Compositions And Methods For The Detection Diagnosis And Therapy Of Hematological Malignancies

Abstract

Disclosed are methods and compositions for the detection, diagnosis, prognosis, and therapy of hematological malignancies, and in particular, B cell leukemias, lymphomas and multiple myelomas. Disclosed are compositions, methods and kits for eliciting immune and T cell responses to specific malignancy-related antigenic polypeptides and antigenic polypeptide fragments thereof in an animal. Also disclosed are compositions and methods for use in the identification of cells and biological samples containing one or more hematological malignancy-related compositions, and methods for the detection and diagnosis of such diseases and affected cell types. Also disclosed are diagnostic and therapeutic kits, as well as methods for the diagnosis, therapy and/or prevention of a variety of leukemias and lymphomas.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. patent application Ser. No. 10/057,475, filed Jan. 22, 2002, which is a continuation in part of U.S. patent application Ser. No. 10/040,862, filed Nov. 6, 2001, which is a continuation in part of U.S. Ser. No. 09/796,692 filed Mar. 1, 2001, which claims priority to U.S. Provisional Patent Application Ser. No. 60/186,126, filed Mar. 1, 2000; Ser. No. 60/190,479, filed Mar. 17, 2000; Ser. No. 60/200,545, filed Apr. 27, 2000; Ser. No. 60/200,303, filed Apr. 28, 2000; Ser. No. 60/200,779, filed Apr. 28, 2000; Ser. No. 60/200,999; filed May 1, 2000; Ser. No. 60/202,084, filed May 4, 2000; Ser. No. 60/206,201, filed May 22, 2000; Ser. No. 60/218,950, filed Jul. 14, 2000; Ser. No. 60/222,903, filed Aug. 3, 2000; Ser. No. 60/223,416, filed Aug. 4, 2000; and Ser. No. 60/223,378, filed Aug. 7, 2000; the entire specification, claims, sequences and figures of each of which is specifically incorporated herein by reference in its entirety without disclaimer and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] Not applicable.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

[0003] Not applicable.

1. BACKGROUND OF THE INVENTION

[0004] 1.1 Field of the Invention

[0005] The present invention relates generally to the fields of cancer diagnosis and therapy. More particularly, it concerns the surprising discovery of compositions and methods for the detection and immunotherapy of hematological malignancies, and particularly, B cell leukemias, and lymphomas and multiple myelomas. The invention provides new, effective methods, compositions and kits for eliciting immune and T-cell response to antigenic polypeptides, and antigenic peptide fragments isolated therefrom, and methods for the use of such compositions for diagnosis, detection, treatment, monitoring, and/or prevention of various types of human hematological malignancies. In particular, the invention provides polypeptide, peptide, antibody, antigen binding fragment, hybridoma, host cell, vector, and polynucleotide compounds and compositions for use in identification and discrimination between various types of hematological malignancies, and methods for the detection, diagnosis, prognosis, monitoring, and therapy of such conditions in an affected animal.

[0006] 1.2 Description of Related Art

[0007] 1.2.1 Hematological Malignancies

[0008] Hematological malignancies, such as leukemias and lymphomas, are conditions characterized by abnormal growth and maturation of hematopoietic cells. Leukemias are generally neoplastic disorders of hematopoietic stem cells, and include adult and pediatric acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and secondary leukemia. Among lymphomas, there are two distinct groups: non-Hodgkin's lymphoma (NHL) and Hodgkin's disease. NHLs are the result of a clonal expansion of B- or T-cells, but the molecular pathogenesis of Hodgkin's disease, including lineage derivation and clonality, remains obscure. Other hematological malignancies include myelodysplastic syndromes (MDS), myeloproliferative syndromes (MPS) and multiple myeloma. Hematological malignancies are generally serious disorders, resulting in a variety of symptoms, including bone marrow failure and organ failure.

[0009] NHLs are the sixth most common cause of cancer related deaths in the United States. Only prostate, breast, lung, colorectal and bladder cancer currently exceed lymphoma in annual incidence. In 1995, more than 45,000 new NHLs were diagnosed, and over 21,000 patients died of these diseases. The average age of lymphoma patients is relatively young (42 years), and the resulting number of years of life lost to these diseases renders NHLs fourth in economic impact among cancers in the United States. In the past 15 years, the American Cancer Society reported a 50% increase in the incidence of NHLs, one of the largest increases for any cancer group. Much of this increase has been attributed to the development of lymphomas in younger men who have acquired AIDS. Lymphomas are also the third most common childhood malignancy and account for approximately 10% of cancers in children. The survival rate (all ages) varies from 73% (low risk) to 26% (high risk).

[0010] 1.3 Deficiencies in the Prior Art

[0011] Treatment for many hematological malignancies, including leukemias and lymphomas, remains difficult, and existing therapies are not universally effective. While treatments involving specific immunotherapy appear to have considerable potential, such treatments have been limited by the small number of known malignancy-associated antigens. Moreover the ability to detect such hematological malignancies in their early stages can be quite difficult depending upon the particular malady. The lack of a sufficient number of specific diagnostic and prognostic markers of the diseases, and identification of cells and tissues that can be affected, has significantly limited the field of oncology.

[0012] Accordingly, there remains a need in the art for improved methods for detecting, screening, diagnosis and treatment of hematological malignancies such as B cell leukemias and lymphomas and multiple myelomas. The present invention fulfills these and other inherent needs in the field, and provides significant advantages in the detection of cells, and cell types that express one or more polypeptides that have been shown to be over-expressed in one or more of such hematological malignancies.

