVGTLDREDEQFVRNNIYEVMVLAMDNGSPPTTGTGTLLLTLIDV
NDHGPVPEPRQITICNQSPVRQVLNITDKDLSPHTSPFQAQLTDDSD
IYWTAEVNEEGDTVVLSLKKFLKQDTYDVHLSLSDHGNKEQLTVIRA
TVCDCHGHVETCPGPWKGG
HER2
(SEQ ID NO: 15)
TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNA
SLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAV
LDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCY
QDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSE
DCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLH
FNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYL
STDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLR
EVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQ
VFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTL
QGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQA
LLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQE
CVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVA
CAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS
CVDLDDKGCPAEQRASPLT
IGFBP2
(SEQ ID NO: 6)
EVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPG
CGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGT
CEKRRDAEYGASPEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAG
RKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPAR
TPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQ
CKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARG
VHTQRMQ
LRP10
(SEQ ID NO: 17)
HPDRIIFPNHACEDPPAVLLEVQGTLQRPLVRDSRTSPANCTWLILG
SKEQTVTIRFQKLHLACGSERLTLRSPLQPLISLCEAPPSPLQLPGG
NVTITYSYAGARAPMGQGFLLSYSQDWLMCLQEEFQCLNHRCVSAVQ
RCDGVDACGDGSDEAGCSSDPFPGLTPRPVPSLPCNVTLEDFYGVFS
SPGYTHLASVSHPQSCHWLLDPHDGRRLAVRFTALDLGFGDAVHVYD
GPGPPESSRLLRSLTHFSNGKAVTVETLSGQAVVSYHTVAWSNGRGF
NATYHVRGYCLPWDRPCGLGSGLGAGEGLGERCYSEAQRCDGSWDCA
DGTDEEDCPGCPPGHFPCGAAGTSGATACYLPADRCNYQTFCADGAD
ERRCRHCQPGNFRCRDEKCVYETWVCDGQPDCADGSDEWDCSYVLPR
K
SPINT2
(SEQ ID NO: 18)
ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGN
SNNYLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSED
HSSDMFNYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRG
NKNSYRSEEACMLRCFRQQENPPLPLGSKV
SUSD2
(SEQ ID NO: 19)
QESCSMRCGALDGPCSCHPTCSGLGTCCLDFRDFCLEILPYSGSMMG
GKDFVVRHFKMSSPTDASVICRFKDSIQTLGHVDSSGQVHCVSPLLY
ESGRIPFTVSLDNGHSFPRAGTWLAVHPNKVSMMEKSELVNETRWQY
YGTANTSGNLSLTWHVKSLPTQTITIELWGYEETGMPYSQEWTAKWS
YLYPLATHIPNSGSFTFTPKPAPPSYQRWRVGALRIIDSKNYAGQKD
VQALWTNDHALAWHLSDDFREDPVAWARTQCQAWEELEDQLPNFLEE
LPDCPCTLTQARADSGRFFTDYGCDMEQGSVCTYHPGAVHCVRSVQA
SLRYGSGQQCCYTADGTQLLTADSSGGSTPDRGHDWGAPPFRTPPRV
PSMSHWLYDVLSFYYCCLWAPDCPRYMQRRPSNDCRNYRPPRLASAF
GDPHFVTFDGTNFTFNGRGEYVLLEAALTDLRVQARAQPGTMSNGTE
TRGTGLTAVAVQEGNSDVVEVRLANRTGGLEVLLNQEVLSFTEQSWM
DLKGMFLSVAAGDRVSIMLASGAGLEVSVQGPFLSVSVLLPEKFLTH
THGLLGTLNNDPTDDFTLHSGRVLPPGTSPQELFLFGANWTVHNASS
LLTYDSWFLVHNFLYQPKHDPTFEPLFPSETTLNPSLAQEAAKLCGD
DHFCNFDVAATGSLSTGTATRVAHQLHQRRMQSLQPVVSCGWLAPPP
NGQKEGNRYLAGSTIYFHCDNGYSLAGAETSTCQADGTWSSPTPKCQ
PGRSYA
CST2
(SEQ ID NO: 20)
WSPQEEDRIIEGGIYDADLNDERVQRALHFVISEYNKATEDEYYRRL
LRVLRAREQIVGGVNYFFDIEVGRTICTKSQPNLDTCAFHEQPELQK
KQLCSFQIYEVPWEDRMSLVNSRCQEA

In a further embodiment, the antibody detection marker is a protein, such as those disclosed above, that is in its native form. As disclosed in the accompanying examples, the inventors utilized a eukaryotic expression system to generate conformation-carrying tumor antigens that are properly folded and contain non-continuous epitopes for use in the detection of autoantibodies. The protein may be used in any suitable format; in one non-limiting embodiment, the protein may be an Fc fusion protein.

In all of the above embodiments, the antibody detection reagents can be labeled with a detectable label. In one embodiment, the detectable labels for reagents to detect autoantibodies against one protein are distinguishable from the detectable labels to detect autoantibodies against the other protein. Methods for detecting the label include, but are not limited to spectroscopic, photochemical, biochemical, immunochemical, physical or chemical techniques. Any suitable detectable label can be used.

The compositions can be stored frozen, in lyophilized form, or as a solution. In one embodiment, the compositions can be placed on a solid support, such as in a microarray or microplate format; this embodiment facilitates use of the compositions in various detection assays. For example, anti-IgG can be used to precoat the wells of a microwell plate and the antibody detection reagents (such as the proteins discussed herein) can be added to the precoated wells.

In a second aspect, the present invention provides methods for detecting breast cancer or breast cancer recurrence, comprising contacting a bodily fluid sample from a subject at risk of having breast cancer or breast cancer recurrence with one or more reagents for detecting autoantibodies against one or more of human ANGPTL4, DKK1, EPHA2, LAMC2, SPON2, SSR2, GAL1, GFRA1, LRRC15, CD147, CD320, CDH3, LRP10, SPINT2, SUSD2, and CST2, wherein the presence of autoantibodies against the one or more proteins correlates with a likelihood of the subject having breast cancer or breast cancer recurrence.

In one embodiment, the composition includes reagents for detecting human autoantibodies against at least two proteins selected from the group consisting of human ANGPTL4, DKK1, EPHA2, GAL1, LAMC2, SPON2, CST2, SPINT2 and SSR2.

As will be understood by those of skill in the art, the methods may include the use of additional antibody detection markers and controls as is appropriate for an intended use of the composition. In one non-limiting embodiment, the compositions may further comprise reagents for detecting antibodies against one or both of mucin-1(MUC1), HER-2 (41), IGFBP2, and GRANULIN.

In another embodiment of the methods of the invention, the compositions comprise or consist of reagents for detecting human autoantibodies against one of the following marker sets:

  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, and LRRC15;
  • ANGPTL4, DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GFRA1, GRANULIN, LRRC1, and 5 MUC1;
  • ANGPTL4, DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GRANULIN, LRRC15, and MUC1;
  • DKK1, GAL1, GFRA1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, DKK1, GAL1, GRANULIN, and LRRC15;
  • DKK1, GAL1, GFRA1, and LRRC15;
  • DKK1, GAL1, GRANULIN, and LRRC15;
  • ANGPTL4, DKK1, GAL1, LRRC15, and MUC1;
  • DKK1, GAL1, and LRRC15;
  • ANGPTL4, GAL1, LRRC15, and MUC1;
  • GAL1, GFRA1, LRRC15, and MUC1;
  • GAL1, GFRA1, and LRRC15;
  • ANGPTL4, GAL1, and LRRC15;
  • DKK1, GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, and LRRC15;
  • GAL1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, GFRA1, LRRC15, and MUC1;
  • ANGPTL4, GAL1, and GFRA1;
  • DKK1, GAL1, and GFRA1; and
  • GAL1, GFRA1, and MUC1.

In another embodiment, the compositions comprise or consist of reagents for detecting human autoantibodies against ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15.

The antibody detection markers may be any suitable reagents that can be used to detect antibodies against the recited proteins, including but not limited to the recited protein, a secreted version of the protein (such as a native secreted form of the protein), or an extracellular domain of the protein. Secreted proteins are more easily delivered from tumor cells to lymph nodes, where interactions of immune cells take place resulting in abundant high-affinity antibodies. Membrane surface proteins are commonly released in a soluble form from tumor cells through metalloproteinase-dependent cleavage. The shed proteins are more easily transferred to the lymph nodes than intracellular protein. Thus, in one embodiment the antibody detection marker is a secreted or membrane portion of the recited protein. Exemplary amino acid sequences of the secreted or membrane portion of the recited proteins are as disclosed herein.

In another embodiment, the antibody detection marker comprises or consists of a composition of the invention.

The contacting can be carried out under any suitable conditions for promoting binding between the autoantibodies in the bodily fluid sample and the reagent to forma binding complex that can be detected. Appropriate such conditions can be determined by those of skill in the art based on the intended assay, in light of the teachings herein. Similarly, any suitable additional steps can be used in the methods, such as one or more wash or other steps to remove unbound reagents.

Any suitable detection technique can be used, including but not limited to enzyme linked immunosorbent assays (ELISA), bead based assay platforms such as the Luminex systems, 2-D array based assay platforms such as SearchLight®, and the Inanovate®‘Longitudinal Assay Screening’ platform which may be capable of quantitating all the listed breast cancer biomarker from patient samples at their clinically relevant concentrations in a single test and dilution. In one embodiment, the compositions can be placed on a solid support, such as in a microarray, glass slide, membrane, microplate format or beads. The embodiment facilitates use of the compositions. Exemplary such assays are provided in the examples.

Similarly, any suitable bodily fluid can be used, including but not limited to a serum sample, plasma sample or blood sample from the subject. The subject may be any subject at risk of breast cancer, such as a human subject.

In a further embodiment, method identifies the subject as likely to have breast cancer, and wherein the method further comprises treating the subject with an amount of a therapeutic sufficient to treat the breast cancer.

In one non-limiting embodiment of any of the above embodiments, ANGPTL4, DKK1, GAL1, and MUC1 autoantibody response are correlated with BCa; and autoantibody responses against GFRA1, GRN and LRRC15 are inversely correlated with BCa.

In one specific embodiment, the reagents include ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15; where, autoantibody responses against GFRA1, GRN and LRRC15 are inversely correlated with BCa. As detailed in the examples, when the autoantibody responses against the 7 antigens were added to the base model, including age, body mass index (BMI), race and current smoking status, the assay had the following diagnostic capabilities: c-stat (95% CI), 0.82 (0.78 to 0.86); sensitivity, 73%; specificity, 76%; and PLR (95% CI), 3.04 (2.34 to 3.94). The model was calibrated across risk deciles (Hosmer-Lemeshow, p=0.13) and performed well in specific subtypes of BCa including estrogen receptor positive, HER-2 positive, invasive, in situ and tumor sizes >1 cm. Diagnostic capabilities of other exemplary marker sets are provided in Table 5.

In a third aspect, the invention provides methods for treating a subject with breast cancer, comprising:

    • (a) testing a bodily fluid sample from a subject at risk of breast cancer, and identifying candidate subjects that:
      • (i) have autoantibodies against at least one of ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15; and/or
      • (b) do not have autoantibodies against GFRA1, GRN and/or LRRC15; and
    • (b) treating the candidate subjects with an amount of a therapeutic sufficient to treat the breast cancer.

EXAMPLE 1

Breast cancer (BCa) patients elicit an autoantibody response against cancer proteins, which reflects and amplifies the cellular changes associated with tumorigenesis. Detection of autoantibodies in plasma may provide a minimally invasive mechanism for early detection of BCa. To identify cancer proteins that elicit a humoral response, we generated a cDNA library enriched for BCa genes that encode membrane and secreted proteins, which are more likely to induce an antibody response compared to intracellular proteins. To generate conformation-carrying antigens that are efficiently recognized by patients' antibodies, a eukaryotic expression strategy was established. Plasma from 200 BCa patients and 200 age-matched healthy controls were measured for autoantibody activity against 20 different antigens designed to have conformational epitopes using ELISA. A conditional logistic regression model was used to select a combination of autoantibody responses against the 20 different antigens to classify BCa patients from healthy controls. The best combination included ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15; however, autoantibody responses against GFRA1, GRN and LRRC15 were inversely correlated with BCa. When the autoantibody responses against the 7 antigens were added to the base model, including age, BMI, race and current smoking status, the assay had the following diagnostic capabilities: c-stat (95% CI), 0.82 (0.78 to 0.86); sensitivity, 73%; specificity, 76%; and PLR (95% CI), 3.04 (2.34 to 3.94). The model was calibrated across risk deciles (Hosmer-Lemeshow, p=0.13) and performed well in specific subtypes of BCa including estrogen receptor positive, HER-2 positive, invasive, in situ and tumor sizes >1 cm.