2. SUMMARY OF THE INVENTION

[0013] The present invention addresses the foregoing long-felt need and other deficiencies in the art by identifying new and effective strategies for the identification, detection, screening, diagnosis, prognosis, prophylaxis, therapy, and immunomodulation of one or more hematological malignancies, and in particular, B cell leukemias and lymphomas, and multiple myelomas.

[0014] The present invention is based, in part, upon the surprising and unexpected discovery that certain previously unknown or unidentified human polypeptides, peptides, and antigenic fragments derived therefrom have now been identified that are overexpressed in one or more types of hematological malignancies. The genes encoding several of these polypeptides are now identified and obtained in isolated form, and have been characterized using a series of molecular biology methodologies including subtractive library analysis, microarray screening, polynucleotide sequencing, peptide and epitopic identification and characterization, as well as expression profiling, and in vitro whole gene cell priming. A set of these polynucleotides, and the polypeptides, peptides, and antigenic fragments they encode are now identified and implicated in the complex processes of hematological malignancy disease onset, progression, and/or outcome, and in particular, diseases such as leukemias and lymphomas.

[0015] The inventors have further demonstrated that a number of these polynucleotides, and their encoded polypeptides, as well as antibodies, antigen presenting cells, T cells, and the antigen binding fragments derived from such antibodies are useful in the development of particularly advantageous compositions and methods for the detection, diagnosis, prognosis, prophylaxis and/or therapy of one or more of these diseases, and particularly those conditions that are characterized by (a) an increased, altered, elevated, or sustained expression of one or more polynucleotides that comprise at least a first sequence region that comprises a nucleic acid sequence as disclosed in any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or (b) an increased, altered, elevated, or sustained biological activity of one or more polypeptides that comprise at least a first sequence region that comprises an amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0016] The present invention also provides methods and uses for one or more of the disclosed peptide, polypeptide, antibody, antigen binding fragment, and polynucleotide compositions of the present invention in generating an immune response or in generating a T-cell response in an animal, and in particular in a mammal such as a human. The invention also provides methods and uses for one or more of these compositions in the identification, detection, and quantitation of hematological malignancy compositions in clinical samples, isolated cells, whole tissues, and even affected individuals. The compositions and methods disclosed herein also may be used in the preparation of one or more diagnostic reagents, assays, medicaments, or therapeutics, for diagnosis and/or therapy of such diseases.

[0017] In a first important embodiment, there is provided a composition comprising at least a first isolated peptide or polypeptide that comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. Exemplary preferred sequences are those that comprise at least a first coding region that comprises an amino acid sequence that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, or about 94% identical to the amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, with those sequences that comprise at least a first coding region that comprises an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121 being examples of particularly preferred sequences in the practice of the present invention. Likewise, peptide and polypeptide compounds and compositions are also provided that comprise, consist essentially of, or consist of the amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0018] In a similar fashion, there are also embodiments disclosed herein that provide compositions and methods for the detection, diagnosis, prognosis, prophylaxis, treatment, and therapy of B cell leukemia, lymphoma and multiple myeloma. Exemplary preferred peptide and polypeptide compounds and compositions relating to this aspect of the invention include, but are not limited to, those peptide and polypeptide compounds or compositions that comprise at least a first isolated peptide or polypeptide that comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, and those that comprise at least a first coding region that comprises an amino acid sequence that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, or about 94% identical to the amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, and even those sequences that comprise at least a first coding region that comprises an amino acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0019] Exemplary peptides of the present invention may be of any suitable length, depending upon the particular application thereof; and encompass those peptides that are about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 or so amino acids in length. Of course, the peptides of the invention may also encompass any intermediate lengths or integers within the stated ranges.

[0020] Exemplary polypeptides and proteins of the present invention may be of any suitable length, depending upon the particular application thereof, and encompass those polypeptides and proteins that are about 100, about 150, about 200, about 250, about 300, about 350, or about 400 or so amino acids in length. Of course, the polypeptides and proteins of the invention may also encompass any intermediate lengths or integers within the stated ranges.

[0021] The peptides, polypeptides, proteins, antibodies, and antigen binding fragments of the present invention will preferably comprise a sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 contiguous amino acids from any one of the peptides as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0022] Furthermore, the polypeptides, proteins, antibodies, and antigen binding fragments of the present invention will even more preferably comprise at least a first isolated coding region that comprises a sequence of at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 contiguous amino acids from any one of the peptides as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0023] Likewise, the polypeptides, proteins, antibodies, and antigen binding fragments of the present invention may comprise at least a first isolated coding region that comprises a substantially longer sequence, such as for example, one of at least about 200, 220, 240, 260, 280, or 300 or more contiguous amino acids from any one of the peptides as encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0024] In illustrative embodiments, and particularly in those embodiments concerning methods and compositions relating to B cell leukemias, lymphomas and multiple myelomas, the polypeptides of the invention comprise an amino acid sequence that (a) comprises, (b) consists essentially of, or (c) consists of, the amino acid sequence encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121.

[0025] The polypeptides and proteins of the invention preferably comprise an amino acid sequence that is encoded by at least a first nucleic acid segment that comprises an at least 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124.

[0026] The polypeptides and proteins of the invention may also preferably comprise an amino acid sequence encoded by at least a first nucleic acid segment that comprises an at least about 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. The polypeptides and proteins of the invention may also preferably comprise one or more coding regions that comprise an amino acid sequence encoded by at least a first nucleic acid segment that comprises an at least about 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. The polypeptides and proteins of the invention may also preferably comprise one or more coding regions that comprise an amino acid sequence encoded by at least a first nucleic acid segment that comprises an at least about 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. The polypeptides and proteins of the invention may also preferably comprise one or more coding regions that comprise an amino acid sequence encoded by at least a first nucleic acid segment that comprises an at least about 70, 80, 90, 100, 110, 120, 130, 140 or 150 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. The polypeptides and proteins of the invention may also preferably comprise one or more coding regions that comprise an amino acid sequence encoded by at least a first nucleic acid segment that comprises an at least about 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. The polypeptides and proteins of the invention may also preferably comprise one or more coding regions that comprise an amino acid sequence encoded by at least a first nucleic acid segment that comprises an at least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 contiguous nucleotide sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124.

[0027] In a second important embodiment, there is provided a composition comprising at least a first isolated polynucleotide that comprises a nucleic acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleic acid sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. Exemplary preferred sequences are those that comprise a nucleic acid sequence that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, or about 94% identical to the nucleic acid sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124, with those sequences that comprise at least a nucleic acid sequence that is at least about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleic acid sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 being examples of particularly preferred sequences in the practice of the present invention.

[0028] In embodiments that relate particularly to compositions and methods for the detection, diagnosis, prognosis, prophylaxis, treatment, and therapy of B cell leukemias, lymphomas, and multiple myelomas exemplary preferred polynucleotide compositions include those compositions that comprise at least a first isolated nucleic acid segment that comprises a sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the nucleic acid sequence of any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124. Such polynucleotides will preferably comprise one or more isolated coding region, each of which may (a) comprise, (b) consist essentially of, or (c) consist of, the nucleic acid sequence of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124.

[0029] Exemplary polynucleotides of the present invention may be of any suitable length, depending upon the particular application thereof, and encompass those polynucleotides that (a) are at least about, or (b) comprise at least a first isolated nucleic acid segment that is at least about 27, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 120, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 625, 650, 675, 700, 750, 800, 850, 900, 950, or 1000 or so nucleic acids in length, as well as longer polynucleotides that (a) are at least about, or (b) comprise at least a first isolated nucleic acid segment that is at least about 1000, 1025, 1050, 1075, 1100, 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 or so nucleic acids in length, as well as substantially larger polynucleotides. Of course, the polynucleotides and nucleic acid segments of the invention may also encompass any intermediate lengths or integers within the stated ranges.

[0030] The compositions of the present invention may comprise a single polypeptide or polynucleotide, or alternatively, may comprise two or more such hematological malignancy compounds, such as for example, two or more polypeptides, two or more polynucleotides, or even combinations of one or more peptides or polypeptides, along with one or more polynucleotides. When two or more polypeptides are contemplated for particular applications, the second and/or third and/or fourth, etc. isolated peptides and/or polypeptides will preferably comprise an amino acid sequence that is at least about 91%, 93%, 95%, 97%, or 99% identical to the amino acid sequence encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. Alternatively, the polynucleotides of the invention may comprise one or more coding regions that encode a first fusion protein or peptide, such as an adjuvant-coding region fused in correct reading frame to one or more of the disclosed hematological malignancy peptides or polypeptides. Alternatively, the fusion protein may comprise a hematological malignancy polypeptide or peptide fused, in correct reading frame, to a detectable protein or peptide, or to an immunostimulant protein or peptide, or other such construct. Fusion proteins such as these are particularly useful in those embodiments relating to diagnosis, detection, and therapy of one or more of the hematological malignancies as discussed herein.

[0031] The invention also provides a composition comprising at least a first hybridoma cell line that produces a monoclonal antibody having immunospecificity for one or more of the peptides or polypeptides as disclosed herein, or at least a first monoclonal antibody, or an antigen-binding fragment thereof, that has immunospecificity for such a peptide or polypeptide. The antigen binding fragments may comprise a light chain variable region, a heavy-chain variable region, a Fab fragment, a F(ab).sub.2 fragment, an Fv fragment, an scFv fragment, or an antigen-binding fragment of such an antibody.

[0032] The invention also provides a composition comprising at least a first isolated antigen-presenting cell that expresses a peptide or polypeptide as disclosed herein, or a plurality of isolated T cells that specifically react with such a peptide or polypeptide. Such pluralities of isolated T cells may be stimulated or expanded by contacting the T cells with one or more peptides or polypeptides as described herein. The T cells may be cloned prior to expansion, and may be obtained from bone marrow, a bone marrow fraction, peripheral blood, or a peripheral blood fraction from a healthy mammal, or from a mammal that is afflicted with at least a first hematological malignancy such as leukemia or lymphoma.

[0033] As described above, the isolated polypeptides of the invention may be on the order of from 9 to about 1000 amino acids in length, or alternatively, may be on the order of from 50 to about 900 amino acids in length, from 75 to about 800 amino acids in length, from 100 to about 700 amino acids in length, or from 125 to about 600 amino acids in length, or any other such suitable range.

[0034] The isolated nucleic acid segments that encode such isolated polypeptides may be on the order of from 27 to about 10,000 nucleotides in length, from 150 to about 8000 nucleotides in length, from 250 to about 6000 nucleotides in length, from 350 to about 4000 nucleotides in length, or from 450 to about 2000 nucleotides in length, or any other such suitable range.