Introduction

For patients with breast cancer (BCa), early and personalized diagnosis is crucial for optimizing treatments leading to long-term survival. Although mammography is the most widely used method to detect BCa, approximately 20% of screening mammograms result in a false negative diagnosis largely due to high breast density (1). Additionally, 1 in 10 women who get a mammogram will need additional imaging (2). Yet, the overwhelming majority of these women will not have BCa, as only 2 to 4 of every 1,000 screening mammograms leads to a cancer diagnosis (3). Therefore, there is an urgent clinical need to develop a novel, minimally invasive diagnostic strategy for the early diagnosis of BCa.

At present, there is no established tumor marker that is secreted into the peripheral circulation that can be measured by a blood test for the diagnosis of BCa. Currently, tumor markers that are accepted in clinical practice are tissue-based prognostic markers, such as the estrogen receptor (ER), HER-2 amplification, 21-gene Oncotype DX and 70-gene MammaPrint (6-12). All require an invasive biopsy or surgical procedure to acquire tumor tissue for assessment, bearing a heavy burden on patients. Serum tumor markers are valuable tools that allow minimally invasive procedures for sampling to promote the early diagnosis of cancer as well as following the prognosis after treatment (4, 5). However, tumor markers produced by tumor cells usually have relatively low concentrations in the peripheral circulation, especially in early stage disease.

Here we report the use of a molecular approach to identify tumor antigen candidates that elicit an antibody response in BCa patients. Previously, we generated a BCa cDNA library from membrane-associated polyribosomal (MAP) RNA, which encodes secreted and membrane proteins, and subtracted the library with RNA from normal tissues (29). Secreted proteins are more easily delivered from tumor cells to lymph nodes, where interactions of immune cells take place resulting in abundant high-affinity antibodies. Membrane surface proteins are commonly released in a soluble form from tumor cells through metalloproteinase-dependent cleavage. The shed proteins are more easily transferred to the lymph nodes than intracellular proteins (30, 31). Consequently, the obtained subtracted library, referred to as the membrane-associated polyribosomal cDNA library (MAPcL), is enriched with clones encoding membrane and secreted TAA that are highly abundant in BCa and should preferentially induce an antibody response in patients (29). In addition, we have established a method for producing recombinant antigens as Fc fusion proteins designed to have native conformations, which is essential for the expression of membrane and secreted proteins that may induce an antibody response in patients.

We have developed a conformation-carrying antigen ELISA-based strategy to discriminate between BCa and healthy patients by the detection of autoantibodies against a panel of TAAs. Twenty antigens were selected from the most abundant genes represented in the MAPcL, and Fc fusion proteins were generated. Blood was collected from 200 newly diagnosed BCa patients and 200 healthy women as age-matched controls. The 400 plasma samples were screened for the presence of autoantibodies against the 20 different MAPcL-derived antigens using ELISA. A combination of seven antigens with patient demographics yielded the best positive likelihood ratio to discriminate between healthy and BCa patients.

Materials and Methods

Plasmid Construction

For production of MAPcL-rabbit Fc-tagged antigens, two constructs, pSecTag2 (Invitrogen, Carlsbad, Calif.) and pFUSE-rIgG-Fc1 (InvivoGen, San Diego, Calif.), were both utilized to generate the 20 MAPcL-rFc expression constructs because of restriction site availability for cloning. pSecTag2 was modified by amplifying the Fc portion of rabbit IgG using primers


(SEQ ID NO: 21)
5′-CCGGATATCAGCAAGCCCACGTGCCCACC-3′
and
(SEQ ID NO: 22)
5′-AAGGAAAAAAGCGGCCGCTC-ATTTACCCGGAGAGCGGGAG-3′

(Integrated DNA Technologies, Coralville, Iowa) using pFUSE-rIgG-Fc1 as a template. The rFc PCR product was digested with EcoRV and NotI and inserted into pSecTag2, referred to as pSecTag2-rFc, which contains an IgK signal sequence for secretion. The pFUSE-rIgG-Fc1 contains an IL2 signal sequence. To keep the signal sequence consistent between the two plasmids, the IgK leader sequence was amplified via PCR using pSecTag2 as a template. The IL2 leader sequence was then replaced with the IgK signal sequence, creating pFUSE-IgK-rFc.

The accession numbers of the 20 MAPcL genes used as templates for cloning and predicted signal sequences are indicated in Table 1. The signal sequences of each encoded protein were determined using SignalP (32, 33). If a protein contained a transmembrane domain, only the encoded extracellular portion was included. The transmembrane domains were predicted using the TMHMM database (34). The amino acid numbers encoded by the cloned fragment are shown in Table 1. ANGPTL4, CDH3, DKK1, SPON2, SSR2, CST2, GFRA1 and GAL1 were custom cloned into pSecTag2-rFc using the SfiI and KpnI restriction sites (Genscript, Piscataway, N.J.). EPHA2, IGFBP2 and LAMC2 were custom cloned into pSecTag2-rFc using the KpnI and BamHI restriction sites. GRN, MUC1 and LRRC15 were custom cloned into pSecTag2-rFc using the SfiI and BamHI restriction sites. HER-2, LRP10, SPINT2 and SUSD2 were cloned into pFUSE-IgK-rFc using the SfiI and XhoI restriction sites. CD147 was cloned into pFUSE-IgK-rFc using the BamHI and SacII restriction sites. CD320 was cloned into pFUSE-IgK-rFc using the EcoRI and XhoI restriction sites.


TABLE 1
MAPcL Candidates for Generation of rFc Fusion Proteins
Signal Sequence*
Encoded Amino
Gene from MAPcL
Accession #
Amino Acids
Acid Fragment†
ANGPTL4 (angiopoietin-like 4)
NM_139314
1-30
31-406
CD147
NM_198589
1-21
22-162
CD320
NM_016579
1-46
47-230
CDH3 (cadherin 3)
NM_001793
1-24
25-654
CST2 (cystatin SA)
NM_001322
1-20
21-141
DKK1 (dickkopf WNT signaling
NM_012242
1-28
29-266
pathway inhibitor 1)
EPHA2 (EPH receptor A2)
NM_004431
1-26
27-535
GAL1 (lectin, galactoside-binding,
NM_002305
1-17
18-135
soluble, 1)
GFRA1 (GPI-linked anchor protein)
AF038421
1-24
25-465
GRN (granulin)
NM_002087
1-17
18-593
HER-2
NM_004448
1-22
23-652
IGFBP2 (insulin-like growth factor
NM_000597
1-39
40-328
binding protein 2)
LAMC2 (laminin, gamma 2)
NM_005562
1-21
22-1111
LRP10 (low density lipoprotein receptor-
NM_014045
1-16
17-440
related protein 10)
LRRC15 (leucine rich repeat containing
NM_001135057
1-27
28-544
15)
MUC1 (mucin 1)
NM_002456
1-22
23-167
SPINT2 (serine peptidase inhibitor,
NM_021102
1-27
28-198
Kunitz type, 2)
SPON2 (sporadin 2)
NM_012445
1-26
27-331
SSR2 (signal sequence receptor, beta
NM_003145
1-17
18-146
(translocon-associated protein beta))
SUSD2 (sushi domain containing 2)
NM_019601
1-27
28-785
*The signal sequences of each encoded protein were determined using SignalP (32, 33) and were not included in the expression constructs.
†The amino acid numbers indicate the encoded portion of the proteins cloned between the Ig signal sequence and the Fc portion of rabbit IgG to generate the secreted MAPcL-rFc fusion proteins.

For production of His-tagged HER-2, HER-2 was amplified via PCR using primers 5′-CCCAAGCTTGCAGCACCCAAGTGTGCACCGGCAC-3′ (SEQ ID NO: 23) and 5′-GTGCTCGAGTCACGTC-AGAGGGCTGGCTCTCTGCTCG-3′(SEQ ID NO: 24). The product was digested with HindIII and XhoI and cloned directionally into the pET-28a expression vector.

Cell Culture

293T and SKBR3 cell lines were cultured in DMEM with 10% FBS. Cultures were maintained at 37° C. with 5% CO2 in a humidified incubator. All cell lines were authenticated and tested negatively for mycoplasma.

Protein Production

The MAPcL-rFc fusion proteins were produced in 293T cells. Briefly, 293T cells were transfected using Effectene (Qiagen, Valencia, Calif.) according to manufacturer's specifications. During transfection, the cells were cultured in DMEM with 2% FBS. Supernatants containing the secreted fusion proteins were harvested, centrifuged to clear cell debris and supplemented with 0.1% sodium azide. His-HER-2 was produced in E. coli BL21 (Invitrogen, Carlsbad, Calif.) and purified using IMAC affinity chromatography.

Sandwich ELISA

Microtiter plates (Nalge Nunc, Rochester, N.Y.) were coated overnight with 2 μg/ml goat anti-rabbit Fc (Jackson Immunoresearch, West Grove, Pa.) diluted with phosphate buffered saline. The supernatants containing the rFc fusion proteins were diluted 1:3 serially in standard blocking buffer (0.5% bovine serum albumin and 0.1% sodium azide in phosphate buffered saline). Plates were washed once, and the serially diluted supernatants were transferred to the microtiter plates. Rabbit IgG of known concentration was diluted similarly and added to one row of the microtiter plate in order to quantify the amount of fusion protein present in the culture media. After incubating for two hours, plates were washed twice and 50 μl of HRP-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, Pa.) diluted 1:3000 in standard blocking buffer with 0.05% Tween 20 added. After a 2-hour incubation, plates were washed 4 times and developed with 100 μl/well of TMB substrate (Pierce, Rockford, Ill.). The development reaction was stopped after five minutes with 50 μl/well of 2N H2SO4, and the absorbance was measured at 450 nm to determine the concentration. The absorbance at 690 nm was subtracted to remove background signal.

Antibody Recognition of Conformational Versus Denatured HER-2 Protein

For the conformational HER-2 assay, microtiter plates were coated with 2 μg/ml goat anti-rabbit Fc (Jackson Immunoresearch, West Grove, Pa.) in PBS overnight. HER-2-ECD-rFc was then added to each well, 100 μl/well. For denatured HER-2, microtiter plates were coated with 2 μg/ml His-HER-2-ECD in PBS overnight.

Three HER-2 antibodies were used in the assay: anti-HER-2 3F27 (US Biological, Swampscott, Mass.), anti-HER-2 3F32 (US Biological, Swampscott, Mass.) and Herceptin (Genentech, South San Francisco, Calif.). Each antibody was diluted to 1 μg/ml in standard blocking buffer with 0.05% Tween 20. The antibodies were then serially diluted. After washing once, 50 μl/well of the serially diluted antibodies was added to the plates and incubated for 2 hours at room temperature. The plates were washed three times, and species appropriate HRP-conjugated secondary antibodies were added at a 1:3000 dilution. Plates were washed four times and developed with 100 μl/well TMB substrate for five minutes. Development was stopped with 50 μl/well 2N H2SO4. Absorbance was measured at 450 nm, and the 690 nm absorbance was subtracted to account for background.

The same antibodies were used to stain HER-2 in SKBR3 BCa cells via flow cytometry. SKBR3 cells were detached from dish using Cell Dissociation Solution Non-enzymatic 1× (Sigma, St. Louis, Mo., catalog # C5914). 2×105 cells were incubated with 0.5 μg/ml of each antibody for 1 hour at room temperature. The cells were then washed, and a 1:200 dilution of PE-conjugated antibody for the appropriate species was added. The cells were again washed, resuspended in FACS buffer (PBS with 5% bovine serum albumin and 0.1% sodium azide) and analyzed by flow cytometry.

Competition of Herceptin Binding

Microtiter plates were coated with 4 μg/ml goat anti-rabbit Fc and incubated overnight. After one wash, 100 μl/well HER-2-ECD-rFc was added to each well and incubated overnight. HER-2-Fc and CD30-Fc chimeric proteins (R&D Systems, Minneapolis, Minn.) were serially diluted from a starting concentration of 10 ug/ml. Herceptin was added to a final concentration of 10 ng/ml in each of the serial chimeric protein dilutions. Plates were washed twice, and 50 μl/well of chimeric protein/Herceptin mixture was applied to the plate. Plates were then washed three times, and a 1:3000 dilution of HRP goat anti-human IgG was applied to each well, 50 μl/well. After four washes, 100 μl/well TMB substrate was added to each well. Development was stopped with 50 μl/well 2N H2SO4 after 5 minutes. Absorbance was measured at 450 nm with 690 nm absorbance subtracted.