[0035] The nucleic acid segment may be operably positioned under the control of at least a first heterologous, recombinant promoter, such as a tissue-specific, cell-specific, inducible, or otherwise regulated promoter. Such promoters may be further controlled or regulated by the presence of one or more additional enhancers or regulatory regions depending upon the particular cell type in which expression of the polynucleotide is desired. The polynucleotides and nucleic acid segments of the invention may also be comprised within a vector, such as a plasmid, or viral vector. The polypeptides and polynucleotides of the invention may also be comprised within a host cell, such as a recombinant host cell, or a human host cell such as a blood or bone marrow cell.

[0036] The polynucleotides of the invention may comprise at least a first isolated nucleic acid segment operably attached, in frame, to at least a second isolated nucleic acid segment, such that the polynucleotide encodes a fusion protein in which the first peptide or polypeptide is linked to the second peptide or polypeptide.

[0037] The polypeptides of the present invention may comprise a contiguous amino acid of any suitable length, such as for example, those of about 2000, about 1900, about 1850, about 1800, about 1750, about 1700, about 1650, about 1600, about 1550, about 1500, about 1450, about 1400, about 1350, about 1300, about 1250, about 1200, about 1150, about 1100 amino acids, or about 1000 or so amino acids in length. Likewise, the polypeptides and peptides of the present invention may comprise slightly shorter contiguous amino acid coding regions, such as for example, those of about 950, about 900, about 850, about 800, about 750, about 700, about 650, about 600, about 550, about 500, about 450, about 400, about 350, about 300, about 250, about 200, about 150, or even about 100 amino acids or so in length.

[0038] In similar fashion, the polypeptides and peptides of the present invention may comprise even smaller contiguous amino acid coding regions, such as for example, those of about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, or even about 9 amino acids or so in length.

[0039] In all such embodiments, those peptides and polypeptides having intermediate lengths including all integers within the preferred ranges (e.g., those peptides and polypeptides that comprise at least a first coding region of at least about 94, about 93, about 92, about 91, about 89, about 88, about 87, about 86, about 84, about 83, about 82, about 81, about 79, about 78, about 77, about 76, about 74, about 73, about 72, about 71, about 69, about 68, about 67, about 66 or so amino acids in length, etc.) are all contemplated to fall within the scope of the present invention.

[0040] In particular embodiments, the peptides and polypeptides of the present invention may comprise a sequence of at least about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20, or about 21, or about 22, or about 23, or about 24, or about 25, or about 26, or about 27, or about 28, or about 29, or about 30, or about 31, or about 32, or about 33, or about 34, or about 35, or about 36, or about 37, or about 38, or about 39, or about 40, or about 41, or about 42, or about 43, or about 44, or about 45, or about 46, or about 47, or about 48, or about 49, or about 50 contiguous amino acids as disclosed in any one or more of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121 herein.

[0041] In other embodiments, the peptides and polypeptides of the present invention may comprise a sequence of at least about 51, or about 52, or about 53, or about 54, or about 55, or about 56, or about 57, or about 58, or about 59, or about 60, or about 61, or about 62, or about 63, or about 64, or about 65, or about 66, or about 67, or about 68, or about 69, or about 70, or about 71, or about 72, or about 73, or about 74, or about 75, or about 76, or about 77, or about 78, or about 79, or about 80, or about 81, or about 82, or about 83, or about 84, or about 85, or about 86, or about 87, or about 88, or about 89, or about 90, about 91, or about 92, or about 93, or about 94, or about 95, or about 96, or about 97, or about 98, or about 99, or 100 contiguous amino acids as disclosed in any one or more of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 1.2, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121 herein.

[0042] In still other embodiments, the preferred peptides and polypeptides of the present invention comprise a sequence of at least about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 or more contiguous amino acids as disclosed in any one or more of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121 herein.

[0043] The polypeptides of the invention typically will comprise at least a first contiguous amino acid sequence according to any one of the peptides encoded by any one of the above polynucleotides or disclosed in any one of SEQ ID NOs:10,471-10,474; SEQ ID NO: 10,481; SEQ ID NOs:10,599-10,819; SEQ ID NOs:10,820-10,842; SEQ ID NOs:10,849-10,908; and SEQ ID NOs:10,909-10,968, but may also, optionally comprise at least a second, at least a third, or even at least a fourth or greater contiguous amino acid sequence according to any one of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. A single polypeptide may contain only a single coding region, or alternatively, a single polypeptide may comprise a plurality of identical or distinctly different contiguous amino acid sequences in accordance with any one of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. In fact, the polypeptide may comprise a plurality of the same contiguous amino acid sequences, or they may comprise one or more different contiguous amino acid sequences of any of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. For example, a single polypeptide can comprise a single contiguous amino acid sequence from one or more of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, or alternatively, may comprise two or more distinctly different contiguous amino acid sequences from one or more of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. In fact, the polypeptide may comprise 2, 3, 4, or even 5 distinct contiguous amino sequences of any one of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. Alternatively, a single polypeptide may comprise 2, 3, 4, or even 5 distinct coding regions. For example, a polypeptide may comprise at least a first coding region that comprises a first contiguous amino acid sequence of any one of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, and at least a second coding region that comprises a second contiguous amino acid sequence of any one of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121. In contrast, a polypeptide may comprise at least a first coding region that comprises a first contiguous amino acid sequence of any one of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, and at least a second coding region that comprises a second distinctly different peptide or polypeptide, such as for example, an adjuvant or an immunostimulant peptide or polypeptide.

[0044] In such cases, the two coding regions may be separate on the same polypeptide, or the two coding regions may be operatively attached, each in the correct reading frame, such that a fusion polypeptide is produced, in which the first amino acid sequence of the first coding region is linked to the second amino acid sequence of the second coding region.

[0045] Throughout this disclosure, a phrase such as "a sequence as disclosed in SEQ ID NO:1 to SEQ ID NO:4" is intended to encompass any and all contiguous sequences disclosed by any one of these sequence identifiers. That is to say, "a sequence as disclosed in any of SEQ ID NO:1 through SEQ ID NO:4" means any sequence that is disclosed in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. Likewise, "a sequence as disclosed in any of SEQ ID NOs:25 to 37" means any sequence that is disclosed in any one of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, and so forth.

[0046] Likewise, a phrase such as "at least a first sequence from any one of SEQ ID NO:55 to SEQ ID NO:62" is intended to refer to a first sequence that is disclosed in any one of SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:62.

[0047] It will also be understood that the kits, and compositions of the present invention comprise in an overall and general sense at least one or more particular polynucleotides, polypeptides, and peptides that comprise one or more contiguous sequence regions from one or more of the nucleic acid sequences disclosed herein in SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or from one or more of the amino acid sequences encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or disclosed in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121, and that such peptide, polypeptide and polynucleotide compositions may be used in one or more of the particular methods and uses disclosed herein for the diagnosis, detection, prophylaxis, and therapy of one or more hematological cancers, and in particular, lymphomas of a variety of specific types. It will also be understood to the skilled artisan having benefit of the teachings of the present specification, that the peptide and polypeptide compositions may be used to generate a T cell or an immune response in an animal, and that such compositions may also be administered to an animal from which immunospecific antibodies and antigen binding fragments may be isolated or identified that specifically bind to such peptides or polypeptides. Such an artisan will also recognize that the polynucleotides identified by the present disclosure may be used to produce such peptides, polypeptides, antibodies, and antigen binding fragments, by recombinant protein production methodologies that are also within the capability of the skilled artisan having benefit of the specific amino acid and nucleic acid sequences provided herein.

[0048] Likewise, it will be understood by a skilled artisan in the field, that one or more of the disclosed compositions may used in one or more diagnostic or detection methodologies to identify certain antibodies, peptides, polynucleotides, or polypeptides in a biological sample, in a host cell, or even within the body or tissues of an animal. It will be understood by a skilled artisan in the field, that one or more of the disclosed nucleic acid or amino acid compositions may used in the preparation or manufacture of one or more medicaments for use in the diagnosis, detection, prognosis, prophylaxis, or therapy of one or more hematological malignancies in an animal, and particularly those malignant conditions disclosed and claimed herein.

[0049] It will also be readily apparent to those of skill in the art, that the methods, kits, and uses, of the present invention preferably employ one or more of the compounds and/or compositions disclosed herein that comprise one or more contiguous nucleotide sequences as may be presented in SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 of the attached sequence listing.

[0050] Likewise, it will also be readily apparent to those of skill in the art, that the methods, kits, and uses, of the present invention may also employ one or more of the compounds and compositions disclosed herein that comprise one or more contiguous amino acid sequences of any of the peptides encoded by any one of SEQ ID NOS: 1-3, 5, 7, 9, 11, 13-14, 16-17, 19-20, 22-25, 27-28, 30-31, 33-34, 36, 38-39, 41-42, 44, 46-47, 49, 51, 53, 55, 57, 59-60, 62, 64-65, 67-70, 72-73, 75, 77-81, 83, 85-86, 88-100, 102-103, 105-106, 108, 110-113, 115-116, 118, or 124 or presented in any one of SEQ ID NOS: 4, 6, 8, 10, 12, 15, 18, 21, 26, 29, 32, 35, 37, 40, 43, 45, 48, 50, 52, 54, 56, 58, 61, 63, 66, 71, 74, 76, 82, 84, 87, 101, 104, 107, 109, 114, 117, or 119-121 of the attached sequence listing.

3. BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

[0051] The invention may be understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0052] FIG. 1 illustrates a schematic outline of the microarray chip technology approach used to identify the cDNA targets of the present invention as described Section 5.1.

[0053] FIG. 2 illustrates a schematic outline of the general protocol for in vitro whole gene CD8.sup.+ T cell priming procedure used to generate antigen-specific lines and to identify clones of interest.

[0054] FIG. 3 illustrates a schematic outline of the general protocol for in vitro whole gene CD4.sup.+ T cell priming procedure used to generate antigen-specific lines and to identify clones of interest.

[0055] FIG. 4 illustrates the panel of probes used to identify cDNAs that are overexpressed in lymphoma cells.

[0056] FIG. 5 lists the antigens that have similar tissue expression profiles as the known therapeutics, CD20 and CD52.

[0057] FIG. 6 illustrates the results of the TMpred report for Ly1484 long and Ly1484 short.

[0058] FIG. 7 illustrates the results of the TSITES analysis of Ly1484 long.

[0059] FIG. 8 illustrates the results of the TSITES analysis of Ly1484 short.