Patients

The inclusion criteria for cases were women over 30 years of age that were newly diagnosed with BCa (any type) at Sanford Health, Sioux Falls, S. Dak. Patients were asked to provide one extra 10 ml EDTA tube of blood prior to mastectomy, lumpectomy, radiation therapy, chemotherapy or other treatment. Case subjects were excluded only if they had a previous history of cancer of any kind Healthy control subjects had a negative mammogram within six months before the blood draw. Healthy subjects were excluded if there was a history of previous cancer of any kind or a history of autoimmune disease. All patients provided written informed consent, and the Sanford Health IRB approved the study protocol. Blood samples from 200 BCa patients were collected from Oct. 8, 2009 to Apr. 17, 2012. In addition, 200 age-matched healthy control blood samples were collected from Oct. 16, 2009 to Jan. 19, 2011. See Table 2 for enrolled patients' characteristics.


TABLE 2
Patient Clinical and Pathological Characteristics
Patients with Breast Cancer
N = 200
Age: Mean (SD)
58.9 (11.4)
White Race: n (%)
 193 (97%)
BMI [kg/m2]: Mean (SD)
29.7 (6.6)
Smoking Status: n (%)
Current
  22 (11%)
Never
 120 (60%)
Past
  58 (29%)
Family History Yes: n (%)
 114 (58%)
Tumor Type: n (%)
Invasive
 148 (74%)
in situ
  52 (26%)
Histology: n (%)
Ductal and Lobular
  3 (2%)
Ductal
 173 (87%)
Lobular
  21 (11%)
Other
  2 (1%)
ER Positive: n (%)
 171 (86%)
PR Positive: n (%)
 147 (74%)
HER-2 Amplification: n (%)
Negative
 156 (78%)
Positive
  33 (17%)
Unknown
  11 (6%)
Triple Negative Yes: n (%)
  18 (12%)
Tumor Max Dimension [cm]: n (%)
≤1
  66 (36%)
>1 to ≤2
  65 (35%)
>2
  53 (29%)
Lymph Node Involvement: n (%)
  47 (24%)
Age-Matched Controls
with Negative Mammogram
N = 200
Age: Mean (SD)
58.8 (11.3)
White Race: n (%)
 192 (97%)
BMI [kg/m2]: Mean (SD)
27.1 (5.5)
Smoking Status: n (%)
Current
  7 (4%)
Never
 125 (63%)
Past
  67 (34%)

Serum Collection

Blood was collected in a 10 ml EDTA tube and centrifuged at 2000×g for 10 minutes. Plasma was removed from the tube, aliquoted and stored at −80 degrees Celsius until screening for the presence of autoantibodies.

Conformation-Carrying Antigen ELISA

Microtiter plates (Nalge Nunc, Rochester, N.Y.) were coated overnight with 4 μg/ml goat anti-rabbit Fc (Jackson Immunoresearch, West Grove, Pa.) in phosphate buffered saline. Plates were washed once, and 100 μl/well of MAPcL-rFc fusion protein was added. Plates were incubated for 2 hours and washed twice. The plates were then coated with 50 μl/well of optimized blocking buffer (phosphate buffered saline with 0.5% bovine serum albumin, 0.2% dry milk, 0.1% polyvinylpyrrolidone, 20 mM L-Glutamine, 20 mM L-Arginine, 0.1% sodium azide, 10% goat serum, and 0.05% Tween 20). The plates were incubated for 1 hour at 37° C. and washed once. Serum samples diluted 1:100 in optimized blocking buffer were added and incubated for 2 hours at room temperature. Plates were then washed three times, and autoantibodies were detected using an HRP-conjugated goat anti-human IgG (Jackson Immunoresearch, West Grove, Pa.) diluted 1:3000 in standard blocking buffer with 0.05% Tween 20. Plates were incubated for 1 hour at room temperature, washed four times and developed with 100 μl/well of TMB substrate (Pierce, Rockford, Ill.) for 15 minutes. Development was stopped with 50 μl/well 2N H2SO4, and the absorbance was measured at 450 nm. The absorbance at 690 nm was subtracted to remove background signal. Each 96-well plate included 14 samples from BCa subjects and 14 samples from normal mammogram subjects. Each sample was tested in triplicate within the same plate. One row in each plate was subjected only to blocking buffer as a negative control for the ELISA.

Statistical Methods

Controls were individually matched to 200 BCa patients 1:1 within a 3-year age window using a greedy caliper matching algorithm (35) while blinded to assay data. For each subject the antigen level was transformed by subtracting the mean of the blocking buffer from the mean of the triplicate measurements. If the difference was less than zero, it was set to zero, and the square root was taken to yield a more symmetrical distribution.

Differences in demographics and autoantibody responses between BCa patients and controls were tested using two-sample t-test and Chi-squared test for continuous and categorical data, respectively. The incremental improvement to the c-statistic (i.e. concordance index, area under the receiver operating characteristic (ROC) curve) was tested by adding the autoantibody response to each antigen to a logistic regression model that already included age, BMI, race, and current smoking status. The model calibration was tested using the Hosmer-Lemeshow goodness-of-fit measure, which constructs a Chi-squared statistic by comparing the predicted and observed number of cases by probability decile (36).

After assessing the individual antigens, a multivariable conditional logistic regression analysis with strata for age-matching was used to determine the subset of antigens that minimized Akaike's Information Criterion (37); all models were adjusted for BMI, race, and current smoking status. Exploratory subgroup analyses were performed to determine if the multivariable subset of antigens performed differently in a particular type of BCa. The multivariable model was tested in the following subgroups: invasive, in situ, ER positive, tumor maximum dimension >1 cm, lymph node involvement, and HER-2 positive. The critical level alpha was set to ≤0.05/20 antigens=0.0025 using the Bonferroni correction. SAS® (Cary, N.C.) version 9.3 software was used for all analyses.

Results

Generation of Tumor-associated Antigens Designed to have Native Conformations

To identify TAAs that elicit a humoral response in patients, candidate genes that encode membrane and secreted proteins were selected from the most abundant genes represented in the MAPcL. Because only 10% of epitopes on proteins are in a linear continuous sequence (24), we utilized a eukaryotic expression system to generate conformation-carrying tumor antigens that are properly folded and contain noncontinuous epitopes for use in the detection of autoantibodies. Sequences encoding the extracellular domains (ECD) or the secreted proteins without the signal sequence of the candidate MAPcL genes were cloned 5′ of the Fc region of rabbit IgG (rFc) into the pSecTag2-rFc vector or pFUSE-IgK-rFc, depending on restriction enzyme cloning sites. The IgK leader sequence contained in the vectors directs the fusion proteins to be secreted. The vectors encoding the fusion proteins were transiently transfected into 293T cells, and the corresponding fusion proteins were secreted into the media. Production of the secreted fusion proteins was confirmed using a sandwich ELISA, and the concentrations were determined by comparison to an established CD147-rFc standard (data not shown).

To demonstrate that the generated MAPcL-rFc proteins were designed to be folded into a native conformation, an ELISA analysis was performed using commercially available anti-HER-2 antibodies generated against either native (monoclonal antibody 3F32 and Herceptin) or denatured (monoclonal antibody 3F27) HER-2 protein. Two antigens consisting of the ECD of HER-2 were analyzed: the conformation-carrying HER-2-ECD-rFc protein generated in 293T cells and a His-HER-2-ECD protein that was produced in bacteria and purified over a nickel column. The anti-native HER-2 antibody (3F32) recognized the HER-2-ECD-rFc produced in 293T (FIG. 1A), but was unable to detect the purified His-HER-2-ECD protein produced in bacteria (FIG. 1B). Also, Herceptin was unable to detect the denatured His-HER-2-ECD protein purified from bacteria (FIG. 1B). However, a strong response was observed for Herceptin when HER-2-ECD-rFc protein was used as the antigen for the ELISA analysis (FIG. 1A). Although the 3F27 antibody generated against denatured HER-2 did not detect the HER-2-ECD-rFc protein (FIG. 1A), this antibody had a strong response to bacterial HER-2-ECD (FIG. 1B).

To confirm the specific recognition of native versus denatured epitopes by the purchased antibodies, flow cytometry was performed on unfixed SKBR3 cells, a BCa cell line known to have HER-2 amplification (38). Because surface HER-2 would retain its native confirmation on the unfixed SKBR3 cells, the anti-HER-2 3F27 antibody, specific for denatured HER-2, was unable to detect surface HER-2 on the cell membrane of SKBR3 cells by flow cytometry (FIG. 1C). When anti-HER-2 3F32 antibody and Herceptin, both of which recognize conformational HER-2, were used for flow cytometry analysis, a large shift in fluorescence was observed indicated that the antibodies recognized HER-2 present on the membrane of the SKBR3 cells (FIG. 1C).

A binding competition assay was performed to verify that the conformation-carrying antigen ELISA was recognizing the MAPcL antigen specifically. Wells were precoated with anti-rabbit IgG followed by HER-2-ECD-rFc. Purchased HER-2-Fc and CD30-Fc purified chimeric proteins (R&D Systems) were serially diluted and added to a constant amount of Herceptin (10 ng/ml) in each well. Following the addition of the HRP-conjugated secondary anti-human IgG antibody, the reactions were developed. Herceptin binding to HER-2-ECD-rFc was competed by addition of HER-2-Fc but not the CD30-Fc protein (FIG. 1D). This result indicates that Herceptin is binding specifically to the HER-2-ECD portion of the conformation-carrying fusion protein.

Screening of Patients for Autoantibodies Using the Conformation-carrying Antigen ELISA

Twenty MAPcL-rFc fusion antigens designed to contain their native conformation were generated by cloning the sequences encoding the ECD or secreted proteins 5′ of the rFc sequence (see Table 1 for identity of all 20 antigens). The expression plasmids were individually transfected into 293T cells, and the MAPcL-rFc fusion proteins were secreted into the media. The 20 fusion proteins were quantitated by sandwich ELISA analysis (data not shown). To detect autoantibodies in plasma collected from patients, a conformation-carrying antigen ELISA was developed using the generated MAPcL-rFc antigens. To immobilize the MAPcL-rFc fusion proteins, anti-rabbit IgG was used to precoat the wells of a 96-well plate. The media from the transfected 293T cells, which contains the generated MAPcL-rFc fusion proteins designed to have native conformations, was added to the precoated wells. To reduce plate variation and increase repeatability of the assay, three replicate samples using the plasma from each individual patient were distributed across the 96-well plate. After addition of an HRP-conjugated secondary anti-human IgG antibody, the plates were developed and the absorbance of each well was measured. The 200 plasma samples collected from newly diagnosed BCa patients and plasma from 200 age-matched healthy subjects were evaluated for autoantibody reactivity against the 20 antigens using the conformation-carrying ELISA.

The 200 BCa patients and 200 healthy controls had a mean (SD) age of 59 (11) years and 97% self identified as white race (Table 2). Cancer patients were more overweight (29.7 vs. 27.1 kg/m2, p<0.0001) and had different smoking habits (p=0.014), such that there was a greater prevalence of current smokers (11% vs. 4%) in the cancer subjects versus healthy. The 200 BCa patients represented the heterogeneity of the disease consisting of 74% invasive, 24% lymph node involvement, 86% ER-positive, 17% HER-2 positive and 12% triple negative BCa (Table 2). Analyzing the absorbance reading of the autoantibody responses against the individual antigens, we determined that there were significant Bonferroni adjusted differences between BCa patients and controls in autoantibody responses against 12 TAAs, i.e. ANGPTL4, DKK1, EPHA2, GAL1, HER-2, IGFBP2, LAMC2, MUC1, SPON2, CST2, SPINT2 and SSR2 (Table 3). Higher levels of these autoantibodies were detected in BCa patients. In logistic regression models adjusted for age, race, BMI and current smoking status, autoantibody responses against MUC1 (1.83), DKK1 (1.77) and GAL1 (1.75) (all p<0.0001) had the largest odds ratios (OR), such that a patient was about 1.8 times as likely to have BCa per 1 SD increase in autoantibody response against any of these three antigens (Table 3). Autoantibody responses against six of the twelve antigens (i.e. GAL1, DKK1, MUC1, ANGPTL4, EPHA2 and IGFBP2) also increased the area under the ROC curve when each of them was added individually to the base logistic regression model adjusted for age, BMI, race and current smoking status (all p<0.05). Five of the six models were well calibrated across probability deciles (minimum Hosmer-Lemeshow p=0.13), but the model including IGFBP2 was not calibrated (p=0.016).