[0060] SEQ ID NO:1 is a full-length cDNA for Ly1728P.

[0061] SEQ ID NO:2 is a full-length protein sequence for Ly1728P.

[0062] SEQ ID NO:3 is a full-length cDNA sequence of Ly1732P.

[0063] SEQ ID NO:4 is a full-length protein of Ly1732P.

[0064] SEQ ID NO:5 is a full length cDNA sequence of Ly1888P.

[0065] SEQ ID NO:6 is a full length protein sequence of Ly1888P.

[0066] SEQ ID NO:7 is a full length cDNA sequence of Ly1452_His-tag-fusion.

[0067] SEQ ID NO:8 is a full length protein sequence of Ly1452_His-tag-fusion.

[0068] SEQ ID NO:9 is a full length cDNA sequence of Ly1452P, splice variant 1.

[0069] SEQ ID NO:10 is a full length protein sequence of Ly1452P, splice variant 1.

[0070] SEQ ID NO:11 is a full length cDNA sequence of Ly1452P, splice variant 2.

[0071] SEQ ID NO:12 is a full length protein sequence of Ly1452P, splice variant 2.

[0072] SEQ ID NO:13 is a partial cDNA sequence of Ly1462P.

[0073] SEQ ID NO:14 is a full length cDNA sequence of Ly1462P.

[0074] SEQ ID NO:15 is a full length protein sequence of Ly1462P.

[0075] SEQ ID NO:16 is a partial cDNA sequence of Ly1484P.

[0076] SEQ ID NO:17 is a full length cDNA sequence of Ly1484P.

[0077] SEQ ID NO:18 is a full length protein sequence of Ly1484P.

[0078] SEQ ID NO:19 is a partial cDNA sequence of Ly1486P.

[0079] SEQ ID NO:20 is a full length cDNA sequence of Ly1486P.

[0080] SEQ ID NO:21 is a full length protein sequence of Ly1486P.

[0081] SEQ ID NO:22 is a partial cDNA sequence of Ly1677P.

[0082] SEQ ID NO:23 is a partial cDNA sequence of Ly1682P.

[0083] SEQ ID NO:24 is a partial cDNA sequence of Ly1693P.

[0084] SEQ ID NO:25 is a full-length cDNA sequence of Ly1693P.

[0085] SEQ ID NO:26 is a full-length protein sequence of Ly1693P.

[0086] SEQ ID NO:27 is a partial cDNA sequence of Ly1697P.

[0087] SEQ ID NO:28 is a full-length cDNA sequence of Ly1715P.

[0088] SEQ ID NO:29 is a full-length protein sequence of Ly1715P.

[0089] SEQ ID NO:30 is a partial cDNA sequence of Ly1727P.

[0090] SEQ ID NO:31 is a full-length cDNA sequence of Ly1727P.

[0091] SEQ ID NO:32 is a full-length protein sequence of Ly1727P.

[0092] SEQ ID NO:33 is a partial cDNA sequence of Ly1885P.

[0093] SEQ ID NO:34 is a full-length cDNA sequence of Ly1885P.

[0094] SEQ ID NO:35 is a full-length protein sequence of Ly1885P.

[0095] SEQ ID NO:36 is a partial cDNA sequence of Ly1905P.

[0096] SEQ ID NO:37 is a partial protein sequence of Ly1905P.

[0097] SEQ ID NO:38 is a partial cDNA sequence of Ly1905P.

[0098] SEQ ID NO:39 is a full-length cDNA sequence of Ly1905P.

[0099] SEQ ID NO:40 is a full-length protein sequence of Ly1905P.

[0100] SEQ ID NO:41 is a partial cDNA sequence of Ly663S.

[0101] SEQ ID NO:42 is a full-length cDNA sequence of Ly663S.

[0102] SEQ ID NO:43 is a full-length protein sequence of Ly663S.

[0103] SEQ ID NO:44 is a full-length cDNA sequence of Ly664S.

[0104] SEQ ID NO:45 is a full-length protein sequence of Ly664S.

[0105] SEQ ID NO:46 is a partial cDNA sequence of Ly667S.

[0106] SEQ ID NO:47 is a full-length cDNA sequence of Ly667S.

[0107] SEQ ID NO:48 is a full-length protein sequence of Ly667S.

[0108] SEQ ID NO:49 is a partial cDNA sequence of Ly677S.

[0109] SEQ ID NO:50 is a partial protein sequence of Ly677S.

[0110] SEQ ID NO:51 is a partial cDNA sequence of Ly677S.

[0111] SEQ ID NO:52 is a partial protein sequence of Ly677S.

[0112] SEQ ID NO:53 is a full-length cDNA sequence of Ly677S.

[0113] SEQ ID NO:54 is a full-length protein sequence of Ly677S.

[0114] SEQ ID NO:55 is a full-length cDNA sequence of Ly1891P.

[0115] SEQ ID NO:56 is a full-length protein sequence of Ly1891P.

[0116] SEQ ID NO:57 is a full-length cDNA sequence of CD138.

[0117] SEQ ID NO:58 is a full-length protein sequence of CD138.

[0118] SEQ ID NO:59 is a partial cDNA sequence of CD22.

[0119] SEQ ID NO:60 is a full-length cDNA sequence of CD22.

[0120] SEQ ID NO:61 is a full-length protein sequence of CD22.

[0121] SEQ ID NO:62 is a partial cDNA sequence of CD79beta.

[0122] SEQ ID NO:63 is a partial protein sequence of CD79beta.

[0123] SEQ ID NO:64 is a partial cDNA sequence of CD79beta.

[0124] SEQ ID NO:65 is a full-length cDNA sequence of CD79beta.

[0125] SEQ ID NO:66 is a full-length protein sequence of CD79beta.

[0126] SEQ ID NO:67 is a partial cDNA sequence of Ly1450P.

[0127] SEQ ID NO:68 is a partial cDNA sequence of Ly1450P.

[0128] SEQ ID NO:69 is a partial cDNA sequence of Ly1451P.

[0129] SEQ ID NO:70 is a partial cDNA sequence of Ly1451P.

[0130] SEQ ID NO:71 is a partial protein sequence of Ly1451P.

[0131] SEQ ID NO:7272>Ly1454P, Old-SEQ-ID_3577, partial cDNA

[0132] SEQ ID NO:73 is a full-length cDNA sequence of Ly1454P.

[0133] SEQ ID NO:74 is a full-length protein sequence of Ly1454P.

[0134] SEQ ID NO:75 is a partial cDNA sequence of Ly1485P.

[0135] SEQ ID NO:76 is a partial protein sequence of Ly1485P.

[0136] SEQ ID NO:77 is a partial cDNA sequence of Ly1485P.

[0137] SEQ ID NO:78 is a partial cDNA sequence of Ly1500P.

[0138] SEQ ID NO:79 is a full-length cDNA sequence of Ly1500P, splice variant 1.

[0139] SEQ ID NO:80 is a full-length protein sequence of Ly500P, splice variant 1.

[0140] SEQ ID NO:81 is a full-length cDNA sequence of Ly1500P, splice variant 2.

[0141] SEQ ID NO:82 is a full-length protein sequence of Ly1500P, splice variant 2.

[0142] SEQ ID NO:83 is a full-length cDNA sequence of Ly1500P, splice variant 3.

[0143] SEQ ID NO:84 is a full-length protein sequence of Ly1500P, splice variant 3.

[0144] SEQ ID NO:85 is a partial cDNA sequence of Ly1516P.

[0145] SEQ ID NO:86 is a full-length cDNA sequence of Ly1516P, splice variant 1.

[0146] SEQ ID NO:87 is a full-length protein sequence of Ly1516P, splice variant 1.

[0147] SEQ ID NO:88 is a partial cDNA sequence of Ly1516P, splice variant 2.

[0148] SEQ ID NO:89 is a partial cDNA sequence of Ly1516P, splice variant 3.

[0149] SEQ ID NO:90 is a partial cDNA sequence of Ly1678P.

[0150] SEQ ID NO:91 is a partial cDNA sequence of Ly1678P.

[0151] SEQ ID NO:92 is a partial cDNA sequence of Ly1678P.

[0152] SEQ ID NO:93 is a partial cDNA sequence of Ly1678P.

[0153] SEQ ID NO:94 is a partial cDNA sequence of Ly1680P.

[0154] SEQ ID NO:95 is a partial cDNA sequence of Ly1686P.

[0155] SEQ ID NO:96 is a partial cDNA sequence of Ly1687P.

[0156] SEQ ID NO:97 is a partial cDNA sequence of Ly1706P.

[0157] SEQ ID NO:98 is a partial cDNA sequence of Ly1712P.

[0158] SEQ ID NO:99 is a partial cDNA sequence of Ly1729P.

[0159] SEQ ID NO:100 is a full-length cDNA sequence of Ly1729P.

[0160] SEQ ID NO:101 is a full-length protein sequence of Ly1729P.

[0161] SEQ ID NO:102 is a partial cDNA sequence of Ly1848P.

[0162] SEQ ID NO:103 is a partial cDNA sequence of Ly1859P.

[0163] SEQ ID NO:104 is a partial protein sequence of Ly1859P.

[0164] SEQ ID NO:105 is a partial cDNA sequence of Ly1859P.

[0165] SEQ ID NO:106 is a full-length cDNA sequence of Ly1859P.

[0166] SEQ ID NO:107 is a full-length protein sequence of Ly1859P.

[0167] SEQ ID NO:108 is a full length cDNA sequence for Ly1866P

[0168] SEQ ID NO:109 is a full length protein sequence for Ly1866P

[0169] SEQ ID NO:110 is a partial cDNA sequence for Ly1867P.

[0170] SEQ ID NO:111 is a partial cDNA sequence for Ly1868P.

[0171] SEQ ID NO:112 is a partial cDNA sequence for Ly1886P.

[0172] SEQ ID NO:113 is a full length cDNA sequence for Ly669S.

[0173] SEQ ID NO:114 is a full length protein sequence for Ly669S.

[0174] SEQ ID NO:115 is a partial cDNA sequence for Ly672S.

[0175] SEQ ID NO:116 is a full length cDNA sequence for Ly672S.

[0176] SEQ ID NO:117 is a full length cDNA sequence for Ly672S.

[0177] SEQ ID NO:118 is a partial cDNA sequence of Ly675S.

[0178] SEQ ID NO:119 is a partial protein sequence of Ly675S.

[0179] SEQ ID NO:120 is a partial protein sequence of Ly1484P.

[0180] SEQ ID NO:121 is a partial protein sequence of Ly1484P.

[0181] SEQ ID NO:122 is a PCR primer sequence for His-Ly1452P.