TABLE 3
Absorbance Measurements of Autoantibodies and their Association with Breast Cancer
Normal
Mammogram
Breast Cancer
Odds
Increase in
Autoantibody
(n = 200)
(n = 200)
P-value*
Ratio†
95% CI
c-statistic‡
P-value
CD320
0.15 (0.12)
0.16 (0.12)
0.62
1.10
0.90-1.35
0.000
0.96
EPHA2
0.13 (0.06)
0.16 (0.10)
0.0006
1.64
1.21-2.24
0.034
0.037
GFRA1
0.18 (0.06)
0.20 (0.08)
0.0081
1.28
1.03-1.59
0.013
0.32
IGFBP2
0.21 (0.12)
0.25 (0.13)
0.0006
1.39
1.10-1.75
0.030
0.050
CST2
0.17 (0.09)
0.20 (0.10)
0.0013
1.39
1.12-1.73
0.026
0.13
GAL1
0.17 (0.06)
0.20 (0.07)
<0.0001
1.75
1.37-2.23
0.051
0.021
HER-2
0.13 (0.04)
0.15 (0.06)
<0.0001
1.65
1.28-2.13
0.039
0.054
LAMC2
0.15 (0.05)
0.17 (0.08)
0.0007
1.47
1.16-1.88
0.025
0.13
ANGPTL4
0.18 (0.05)
0.20 (0.06)
0.0001
1.57
1.24-1.99
0.041
0.032
DKK1
0.18 (0.10)
0.24 (0.11)
<0.0001
1.77
1.40-2.24
0.060
0.0093
MUC1
0.14 (0.06)
0.18 (0.08)
<0.0001
1.83
1.41-2.37
0.055
0.012
SSR2
0.14 (0.07)
0.17 (0.08)
0.0007
1.53
1.23-1.92
0.029
0.14
LRP10
0.14 (0.05)
0.15 (0.07)
0.0098
1.35
1.09-1.68
0.011
0.47
LRRC15
0.11 (0.04)
0.12 (0.05)
0.30
1.09
0.89-1.34
0.001
0.82
SPINT2
0.15 (0.07)
0.18 (0.09)
0.0022
1.40
1.13-1.74
0.018
0.31
SPON2
0.14 (0.07)
0.17 (0.08)
<0.0001
1.65
1.31-2.07
0.042
0.052
CD147
0.10 (0.05)
0.12 (0.06)
0.0039
1.43
1.15-1.78
0.016
0.38
CDH3
0.10 (0.04)
0.12 (0.04)
0.0033
1.43
1.14-1.79
0.014
0.40
GRN
0.12 (0.06)
0.13 (0.07)
0.19
1.16
0.94-1.43
0.004
0.65
SUSD2
0.12 (0.04)
0.13 (0.05)
0.0085
1.36
1.10-1.70
0.013
0.38
Data shown as mean (SD) of {square root over (O.D. − Background)};
*Differences between groups were tested using t-tests;
Significant Bonferroni adjusted p-value <0.05/20 = 0.0025 are shown in bold;
†Odds ratio (95% CI) for breast cancer prevalence per 1 SD increase in autoantibody was determined using logistic regression models adjusted for age, race, BMI and current smoking status;
‡Change in area under the ROC curve (i.e. c-statistic) was determined when autoantibody was added to the adjusted logistic regression models.

To increase the predictive ability of the conformation-carrying ELISA, the autoantibody response against a group of antigens was determined using conditional logistic regression analysis incorporating the individual age-matching study design and adjusting for BMI, race and current smoking status. The group with the best model fit (i.e. minimum AIC) contained the autoantibody responses against the following 7 antigens: ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15 (Table 4). Of these 7, only autoantibody responses against ANGPTL4, DKK1, MUC1 and GAL1 individually showed a significant increase in the area under the ROC curve when added to the base model (Table 3). In the fully adjusted logistic regression model including the group of antigens, current smoking had the largest OR (95% CI) of prevalent BCa OR=7.88 (2.68-23.2); and BMI was also a significant risk factor OR=1.09 (1.04-1.13) per 1 kg/m2 increase (Table 4). GAL1 had an OR of 6.73 (3.42-13.3), so a patient was almost 7 times as likely to have BCa per 1 SD increase in autoantibody response against GAL1. The autoantibody responses against GFRA1 (OR=0.41), GRN (OR=0.55) and LRRC15 (OR=0.32) all had inverse associations with odds of prevalent BCa when adjusted for responses against the other antigens (Table 4). Taken together, the autoantibody response against the group of 7 antigens increased the area under the ROC curve from 0.64 to 0.82 (p<0.0001) and had the following diagnostic measures: sensitivity (72.9%), specificity (76.0%), and positive likelihood ratio (95% CI) 3.04 (2.34 to 3.94) (FIG. 2). The model was also calibrated across risk deciles (Hosmer-Lemeshow, p=0.13).


TABLE 4
Multivariable Logistic Regression
Model Odds Ratios for Breast Cancer
Variable
Odds Ratio
95%
CI
Age (per 1 year)
1.00*
0.98
1.02
White Race
0.70
0.19
2.68
BMI (per 1 kg/m2)
1.09
1.04
1.13
Current Smoking
7.88
2.68
23.2
ANGPTL4 (per 1 SD)
1.71
1.16
2.50
DKK1 (per 1 SD)
1.87
1.28
2.73
GAL1 (per 1 SD)
6.73
3.42
13.3
GFRA1 (per 1 SD)
0.41
0.21
0.82
GRN (per 1 SD)
0.55
0.38
0.81
LRRC15 (per 1 SD)
0.32
0.19
0.55
MUC1 (per 1 SD)
1.67
1.16
2.41
*Due to individual 1:1 age-matching.

Because BCa is a heterogeneous disease, it is possible that the autoantibody response against a combination of antigens may categorize a subtype of BCa differently than analyzing all BCa subtypes as a whole. The BCa samples were grouped into individual BCa subtypes: invasive, in situ, ER positive, tumor maximum dimension >1 cm, lymph node involvement and HER-2 positive. The ability to discriminate cases from controls in each subtype was tested using autoantibody reactivity against the 7-antigen combination in addition to age, BMI, race and current smoking status (FIG. 2). The 7-antigen combination model performed similarly in all subtypes of BCa; the c-statistic was 0.81 to 0.85. Of the BCa subtypes, in situ tumors had the greatest area under the ROC curve (0.8520, p<0.0001) when analyzed for autoantibody responses against the 7-antigen combination. The model was not calibrated when considering only those cancers with lymph node involvement due to four unexpected BCas with very low model probabilities (Hosmer-Lemeshow p=0.0036).

Discussion

Early detection of BCa allows a physician to treat the initial stage of the disease before metastasis, thereby allowing for a higher rate of remission or long-term survival for the patient. Detecting the presence of autoantibodies generated against tumor proteins in the blood of patients would be an ideal method for BCa detection. However, the tumor antigens need to be identified before specific autoantibody responses in patients can be ascertained. We generated a library that encodes membrane and secreted proteins that are highly expressed in BCa and may elicit an immune response.

We have shown that antigen conformation alters antibody-binding affinity in our assay, and the detection of autoantibodies is limited by epitope conformation (FIG. 1). We used a robust sample set to develop the conformation-carrying ELISA consisting of 200 plasma samples collected from newly diagnosed BCa patients before surgery, chemotherapy or radiation treatment. In addition, plasma was collected from 200 age-matched subjects defined by a confirmed normal mammogram in the preceding six months (Table 2). All 400 plasma samples were screened individually for autoantibody response against 20 TAAs designed to contain their native conformation using ELISA. Four of the 20 TAAs analyzed in our assay have previously been reported to generate an antibody response in BCa patients: MUC1 (39, 40), HER-2 (41), IGFBP2 (15) and GRN (42). Detection of autoantibodies against 12 of the 20 antigens was statistically significant for discriminating between normal and cancer samples (Table 3, bold). However, we did not observe a significant autoantibody response against GRN in our assay. Of the 12 significant antigens, 9 have not been previously associated with BCa autoantibodies. To our knowledge, this is the first report of the detection of autoantibodies against ANGPTL4, CST2, DKK1, EPHA2, GAL1, LAMC2, SPINT2, SPON2 and SSR2 in BCa patients (Table 3).

Previously it has been shown that screening serum against a panel of antigens to detect autoantibodies compared to only a single antigen increases the sensitivity of the assay (17). This finding is consistent with the fact that BCa is a heterogeneous disease (43), and each individual patient's immune system is distinct. A combination of seven TAAs, consisting of ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN and LRRC15, had the greatest diagnostic capability (Table 4). Compared to previously published multiple antigen panels used to detect BCa autoantibodies (17, 44-46), the combination of these seven TAAs is unique, and our study contains the largest patient population of BCa and healthy samples. Interestingly, in the seven-antigen combination, four of the antigens have statistical significance individually (Table 3), but three of the antigens, GFRA1, GRN and LRRC15, were not statistically significant on their own (Table 3). However, GFRA1, GRN and LRRC15 were inversely associated with BCa, indicating that lower amounts of these autoantibodies in a patient, in combination with higher levels of the directly associated autoantibodies, increased the likelihood of having BCa (Table 4). When the 7 antigens were added to knowledge of current smoking status and BMI, the sensitivity and specificity of the assay was 72.9% and 76.0%, respectively. The area under the ROC curve (95% CI) was 0.82 (0.77 to 0.85), and the positive likelihood ratio was 3.04 for the conformation-carrying ELISA. Because BCa is a heterogeneous disease, patients were grouped into tumor characteristics, including ER positive, HER-2 positive, in situ, invasive, tumor size and lymph node involvement. The 7-antigen combination performed well for all groups (FIG. 2). These results suggest that the assay has potential clinical application. One serum recurrence marker for BCa that is currently used in the clinic is mucin-associated antigen CA27.29. The CA27.29 antigen is detected in the blood of a patient using a monoclonal antibody that recognizes MUC1. Because of the low sensitivity of the CA27.29 tumor marker, the test is used to follow a patient for BCa recurrence (47). Compared to the traditional CA27.29 tumor marker, the conformation-carrying ELISA described here shows great promise.

Currently, mammography is the standard method for BCa screening. However, the machinery necessary to perform a mammogram is expensive, requires specialized medical personnel to operate and is challenging to transport to medically underserved areas. The development of a blood test for the early detection of BCa would greatly advance access to screening. Drawing blood is a common procedure, and blood can easily be mailed to a clinical laboratory for analysis. This study demonstrates that a combination of autoantibody responses against antigens designed to contain conformational epitopes is a promising strategy for BCa detection. Future studies will focus on the identification of additional antigens to improve the sensitivity and specificity of the assay for translation into the clinic.


TABLE 5
Autoantibody combination subsets and their association with breast cancer
ROC
Increase in
Plex
Sets of Autoantibodies
Sensitivity %
Specificity %
PLR
AUC
ROC AUC*
7
ANGPTL4 DKK1 GAL1 GFRA1
72.9
76.0
3.04
0.818
0.181
GRANULIN LRRC15 MUC1
6
ANGPTL4 DKK1 GAL1 GRANULIN
72.4
75.5
2.96
0.810
0.173
LRRC15 MUC1
4
ANGPTL4 DKK1 GAL1 LRRC15
69.8
74.5
2.74
0.790
0.152
5
ANGPTL4 DKK1 GAL1 GFRA1
69.3
74.5
2.72
0.803
0.165
LRRC15
6
DKK1 GAL1 GFRA1 GRANULIN
70.4
74.0
2.71
0.809
0.172
LRRC15 MUC1
6
ANGPTL4 DKK1 GAL1 GFRA1
70.9
73.5
2.68
0.812
0.175
GRANULIN LRRC15
5
DKK1 GAL1 GRANULIN LRRC15
69.8
73.0
2.59
0.805
0.167
MUC1
5
DKK1 GAL1 GFRA1 GRANULIN
68.3
73.5
2.58
0.799
0.162
LRRC15
5
DKK1 GAL1 GFRA1 LRRC15 MUC1
68.8
73.0
2.55
0.797
0.160
5
ANGPTL4 DKK1 GAL1 GRANULIN
68.3
73.0
2.53
0.804
0.166
LRRC15
4
DKK1 GAL1 GFRA1 LRRC15
68.3
73.0
2.53
0.791
0.153
4
DKK1 GAL1 GRANULIN LRRC15
68.8
72.4
2.49
0.796
0.159
5
ANGPTL4 DKK1 GAL1 LRRC15
69.8
71.4
2.44
0.794
0.157
MUC1
3
DKK1 GAL1 LRRC15
66.8
72.4
2.42
0.784
0.147
4
ANGPTL4 GAL1 LRRC15 MUC1
66.8
72.4
2.42
0.784
0.147
4
GAL1 GFRA1 LRRC15 MUC1
68.3
71.4
2.39
0.788
0.151
3
GAL1 GFRA1 LRRC15
66.8
71.9
2.38
0.770
0.132
3
ANGPTL4 GAL1 LRRC15
71.6
69.8
2.37
0.774
0.137
4
DKK1 GAL1 LRRC15 MUC1
67.8
71.4
2.37
0.789
0.152
4
ANGPTL4 GAL1 GFRA1 LRRC15
68.8
70.9
2.36
0.793
0.156
3
GAL1 LRRC15 MUC1
67.3
71.4
2.35
0.778
0.141
5
ANGPTL4 GAL1 GFRA1 LRRC15
68.8
69.9
2.29
0.798
0.161
MUC1
3
ANGPTL4 GAL1 GFRA1
64.8
66.3
1.92
0.753
0.116
3
DKK1 GAL1 GFRA1
65.3
65.8
1.91
0.746
0.109
3
GAL1 GFRA1 MUC1
63.3
64.8
1.80
0.746
0.109
PLR = positive likelihood ratio, sensitivity/(100 − specificity);
ROC = receiver operating characteristic curve;
AUC = area under the curve;
*Change in area under the ROC curve (i.e. c-statistic) was determined when the set of autoantibodies was added to a logistic regression model adjusted for age, race, BMI, and current smoking status (all p-value <0.0001).

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<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 4

Thr Ser Arg Arg Glu Val Cys Asp Cys Asn Gly Lys Ser Arg Gln Cys

1 5 10 15

Ile Phe Asp Arg Glu Leu His Arg Gln Thr Gly Asn Gly Phe Arg Cys

20 25 30

Leu Asn Cys Asn Asp Asn Thr Asp Gly Ile His Cys Glu Lys Cys Lys

35 40 45

Asn Gly Phe Tyr Arg His Arg Glu Arg Asp Arg Cys Leu Pro Cys Asn

50 55 60

Cys Asn Ser Lys Gly Ser Leu Ser Ala Arg Cys Asp Asn Ser Gly Arg

65 70 75 80

Cys Ser Cys Lys Pro Gly Val Thr Gly Ala Arg Cys Asp Arg Cys Leu

85 90 95

Pro Gly Phe His Met Leu Thr Asp Ala Gly Cys Thr Gln Asp Gln Arg

100 105 110

Leu Leu Asp Ser Lys Cys Asp Cys Asp Pro Ala Gly Ile Ala Gly Pro

115 120 125

Cys Asp Ala Gly Arg Cys Val Cys Lys Pro Ala Val Thr Gly Glu Arg

130 135 140

Cys Asp Arg Cys Arg Ser Gly Tyr Tyr Asn Leu Asp Gly Gly Asn Pro

145 150 155 160

Glu Gly Cys Thr Gln Cys Phe Cys Tyr Gly His Ser Ala Ser Cys Arg

165 170 175

Ser Ser Ala Glu Tyr Ser Val His Lys Ile Thr Ser Thr Phe His Gln

180 185 190

Asp Val Asp Gly Trp Lys Ala Val Gln Arg Asn Gly Ser Pro Ala Lys

195 200 205

Leu Gln Trp Ser Gln Arg His Gln Asp Val Phe Ser Ser Ala Gln Arg

210 215 220

Leu Asp Pro Val Tyr Phe Val Ala Pro Ala Lys Phe Leu Gly Asn Gln

225 230 235 240

Gln Val Ser Tyr Gly Gln Ser Leu Ser Phe Asp Tyr Arg Val Asp Arg

245 250 255

Gly Gly Arg His Pro Ser Ala His Asp Val Ile Leu Glu Gly Ala Gly

260 265 270

Leu Arg Ile Thr Ala Pro Leu Met Pro Leu Gly Lys Thr Leu Pro Cys

275 280 285

Gly Leu Thr Lys Thr Tyr Thr Phe Arg Leu Asn Glu His Pro Ser Asn

290 295 300

Asn Trp Ser Pro Gln Leu Ser Tyr Phe Glu Tyr Arg Arg Leu Leu Arg

305 310 315 320

Asn Leu Thr Ala Leu Arg Ile Arg Ala Thr Tyr Gly Glu Tyr Ser Thr

325 330 335

Gly Tyr Ile Asp Asn Val Thr Leu Ile Ser Ala Arg Pro Val Ser Gly

340 345 350

Ala Pro Ala Pro Trp Val Glu Gln Cys Ile Cys Pro Val Gly Tyr Lys

355 360 365

Gly Gln Phe Cys Gln Asp Cys Ala Ser Gly Tyr Lys Arg Asp Ser Ala

370 375 380

Arg Leu Gly Pro Phe Gly Thr Cys Ile Pro Cys Asn Cys Gln Gly Gly

385 390 395 400

Gly Ala Cys Asp Pro Asp Thr Gly Asp Cys Tyr Ser Gly Asp Glu Asn

405 410 415

Pro Asp Ile Glu Cys Ala Asp Cys Pro Ile Gly Phe Tyr Asn Asp Pro

420 425 430

His Asp Pro Arg Ser Cys Lys Pro Cys Pro Cys His Asn Gly Phe Ser

435 440 445

Cys Ser Val Met Pro Glu Thr Glu Glu Val Val Cys Asn Asn Cys Pro

450 455 460

Pro Gly Val Thr Gly Ala Arg Cys Glu Leu Cys Ala Asp Gly Tyr Phe

465 470 475 480

Gly Asp Pro Phe Gly Glu His Gly Pro Val Arg Pro Cys Gln Pro Cys

485 490 495

Gln Cys Asn Asn Asn Val Asp Pro Ser Ala Ser Gly Asn Cys Asp Arg

500 505 510

Leu Thr Gly Arg Cys Leu Lys Cys Ile His Asn Thr Ala Gly Ile Tyr

515 520 525

Cys Asp Gln Cys Lys Ala Gly Tyr Phe Gly Asp Pro Leu Ala Pro Asn

530 535 540

Pro Ala Asp Lys Cys Arg Ala Cys Asn Cys Asn Pro Met Gly Ser Glu

545 550 555 560

Pro Val Gly Cys Arg Ser Asp Gly Thr Cys Val Cys Lys Pro Gly Phe

565 570 575

Gly Gly Pro Asn Cys Glu His Gly Ala Phe Ser Cys Pro Ala Cys Tyr

580 585 590

Asn Gln Val Lys Ile Gln Met Asp Gln Phe Met Gln Gln Leu Gln Arg

595 600 605

Met Glu Ala Leu Ile Ser Lys Ala Gln Gly Gly Asp Gly Val Val Pro

610 615 620

Asp Thr Glu Leu Glu Gly Arg Met Gln Gln Ala Glu Gln Ala Leu Gln

625 630 635 640

Asp Ile Leu Arg Asp Ala Gln Ile Ser Glu Gly Ala Ser Arg Ser Leu

645 650 655

Gly Leu Gln Leu Ala Lys Val Arg Ser Gln Glu Asn Ser Tyr Gln Ser

660 665 670

Arg Leu Asp Asp Leu Lys Met Thr Val Glu Arg Val Arg Ala Leu Gly

675 680 685

Ser Gln Tyr Gln Asn Arg Val Arg Asp Thr His Arg Leu Ile Thr Gln

690 695 700

Met Gln Leu Ser Leu Ala Glu Ser Glu Ala Ser Leu Gly Asn Thr Asn

705 710 715 720

Ile Pro Ala Ser Asp His Tyr Val Gly Pro Asn Gly Phe Lys Ser Leu

725 730 735

Ala Gln Glu Ala Thr Arg Leu Ala Glu Ser His Val Glu Ser Ala Ser

740 745 750

Asn Met Glu Gln Leu Thr Arg Glu Thr Glu Asp Tyr Ser Lys Gln Ala

755 760 765

Leu Ser Leu Val Arg Lys Ala Leu His Glu Gly Val Gly Ser Gly Ser

770 775 780

Gly Ser Pro Asp Gly Ala Val Val Gln Gly Leu Val Glu Lys Leu Glu

785 790 795 800

Lys Thr Lys Ser Leu Ala Gln Gln Leu Thr Arg Glu Ala Thr Gln Ala

805 810 815

Glu Ile Glu Ala Asp Arg Ser Tyr Gln His Ser Leu Arg Leu Leu Asp

820 825 830

Ser Val Ser Arg Leu Gln Gly Val Ser Asp Gln Ser Phe Gln Val Glu

835 840 845

Glu Ala Lys Arg Ile Lys Gln Lys Ala Asp Ser Leu Ser Ser Leu Val

850 855 860

Thr Arg His Met Asp Glu Phe Lys Arg Thr Gln Lys Asn Leu Gly Asn

865 870 875 880

Trp Lys Glu Glu Ala Gln Gln Leu Leu Gln Asn Gly Lys Ser Gly Arg

885 890 895

Glu Lys Ser Asp Gln Leu Leu Ser Arg Ala Asn Leu Ala Lys Ser Arg

900 905 910

Ala Gln Glu Ala Leu Ser Met Gly Asn Ala Thr Phe Tyr Glu Val Glu

915 920 925

Ser Ile Leu Lys Asn Leu Arg Glu Phe Asp Leu Gln Val Asp Asn Arg

930 935 940

Lys Ala Glu Ala Glu Glu Ala Met Lys Arg Leu Ser Tyr Ile Ser Gln

945 950 955 960

Lys Val Ser Asp Ala Ser Asp Lys Thr Gln Gln Ala Glu Arg Ala Leu

965 970 975

Gly Ser Ala Ala Ala Asp Ala Gln Arg Ala Lys Asn Gly Ala Gly Glu

980 985 990

Ala Leu Glu Ile Ser Ser Glu Ile Glu Gln Glu Ile Gly Ser Leu Asn

995 1000 1005

Leu Glu Ala Asn Val Thr Ala Asp Gly Ala Leu Ala Met Glu Lys

1010 1015 1020

Gly Leu Ala Ser Leu Lys Ser Glu Met Arg Glu Val Glu Gly Glu

1025 1030 1035

Leu Glu Arg Lys Glu Leu Glu Phe Asp Thr Asn Met Asp Ala Val

1040 1045 1050

Gln Met Val Ile Thr Glu Ala Gln Lys Val Asp Thr Arg Ala Lys

1055 1060 1065

Asn Ala Gly Val Thr Ile Gln Asp Thr Leu Asn Thr Leu Asp Gly

1070 1075 1080

Leu Leu His Leu Met Gly Met

1085 1090

<210> SEQ ID NO: 5

<211> LENGTH: 305

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 5

Gln Pro Leu Gly Gly Glu Ser Ile Cys Ser Ala Arg Ala Pro Ala Lys

1 5 10 15

Tyr Ser Ile Thr Phe Thr Gly Lys Trp Ser Gln Thr Ala Phe Pro Lys

20 25 30

Gln Tyr Pro Leu Phe Arg Pro Pro Ala Gln Trp Ser Ser Leu Leu Gly

35 40 45

Ala Ala His Ser Ser Asp Tyr Ser Met Trp Arg Lys Asn Gln Tyr Val

50 55 60

Ser Asn Gly Leu Arg Asp Phe Ala Glu Arg Gly Glu Ala Trp Ala Leu

65 70 75 80

Met Lys Glu Ile Glu Ala Ala Gly Glu Ala Leu Gln Ser Val His Glu

85 90 95

Val Phe Ser Ala Pro Ala Val Pro Ser Gly Thr Gly Gln Thr Ser Ala

100 105 110

Glu Leu Glu Val Gln Arg Arg His Ser Leu Val Ser Phe Val Val Arg

115 120 125

Ile Val Pro Ser Pro Asp Trp Phe Val Gly Val Asp Ser Leu Asp Leu

130 135 140

Cys Asp Gly Asp Arg Trp Arg Glu Gln Ala Ala Leu Asp Leu Tyr Pro

145 150 155 160

Tyr Asp Ala Gly Thr Asp Ser Gly Phe Thr Phe Ser Ser Pro Asn Phe

165 170 175

Ala Thr Ile Pro Gln Asp Thr Val Thr Glu Ile Thr Ser Ser Ser Pro

180 185 190

Ser His Pro Ala Asn Ser Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro

195 200 205

Pro Ile Ala Arg Val Thr Leu Leu Arg Leu Arg Gln Ser Pro Arg Ala

210 215 220

Phe Ile Pro Pro Ala Pro Val Leu Pro Ser Arg Asp Asn Glu Ile Val

225 230 235 240

Asp Ser Ala Ser Val Pro Glu Thr Pro Leu Asp Cys Glu Val Ser Leu

245 250 255

Trp Ser Ser Trp Gly Leu Cys Gly Gly His Cys Gly Arg Leu Gly Thr

260 265 270

Lys Ser Arg Thr Arg Tyr Val Arg Val Gln Pro Ala Asn Asn Gly Ser

275 280 285

Pro Cys Pro Glu Leu Glu Glu Glu Ala Glu Cys Val Pro Asp Asn Cys

290 295 300

Val

305

<210> SEQ ID NO: 6

<211> LENGTH: 129

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 6

Glu Glu Gly Ala Arg Leu Leu Ala Ser Lys Ser Leu Leu Asn Arg Tyr

1 5 10 15

Ala Val Glu Gly Arg Asp Leu Thr Leu Gln Tyr Asn Ile Tyr Asn Val

20 25 30

Gly Ser Ser Ala Ala Leu Asp Val Glu Leu Ser Asp Asp Ser Phe Pro

35 40 45

Pro Glu Asp Phe Gly Ile Val Ser Gly Met Leu Asn Val Lys Trp Asp

50 55 60

Arg Ile Ala Pro Ala Ser Asn Val Ser His Thr Val Val Leu Arg Pro

65 70 75 80

Leu Lys Ala Gly Tyr Phe Asn Phe Thr Ser Ala Thr Ile Thr Tyr Leu

85 90 95

Ala Gln Glu Asp Gly Pro Val Val Ile Gly Ser Thr Ser Ala Pro Gly

100 105 110

Gln Gly Gly Ile Leu Ala Gln Arg Glu Phe Asp Arg Arg Phe Ser Pro

115 120 125

His

<210> SEQ ID NO: 7

<211> LENGTH: 118

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 7

Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val Leu

1 5 10 15

Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro Arg

20 25 30

Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys Asp

35 40 45

Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe Gln

50 55 60

Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn Leu

65 70 75 80

Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg Leu

85 90 95

Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys Ile

100 105 110

Lys Cys Val Ala Phe Asp

115

<210> SEQ ID NO: 8

<211> LENGTH: 441

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 8

Asp Arg Leu Asp Cys Val Lys Ala Ser Asp Gln Cys Leu Lys Glu Gln

1 5 10 15

Ser Cys Ser Thr Lys Tyr Arg Thr Leu Arg Gln Cys Val Ala Gly Lys

20 25 30

Glu Thr Asn Phe Ser Leu Ala Ser Gly Leu Glu Ala Lys Asp Glu Cys

35 40 45

Arg Ser Ala Met Glu Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg

50 55 60

Cys Lys Arg Gly Met Lys Lys Glu Lys Asn Cys Leu Arg Ile Tyr Trp

65 70 75 80

Ser Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro

85 90 95

Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp Ile Phe Arg Val Val Pro

100 105 110

Phe Ile Ser Asp Val Phe Gln Gln Val Glu His Ile Pro Lys Gly Asn

115 120 125

Asn Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Ile Cys Lys

130 135 140

Lys Tyr Arg Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser Asn

145 150 155 160

Asp Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe

165 170 175

Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser Cys

180 185 190

Arg Asp Ile Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val Pro Val

195 200 205

Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn Leu Gln Asp

210 215 220

Ser Cys Lys Thr Asn Tyr Ile Cys Arg Ser Arg Leu Ala Asp Phe Phe

225 230 235 240

Thr Asn Cys Gln Pro Glu Ser Arg Ser Val Ser Ser Cys Leu Lys Glu

245 250 255

Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser Gly Leu Ile Gly Thr Val

260 265 270

Met Thr Pro Asn Tyr Ile Asp Ser Ser Ser Leu Ser Val Ala Pro Trp

275 280 285

Cys Asp Cys Ser Asn Ser Gly Asn Asp Leu Glu Glu Cys Leu Lys Phe

290 295 300

Leu Asn Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn Ala Ile Gln Ala

305 310 315 320

Phe Gly Asn Gly Ser Asp Val Thr Val Trp Gln Pro Ala Phe Pro Val

325 330 335

Gln Thr Thr Thr Ala Thr Thr Thr Thr Ala Leu Arg Val Lys Asn Lys

340 345 350

Pro Leu Gly Pro Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu

355 360 365

Pro Pro Cys Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser Asn Val Ser

370 375 380

Gly Asn Thr His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly

385 390 395 400

Leu Gly Ala Ser Ser His Ile Thr Thr Lys Ser Met Ala Ala Pro Pro

405 410 415

Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu Ser

420 425 430

Thr Leu Leu Ser Leu Thr Glu Thr Ser

435 440

<210> SEQ ID NO: 9

<211> LENGTH: 517

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 9

Tyr His Gly Cys Pro Ser Glu Cys Thr Cys Ser Arg Ala Ser Gln Val

1 5 10 15

Glu Cys Thr Gly Ala Arg Ile Val Ala Val Pro Thr Pro Leu Pro Trp

20 25 30

Asn Ala Met Ser Leu Gln Ile Leu Asn Thr His Ile Thr Glu Leu Asn

35 40 45

Glu Ser Pro Phe Leu Asn Ile Ser Ala Leu Ile Ala Leu Arg Ile Glu

50 55 60

Lys Asn Glu Leu Ser Arg Ile Thr Pro Gly Ala Phe Arg Asn Leu Gly

65 70 75 80

Ser Leu Arg Tyr Leu Ser Leu Ala Asn Asn Lys Leu Gln Val Leu Pro

85 90 95

Ile Gly Leu Phe Gln Gly Leu Asp Ser Leu Glu Ser Leu Leu Leu Ser

100 105 110

Ser Asn Gln Leu Leu Gln Ile Gln Pro Ala His Phe Ser Gln Cys Ser

115 120 125

Asn Leu Lys Glu Leu Gln Leu His Gly Asn His Leu Glu Tyr Ile Pro

130 135 140

Asp Gly Ala Phe Asp His Leu Val Gly Leu Thr Lys Leu Asn Leu Gly

145 150 155 160

Lys Asn Ser Leu Thr His Ile Ser Pro Arg Val Phe Gln His Leu Gly

165 170 175

Asn Leu Gln Val Leu Arg Leu Tyr Glu Asn Arg Leu Thr Asp Ile Pro

180 185 190

Met Gly Thr Phe Asp Gly Leu Val Asn Leu Gln Glu Leu Ala Leu Gln

195 200 205

Gln Asn Gln Ile Gly Leu Leu Ser Pro Gly Leu Phe His Asn Asn His

210 215 220

Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His Ile Ser Gln Leu Pro

225 230 235 240

Pro Ser Val Phe Met Gln Leu Pro Gln Leu Asn Arg Leu Thr Leu Phe

245 250 255

Gly Asn Ser Leu Lys Glu Leu Ser Pro Gly Ile Phe Gly Pro Met Pro

260 265 270

Asn Leu Arg Glu Leu Trp Leu Tyr Asp Asn His Ile Ser Ser Leu Pro

275 280 285

Asp Asn Val Phe Ser Asn Leu Arg Gln Leu Gln Val Leu Ile Leu Ser

290 295 300

Arg Asn Gln Ile Ser Phe Ile Ser Pro Gly Ala Phe Asn Gly Leu Thr

305 310 315 320

Glu Leu Arg Glu Leu Ser Leu His Thr Asn Ala Leu Gln Asp Leu Asp

325 330 335

Gly Asn Val Phe Arg Met Leu Ala Asn Leu Gln Asn Ile Ser Leu Gln

340 345 350

Asn Asn Arg Leu Arg Gln Leu Pro Gly Asn Ile Phe Ala Asn Val Asn

355 360 365

Gly Leu Met Ala Ile Gln Leu Gln Asn Asn Gln Leu Glu Asn Leu Pro

370 375 380

Leu Gly Ile Phe Asp His Leu Gly Lys Leu Cys Glu Leu Arg Leu Tyr

385 390 395 400

Asp Asn Pro Trp Arg Cys Asp Ser Asp Ile Leu Pro Leu Arg Asn Trp

405 410 415

Leu Leu Leu Asn Gln Pro Arg Leu Gly Thr Asp Thr Val Pro Val Cys

420 425 430

Phe Ser Pro Ala Asn Val Arg Gly Gln Ser Leu Ile Ile Ile Asn Val

435 440 445

Asn Val Ala Val Pro Ser Val His Val Pro Glu Val Pro Ser Tyr Pro

450 455 460

Glu Thr Pro Trp Tyr Pro Asp Thr Pro Ser Tyr Pro Asp Thr Thr Ser

465 470 475 480

Val Ser Ser Thr Thr Glu Leu Thr Ser Pro Val Glu Asp Tyr Thr Asp

485 490 495

Leu Thr Thr Ile Gln Val Thr Asp Asp Arg Ser Val Trp Gly Met Thr

500 505 510

Gln Ala Gln Ser Gly

515

<210> SEQ ID NO: 10

<211> LENGTH: 576

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 10

Thr Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu Asp

1 5 10 15

Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu Asp Lys Trp

20 25 30

Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln Val Asp Ala

35 40 45

His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser Gly Thr Ser

50 55 60

Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly Asp Gly His His

65 70 75 80

Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp Gly Arg Ser Cys Phe

85 90 95

Gln Arg Ser Gly Asn Asn Ser Val Gly Ala Ile Gln Cys Pro Asp Ser

100 105 110

Gln Phe Glu Cys Pro Asp Phe Ser Thr Cys Cys Val Met Val Asp Gly

115 120 125

Ser Trp Gly Cys Cys Pro Met Pro Gln Ala Ser Cys Cys Glu Asp Arg

130 135 140

Val His Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His Thr Arg

145 150 155 160

Cys Ile Thr Pro Thr Gly Thr His Pro Leu Ala Lys Lys Leu Pro Ala

165 170 175

Gln Arg Thr Asn Arg Ala Val Ala Leu Ser Ser Ser Val Met Cys Pro

180 185 190

Asp Ala Arg Ser Arg Cys Pro Asp Gly Ser Thr Cys Cys Glu Leu Pro

195 200 205

Ser Gly Lys Tyr Gly Cys Cys Pro Met Pro Asn Ala Thr Cys Cys Ser

210 215 220

Asp His Leu His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile Gln

225 230 235 240

Ser Lys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr Lys

245 250 255

Leu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp Met Glu Val Ser

260 265 270

Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser Gly Ala Trp Gly

275 280 285

Cys Cys Pro Phe Thr Gln Ala Val Cys Cys Glu Asp His Ile His Cys

290 295 300

Cys Pro Ala Gly Phe Thr Cys Asp Thr Gln Lys Gly Thr Cys Glu Gln

305 310 315 320

Gly Pro His Gln Val Pro Trp Met Glu Lys Ala Pro Ala His Leu Ser

325 330 335

Leu Pro Asp Pro Gln Ala Leu Lys Arg Asp Val Pro Cys Asp Asn Val

340 345 350

Ser Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu Thr Ser Gly Glu

355 360 365

Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp His Gln

370 375 380

His Cys Cys Pro Gln Gly Tyr Thr Cys Val Ala Glu Gly Gln Cys Gln

385 390 395 400

Arg Gly Ser Glu Ile Val Ala Gly Leu Glu Lys Met Pro Ala Arg Arg

405 410 415

Ala Ser Leu Ser His Pro Arg Asp Ile Gly Cys Asp Gln His Thr Ser

420 425 430

Cys Pro Val Gly Gln Thr Cys Cys Pro Ser Leu Gly Gly Ser Trp Ala

435 440 445

Cys Cys Gln Leu Pro His Ala Val Cys Cys Glu Asp Arg Gln His Cys

450 455 460

Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu Lys

465 470 475 480

Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser Pro His

485 490 495

Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His Phe Cys His Asp

500 505 510

Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln Gly Trp Ala Cys Cys Pro

515 520 525

Tyr Arg Gln Gly Val Cys Cys Ala Asp Arg Arg His Cys Cys Pro Ala

530 535 540

Gly Phe Arg Cys Ala Ala Arg Gly Thr Lys Cys Leu Arg Arg Glu Ala

545 550 555 560

Pro Arg Trp Asp Ala Pro Leu Arg Asp Pro Ala Leu Arg Gln Leu Leu

565 570 575

<210> SEQ ID NO: 11

<211> LENGTH: 145

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 11

Ala Pro Lys Pro Ala Thr Val Val Thr Gly Ser Gly His Ala Ser Ser

1 5 10 15

Thr Pro Gly Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val

20 25 30

Pro Ser Ser Thr Glu Lys Asn Ala Phe Asn Ser Ser Leu Glu Asp Pro

35 40 45

Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser Glu Met Phe

50 55 60

Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser Asn Ile Lys

65 70 75 80

Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala Phe Arg Glu

85 90 95

Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn Gln Tyr Lys

100 105 110

Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp Val Ser Val

115 120 125

Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Ala Gly Val Pro

130 135 140

Gly

145

<210> SEQ ID NO: 12

<211> LENGTH: 141

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 12

Ala Ala Gly Thr Val Phe Thr Thr Val Glu Asp Leu Gly Ser Lys Ile

1 5 10 15

Leu Leu Thr Cys Ser Leu Asn Asp Ser Ala Thr Glu Val Thr Gly His

20 25 30

Arg Trp Leu Lys Gly Gly Val Val Leu Lys Glu Asp Ala Leu Pro Gly

35 40 45

Gln Lys Thr Glu Phe Lys Val Asp Ser Asp Asp Gln Trp Gly Glu Tyr

50 55 60

Ser Cys Val Phe Leu Pro Glu Pro Met Gly Thr Ala Asn Ile Gln Leu

65 70 75 80

His Gly Pro Pro Arg Val Lys Ala Val Lys Ser Ser Glu His Ile Asn

85 90 95

Glu Gly Glu Thr Ala Met Leu Val Cys Lys Ser Glu Ser Val Pro Pro

100 105 110

Val Thr Asp Trp Ala Trp Tyr Lys Ile Thr Asp Ser Glu Asp Lys Ala

115 120 125

Leu Met Asn Gly Ser Glu Ser Arg Phe Phe Val Ser Ser

130 135 140

<210> SEQ ID NO: 13

<211> LENGTH: 184

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 13

Ala Gly Pro Ser Ser Gly Ser Cys Pro Pro Thr Lys Phe Gln Cys Arg

1 5 10 15

Thr Ser Gly Leu Cys Val Pro Leu Thr Trp Arg Cys Asp Arg Asp Leu

20 25 30

Asp Cys Ser Asp Gly Ser Asp Glu Glu Glu Cys Arg Ile Glu Pro Cys

35 40 45

Thr Gln Lys Gly Gln Cys Pro Pro Pro Pro Gly Leu Pro Cys Pro Cys

50 55 60

Thr Gly Val Ser Asp Cys Ser Gly Gly Thr Asp Lys Lys Leu Arg Asn

65 70 75 80

Cys Ser Arg Leu Ala Cys Leu Ala Gly Glu Leu Arg Cys Thr Leu Ser

85 90 95

Asp Asp Cys Ile Pro Leu Thr Trp Arg Cys Asp Gly His Pro Asp Cys

100 105 110

Pro Asp Ser Ser Asp Glu Leu Gly Cys Gly Thr Asn Glu Ile Leu Pro

115 120 125

Glu Gly Asp Ala Thr Thr Met Gly Pro Pro Val Thr Leu Glu Ser Val

130 135 140

Thr Ser Leu Arg Asn Ala Thr Thr Met Gly Pro Pro Val Thr Leu Glu

145 150 155 160

Ser Val Pro Ser Val Gly Asn Ala Thr Ser Ser Ser Ala Gly Asp Gln

165 170 175

Ser Gly Ser Pro Thr Ala Tyr Gly

180

<210> SEQ ID NO: 14

<211> LENGTH: 630

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 14

Glu Pro Cys Arg Ala Val Phe Arg Glu Ala Glu Val Thr Leu Glu Ala

1 5 10 15

Gly Gly Ala Glu Gln Glu Pro Gly Gln Ala Leu Gly Lys Val Phe Met

20 25 30

Gly Cys Pro Gly Gln Glu Pro Ala Leu Phe Ser Thr Asp Asn Asp Asp

35 40 45

Phe Thr Val Arg Asn Gly Glu Thr Val Gln Glu Arg Arg Ser Leu Lys

50 55 60

Glu Arg Asn Pro Leu Lys Ile Phe Pro Ser Lys Arg Ile Leu Arg Arg

65 70 75 80

His Lys Arg Asp Trp Val Val Ala Pro Ile Ser Val Pro Glu Asn Gly

85 90 95

Lys Gly Pro Phe Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp

100 105 110

Arg Asp Thr Lys Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser

115 120 125

Pro Pro Glu Gly Val Phe Ala Val Glu Lys Glu Thr Gly Trp Leu Leu

130 135 140

Leu Asn Lys Pro Leu Asp Arg Glu Glu Ile Ala Lys Tyr Glu Leu Phe

145 150 155 160

Gly His Ala Val Ser Glu Asn Gly Ala Ser Val Glu Asp Pro Met Asn

165 170 175

Ile Ser Ile Ile Val Thr Asp Gln Asn Asp His Lys Pro Lys Phe Thr

180 185 190

Gln Asp Thr Phe Arg Gly Ser Val Leu Glu Gly Val Leu Pro Gly Thr

195 200 205

Ser Val Met Gln Met Thr Ala Thr Asp Glu Asp Asp Ala Ile Tyr Thr

210 215 220

Tyr Asn Gly Val Val Ala Tyr Ser Ile His Ser Gln Glu Pro Lys Asp

225 230 235 240

Pro His Asp Leu Met Phe Thr Ile His Arg Ser Thr Gly Thr Ile Ser

245 250 255

Val Ile Ser Ser Gly Leu Asp Arg Glu Lys Val Pro Glu Tyr Thr Leu

260 265 270

Thr Ile Gln Ala Thr Asp Met Asp Gly Asp Gly Ser Thr Thr Thr Ala

275 280 285

Val Ala Val Val Glu Ile Leu Asp Ala Asn Asp Asn Ala Pro Met Phe

290 295 300

Asp Pro Gln Lys Tyr Glu Ala His Val Pro Glu Asn Ala Val Gly His

305 310 315 320

Glu Val Gln Arg Leu Thr Val Thr Asp Leu Asp Ala Pro Asn Ser Pro

325 330 335

Ala Trp Arg Ala Thr Tyr Leu Ile Met Gly Gly Asp Asp Gly Asp His

340 345 350

Phe Thr Ile Thr Thr His Pro Glu Ser Asn Gln Gly Ile Leu Thr Thr

355 360 365

Arg Lys Gly Leu Asp Phe Glu Ala Lys Asn Gln His Thr Leu Tyr Val

370 375 380

Glu Val Thr Asn Glu Ala Pro Phe Val Leu Lys Leu Pro Thr Ser Thr

385 390 395 400

Ala Thr Ile Val Val His Val Glu Asp Val Asn Glu Ala Pro Val Phe

405 410 415

Val Pro Pro Ser Lys Val Val Glu Val Gln Glu Gly Ile Pro Thr Gly

420 425 430

Glu Pro Val Cys Val Tyr Thr Ala Glu Asp Pro Asp Lys Glu Asn Gln

435 440 445

Lys Ile Ser Tyr Arg Ile Leu Arg Asp Pro Ala Gly Trp Leu Ala Met

450 455 460

Asp Pro Asp Ser Gly Gln Val Thr Ala Val Gly Thr Leu Asp Arg Glu

465 470 475 480

Asp Glu Gln Phe Val Arg Asn Asn Ile Tyr Glu Val Met Val Leu Ala

485 490 495

Met Asp Asn Gly Ser Pro Pro Thr Thr Gly Thr Gly Thr Leu Leu Leu

500 505 510

Thr Leu Ile Asp Val Asn Asp His Gly Pro Val Pro Glu Pro Arg Gln

515 520 525

Ile Thr Ile Cys Asn Gln Ser Pro Val Arg Gln Val Leu Asn Ile Thr

530 535 540

Asp Lys Asp Leu Ser Pro His Thr Ser Pro Phe Gln Ala Gln Leu Thr

545 550 555 560

Asp Asp Ser Asp Ile Tyr Trp Thr Ala Glu Val Asn Glu Glu Gly Asp

565 570 575

Thr Val Val Leu Ser Leu Lys Lys Phe Leu Lys Gln Asp Thr Tyr Asp

580 585 590

Val His Leu Ser Leu Ser Asp His Gly Asn Lys Glu Gln Leu Thr Val

595 600 605

Ile Arg Ala Thr Val Cys Asp Cys His Gly His Val Glu Thr Cys Pro

610 615 620

Gly Pro Trp Lys Gly Gly

625 630

<210> SEQ ID NO: 15

<211> LENGTH: 630

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 15

Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser

1 5 10 15

Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln

20 25 30

Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser

35 40 45

Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile

50 55 60

Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val

65 70 75 80

Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp

85 90 95

Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro

100 105 110

Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys

115 120 125

Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr

130 135 140

Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr

145 150 155 160

Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met

165 170 175

Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser

180 185 190

Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro

195 200 205

Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly

210 215 220

Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly

225 230 235 240

Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr

245 250 255

Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser

260 265 270

Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser

275 280 285

Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp

290 295 300

Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys

305 310 315 320

Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser

325 330 335

Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu

340 345 350

Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala

355 360 365

Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile

370 375 380

Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu

385 390 395 400

Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn

405 410 415

Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly

420 425 430

Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His

435 440 445

Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe

450 455 460

Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp

465 470 475 480

Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly

485 490 495

His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe

500 505 510

Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu

515 520 525

Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu

530 535 540

Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp

545 550 555 560

Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala

565 570 575

Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp

580 585 590

Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys

595 600 605

Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln

610 615 620

Arg Ala Ser Pro Leu Thr

625 630

<210> SEQ ID NO: 16

<211> LENGTH: 289

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 16

Glu Val Leu Phe Arg Cys Pro Pro Cys Thr Pro Glu Arg Leu Ala Ala

1 5 10 15

Cys Gly Pro Pro Pro Val Ala Pro Pro Ala Ala Val Ala Ala Val Ala

20 25 30

Gly Gly Ala Arg Met Pro Cys Ala Glu Leu Val Arg Glu Pro Gly Cys

35 40 45

Gly Cys Cys Ser Val Cys Ala Arg Leu Glu Gly Glu Ala Cys Gly Val

50 55 60

Tyr Thr Pro Arg Cys Gly Gln Gly Leu Arg Cys Tyr Pro His Pro Gly

65 70 75 80

Ser Glu Leu Pro Leu Gln Ala Leu Val Met Gly Glu Gly Thr Cys Glu

85 90 95

Lys Arg Arg Asp Ala Glu Tyr Gly Ala Ser Pro Glu Gln Val Ala Asp

100 105 110

Asn Gly Asp Asp His Ser Glu Gly Gly Leu Val Glu Asn His Val Asp

115 120 125

Ser Thr Met Asn Met Leu Gly Gly Gly Gly Ser Ala Gly Arg Lys Pro

130 135 140

Leu Lys Ser Gly Met Lys Glu Leu Ala Val Phe Arg Glu Lys Val Thr

145 150 155 160

Glu Gln His Arg Gln Met Gly Lys Gly Gly Lys His His Leu Gly Leu

165 170 175

Glu Glu Pro Lys Lys Leu Arg Pro Pro Pro Ala Arg Thr Pro Cys Gln

180 185 190

Gln Glu Leu Asp Gln Val Leu Glu Arg Ile Ser Thr Met Arg Leu Pro

195 200 205

Asp Glu Arg Gly Pro Leu Glu His Leu Tyr Ser Leu His Ile Pro Asn

210 215 220

Cys Asp Lys His Gly Leu Tyr Asn Leu Lys Gln Cys Lys Met Ser Leu

225 230 235 240

Asn Gly Gln Arg Gly Glu Cys Trp Cys Val Asn Pro Asn Thr Gly Lys

245 250 255

Leu Ile Gln Gly Ala Pro Thr Ile Arg Gly Asp Pro Glu Cys His Leu

260 265 270

Phe Tyr Asn Glu Gln Gln Glu Ala Arg Gly Val His Thr Gln Arg Met

275 280 285

Gln

<210> SEQ ID NO: 17

<211> LENGTH: 424

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 17

His Pro Asp Arg Ile Ile Phe Pro Asn His Ala Cys Glu Asp Pro Pro

1 5 10 15

Ala Val Leu Leu Glu Val Gln Gly Thr Leu Gln Arg Pro Leu Val Arg

20 25 30

Asp Ser Arg Thr Ser Pro Ala Asn Cys Thr Trp Leu Ile Leu Gly Ser

35 40 45

Lys Glu Gln Thr Val Thr Ile Arg Phe Gln Lys Leu His Leu Ala Cys

50 55 60

Gly Ser Glu Arg Leu Thr Leu Arg Ser Pro Leu Gln Pro Leu Ile Ser

65 70 75 80

Leu Cys Glu Ala Pro Pro Ser Pro Leu Gln Leu Pro Gly Gly Asn Val

85 90 95

Thr Ile Thr Tyr Ser Tyr Ala Gly Ala Arg Ala Pro Met Gly Gln Gly

100 105 110

Phe Leu Leu Ser Tyr Ser Gln Asp Trp Leu Met Cys Leu Gln Glu Glu

115 120 125

Phe Gln Cys Leu Asn His Arg Cys Val Ser Ala Val Gln Arg Cys Asp

130 135 140

Gly Val Asp Ala Cys Gly Asp Gly Ser Asp Glu Ala Gly Cys Ser Ser

145 150 155 160

Asp Pro Phe Pro Gly Leu Thr Pro Arg Pro Val Pro Ser Leu Pro Cys

165 170 175

Asn Val Thr Leu Glu Asp Phe Tyr Gly Val Phe Ser Ser Pro Gly Tyr

180 185 190

Thr His Leu Ala Ser Val Ser His Pro Gln Ser Cys His Trp Leu Leu

195 200 205

Asp Pro His Asp Gly Arg Arg Leu Ala Val Arg Phe Thr Ala Leu Asp

210 215 220

Leu Gly Phe Gly Asp Ala Val His Val Tyr Asp Gly Pro Gly Pro Pro

225 230 235 240

Glu Ser Ser Arg Leu Leu Arg Ser Leu Thr His Phe Ser Asn Gly Lys

245 250 255

Ala Val Thr Val Glu Thr Leu Ser Gly Gln Ala Val Val Ser Tyr His

260 265 270

Thr Val Ala Trp Ser Asn Gly Arg Gly Phe Asn Ala Thr Tyr His Val

275 280 285

Arg Gly Tyr Cys Leu Pro Trp Asp Arg Pro Cys Gly Leu Gly Ser Gly

290 295 300

Leu Gly Ala Gly Glu Gly Leu Gly Glu Arg Cys Tyr Ser Glu Ala Gln

305 310 315 320

Arg Cys Asp Gly Ser Trp Asp Cys Ala Asp Gly Thr Asp Glu Glu Asp

325 330 335

Cys Pro Gly Cys Pro Pro Gly His Phe Pro Cys Gly Ala Ala Gly Thr

340 345 350

Ser Gly Ala Thr Ala Cys Tyr Leu Pro Ala Asp Arg Cys Asn Tyr Gln

355 360 365

Thr Phe Cys Ala Asp Gly Ala Asp Glu Arg Arg Cys Arg His Cys Gln

370 375 380

Pro Gly Asn Phe Arg Cys Arg Asp Glu Lys Cys Val Tyr Glu Thr Trp

385 390 395 400

Val Cys Asp Gly Gln Pro Asp Cys Ala Asp Gly Ser Asp Glu Trp Asp

405 410 415

Cys Ser Tyr Val Leu Pro Arg Lys

420

<210> SEQ ID NO: 18

<211> LENGTH: 171

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 18

Ala Asp Arg Glu Arg Ser Ile His Asp Phe Cys Leu Val Ser Lys Val

1 5 10 15

Val Gly Arg Cys Arg Ala Ser Met Pro Arg Trp Trp Tyr Asn Val Thr

20 25 30

Asp Gly Ser Cys Gln Leu Phe Val Tyr Gly Gly Cys Asp Gly Asn Ser

35 40 45

Asn Asn Tyr Leu Thr Lys Glu Glu Cys Leu Lys Lys Cys Ala Thr Val

50 55 60

Thr Glu Asn Ala Thr Gly Asp Leu Ala Thr Ser Arg Asn Ala Ala Asp

65 70 75 80

Ser Ser Val Pro Ser Ala Pro Arg Arg Gln Asp Ser Glu Asp His Ser

85 90 95

Ser Asp Met Phe Asn Tyr Glu Glu Tyr Cys Thr Ala Asn Ala Val Thr

100 105 110

Gly Pro Cys Arg Ala Ser Phe Pro Arg Trp Tyr Phe Asp Val Glu Arg

115 120 125

Asn Ser Cys Asn Asn Phe Ile Tyr Gly Gly Cys Arg Gly Asn Lys Asn

130 135 140

Ser Tyr Arg Ser Glu Glu Ala Cys Met Leu Arg Cys Phe Arg Gln Gln

145 150 155 160

Glu Asn Pro Pro Leu Pro Leu Gly Ser Lys Val

165 170

<210> SEQ ID NO: 19

<211> LENGTH: 758

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 19

Gln Glu Ser Cys Ser Met Arg Cys Gly Ala Leu Asp Gly Pro Cys Ser

1 5 10 15

Cys His Pro Thr Cys Ser Gly Leu Gly Thr Cys Cys Leu Asp Phe Arg

20 25 30

Asp Phe Cys Leu Glu Ile Leu Pro Tyr Ser Gly Ser Met Met Gly Gly

35 40 45

Lys Asp Phe Val Val Arg His Phe Lys Met Ser Ser Pro Thr Asp Ala

50 55 60

Ser Val Ile Cys Arg Phe Lys Asp Ser Ile Gln Thr Leu Gly His Val

65 70 75 80

Asp Ser Ser Gly Gln Val His Cys Val Ser Pro Leu Leu Tyr Glu Ser

85 90 95

Gly Arg Ile Pro Phe Thr Val Ser Leu Asp Asn Gly His Ser Phe Pro

100 105 110

Arg Ala Gly Thr Trp Leu Ala Val His Pro Asn Lys Val Ser Met Met

115 120 125

Glu Lys Ser Glu Leu Val Asn Glu Thr Arg Trp Gln Tyr Tyr Gly Thr

130 135 140

Ala Asn Thr Ser Gly Asn Leu Ser Leu Thr Trp His Val Lys Ser Leu

145 150 155 160

Pro Thr Gln Thr Ile Thr Ile Glu Leu Trp Gly Tyr Glu Glu Thr Gly

165 170 175

Met Pro Tyr Ser Gln Glu Trp Thr Ala Lys Trp Ser Tyr Leu Tyr Pro

180 185 190

Leu Ala Thr His Ile Pro Asn Ser Gly Ser Phe Thr Phe Thr Pro Lys

195 200 205

Pro Ala Pro Pro Ser Tyr Gln Arg Trp Arg Val Gly Ala Leu Arg Ile

210 215 220

Ile Asp Ser Lys Asn Tyr Ala Gly Gln Lys Asp Val Gln Ala Leu Trp

225 230 235 240

Thr Asn Asp His Ala Leu Ala Trp His Leu Ser Asp Asp Phe Arg Glu

245 250 255

Asp Pro Val Ala Trp Ala Arg Thr Gln Cys Gln Ala Trp Glu Glu Leu

260 265 270

Glu Asp Gln Leu Pro Asn Phe Leu Glu Glu Leu Pro Asp Cys Pro Cys

275 280 285

Thr Leu Thr Gln Ala Arg Ala Asp Ser Gly Arg Phe Phe Thr Asp Tyr

290 295 300

Gly Cys Asp Met Glu Gln Gly Ser Val Cys Thr Tyr His Pro Gly Ala

305 310 315 320

Val His Cys Val Arg Ser Val Gln Ala Ser Leu Arg Tyr Gly Ser Gly

325 330 335

Gln Gln Cys Cys Tyr Thr Ala Asp Gly Thr Gln Leu Leu Thr Ala Asp

340 345 350

Ser Ser Gly Gly Ser Thr Pro Asp Arg Gly His Asp Trp Gly Ala Pro

355 360 365

Pro Phe Arg Thr Pro Pro Arg Val Pro Ser Met Ser His Trp Leu Tyr

370 375 380

Asp Val Leu Ser Phe Tyr Tyr Cys Cys Leu Trp Ala Pro Asp Cys Pro

385 390 395 400

Arg Tyr Met Gln Arg Arg Pro Ser Asn Asp Cys Arg Asn Tyr Arg Pro

405 410 415

Pro Arg Leu Ala Ser Ala Phe Gly Asp Pro His Phe Val Thr Phe Asp

420 425 430

Gly Thr Asn Phe Thr Phe Asn Gly Arg Gly Glu Tyr Val Leu Leu Glu

435 440 445

Ala Ala Leu Thr Asp Leu Arg Val Gln Ala Arg Ala Gln Pro Gly Thr

450 455 460

Met Ser Asn Gly Thr Glu Thr Arg Gly Thr Gly Leu Thr Ala Val Ala

465 470 475 480

Val Gln Glu Gly Asn Ser Asp Val Val Glu Val Arg Leu Ala Asn Arg

485 490 495

Thr Gly Gly Leu Glu Val Leu Leu Asn Gln Glu Val Leu Ser Phe Thr

500 505 510

Glu Gln Ser Trp Met Asp Leu Lys Gly Met Phe Leu Ser Val Ala Ala

515 520 525

Gly Asp Arg Val Ser Ile Met Leu Ala Ser Gly Ala Gly Leu Glu Val

530 535 540

Ser Val Gln Gly Pro Phe Leu Ser Val Ser Val Leu Leu Pro Glu Lys

545 550 555 560

Phe Leu Thr His Thr His Gly Leu Leu Gly Thr Leu Asn Asn Asp Pro

565 570 575

Thr Asp Asp Phe Thr Leu His Ser Gly Arg Val Leu Pro Pro Gly Thr

580 585 590

Ser Pro Gln Glu Leu Phe Leu Phe Gly Ala Asn Trp Thr Val His Asn

595 600 605

Ala Ser Ser Leu Leu Thr Tyr Asp Ser Trp Phe Leu Val His Asn Phe

610 615 620

Leu Tyr Gln Pro Lys His Asp Pro Thr Phe Glu Pro Leu Phe Pro Ser

625 630 635 640

Glu Thr Thr Leu Asn Pro Ser Leu Ala Gln Glu Ala Ala Lys Leu Cys

645 650 655

Gly Asp Asp His Phe Cys Asn Phe Asp Val Ala Ala Thr Gly Ser Leu

660 665 670

Ser Thr Gly Thr Ala Thr Arg Val Ala His Gln Leu His Gln Arg Arg

675 680 685

Met Gln Ser Leu Gln Pro Val Val Ser Cys Gly Trp Leu Ala Pro Pro

690 695 700

Pro Asn Gly Gln Lys Glu Gly Asn Arg Tyr Leu Ala Gly Ser Thr Ile

705 710 715 720

Tyr Phe His Cys Asp Asn Gly Tyr Ser Leu Ala Gly Ala Glu Thr Ser

725 730 735

Thr Cys Gln Ala Asp Gly Thr Trp Ser Ser Pro Thr Pro Lys Cys Gln

740 745 750

Pro Gly Arg Ser Tyr Ala

755

<210> SEQ ID NO: 20

<211> LENGTH: 121

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 20

Trp Ser Pro Gln Glu Glu Asp Arg Ile Ile Glu Gly Gly Ile Tyr Asp

1 5 10 15

Ala Asp Leu Asn Asp Glu Arg Val Gln Arg Ala Leu His Phe Val Ile

20 25 30

Ser Glu Tyr Asn Lys Ala Thr Glu Asp Glu Tyr Tyr Arg Arg Leu Leu

35 40 45

Arg Val Leu Arg Ala Arg Glu Gln Ile Val Gly Gly Val Asn Tyr Phe

50 55 60

Phe Asp Ile Glu Val Gly Arg Thr Ile Cys Thr Lys Ser Gln Pro Asn

65 70 75 80

Leu Asp Thr Cys Ala Phe His Glu Gln Pro Glu Leu Gln Lys Lys Gln

85 90 95

Leu Cys Ser Phe Gln Ile Tyr Glu Val Pro Trp Glu Asp Arg Met Ser

100 105 110

Leu Val Asn Ser Arg Cys Gln Glu Ala

115 120

<210> SEQ ID NO: 21

<211> LENGTH: 29

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 21

ccggatatca gcaagcccac gtgcccacc 29

<210> SEQ ID NO: 22

<211> LENGTH: 40

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 22

aaggaaaaaa gcggccgctc atttacccgg agagcgggag 40

<210> SEQ ID NO: 23

<211> LENGTH: 34

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 23

cccaagcttg cagcacccaa gtgtgcaccg gcac 34

<210> SEQ ID NO: 24

<211> LENGTH: 37

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 24

gtgctcgagt cacgtcagag ggctggctct ctgctcg 37

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Patent Valuation

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Reveal the value <>

32.68/100 Score

Market Attractiveness

It shows from an IP point of view how many competitors are active and innovations are made in the different technical fields of the company. On a company level, the market attractiveness is often also an indicator of how diversified a company is. Here we look into the commercial relevance of the market.

60.0/100 Score

Market Coverage

It shows the sizes of the market that is covered with the IP and in how many countries the IP guarantees protection. It reflects a market size that is potentially addressable with the invented technology/formulation with a legal protection which also includes a freedom to operate. Here we look into the size of the impacted market.

75.71/100 Score

Technology Quality

It shows the degree of innovation that can be derived from a company’s IP. Here we look into ease of detection, ability to design around and significance of the patented feature to the product/service.

65.0/100 Score

Assignee Score

It takes the R&D behavior of the company itself into account that results in IP. During the invention phase, larger companies are considered to assign a higher R&D budget on a certain technology field, these companies have a better influence on their market, on what is marketable and what might lead to a standard.

22.43/100 Score

Legal Score

It shows the legal strength of IP in terms of its degree of protecting effect. Here we look into claim scope, claim breadth, claim quality, stability and priority.

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