[0182] SEQ ID NO:123 is a PCR primer sequence for His-Ly1452P.

[0183] SEQ ID NO:124 is an open reading frame sequence for Ly1451P.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0184] In order that the invention herein described may be more fully understood, the following description of various illustrative embodiments is set forth.

[0185] The present invention is generally directed to compositions and methods for the immunotherapy and diagnosis of Hematological malignancies, such as B cell leukemias and lymphomas and multiple myelomas.

[0186] 4.1 Methods of Nucleic Acid Delivery and DNA Transfection

[0187] In certain embodiments, it is contemplated that one or more RNA or DNA and/or substituted polynucleotide compositions disclosed herein will be used to transfect an appropriate host cell. Technology for introduction of RNAs and DNAs, and vectors comprising them into suitable host cells is well known to those of skill in the art. In particular, such polynucleotides may be used to genetically transform one or more host cells, when therapeutic administration of one or more active peptides, compounds or vaccines is achieved through the expression of one or more polynucleotide constructs that encode one or more therapeutic compounds of interest.

[0188] A variety of means for introducing polynucleotides and/or polypeptides into suitable target cells is known to those of skill in the art. For example, when polynucleotides are contemplated for delivery to cells, several non-viral methods for the transfer of expression constructs into cultured mammalian cells are available to the skilled artisan for his use. These include, for example, calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); DEAE-dextran precipitation (Gopal, 1985); electroporation (Wong and Neumann, 1982; Fromm et al., 1985; Tur-Kaspa et al., 1986; Potter et al., 1984; Suzuki et al., 1998; Vanbever et al., 1998), direct microinjection (Capecchi, 1980; Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979; Takakura, 1998) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990; Klein et al., 1992), and receptor-mediated transfection (Curiel et al., 1991; Wagner et al., 1992; Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

[0189] A bacterial cell, a yeast cell, or an animal cell transformed with one or more of the disclosed expression vectors represent an important aspect of the present invention. Such transformed host cells are often desirable for use in the expression of the various DNA gene constructs disclosed herein. In some aspects of the invention, it is often desirable to modulate, regulate, or otherwise control the expression of the gene segments disclosed herein. Such methods are routine to those of skill in the molecular genetic arts. Typically, when increased or over-expression of a particular gene is desired, various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, and in particular, a tissue-specific promoter such as those disclosed herein, as well as by employing sequences, which enhance the stability of the messenger RNA in the particular transformed host cell.

[0190] Typically, the initiation and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal. In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism or eukaryotic host, but will usually be included with a DNA sequence involving a marker, where the second DNA sequence may be joined to the expression construct during introduction of the DNA into the host.

[0191] Where no functional replication system is present, the construct will also preferably include a sequence of at least about 30 or about 40 or about 50 base pairs (bp) or so, preferably at least about 60, about 70, about 80, or about 90 to about 100 or so bp, and usually not more than about 500 to about 1000 or so bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the regulatory regions of the expression construct will be in close proximity to (and also operably positioned relative to) the selected therapeutic gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that the therapeutic gene is lost, the resulting organism will be likely to also lose the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.

[0192] The selected therapeutic gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct may be included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host, in this case, a mammalian host cell. In addition, one or more markers may be present, which have been described previously. Where integration is desired, the plasmid will desirably include a sequence homologous with the host genome.

[0193] Genes or other nucleic acid segments, as disclosed herein, can be inserted into host cells using a variety of techniques that are well known in the art. Five general methods for delivering a nucleic segment into cells have been described: (1) chemical methods (Graham and Van Der Eb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (U.S. Pat. No. 5,472,869; Wong and Neumann, 1982; Fromm et al., 1985), microprojectiles bombardment (U.S. Pat. No. 5,874,265, specifically incorporated herein by reference in its entirety), "gene gun" (Yang et al., 1990); (3) viral vectors (Eglitis and Anderson, 1988); (4) receptor-mediated mechanisms (Curiel et al., 1991; Wagner et al., 1992); and (5) bacterial-mediated transformation.

[0194] 4.2 Hematological Malignancy Related-Specific Antibodies and Antigen-Binding Fragments Thereof

[0195] The present invention further provides antibodies and antigen-binding fragments thereof, that specifically bind to (or are immunospecific for) at least a first peptide or peptide variant as disclosed herein. As used herein, an antibody or an antigen-binding fragment is said to "specifically bind" to a peptide if it reacts at a detectable level (within, for example, an ELISA) with the peptide, and does not react detectably with unrelated peptides or proteins under similar conditions. As used herein, "binding" refers to a non-covalent association between two separate molecules such that a "complex" is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In the context of the present invention, in general, two compounds are said to "bind" when the binding constant for complex formation exceeds about 10.sup.3 L/mol. The binding constant maybe determined using methods well known in the art.

[0196] Any agent that satisfies the above requirements may be a binding agent. In illustrative embodiments, a binding agent is an antibody or an antigen-binding fragment thereof. Such antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (Harlow and Lane, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the peptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the peptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short peptides, a superior immune response may be elicited if the peptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the peptide may then be purified from such antisera by, for example, affinity chromatography using the peptide coupled to a suitable solid support.

[0197] "Antibody" refers to a polypeptide encoded by an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0198] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, i.e., an antigen recognition domain. As used herein, "antigen recognition domain" means that part of the antibody, recombinant molecule, the fusion protein, or the immunoconjugate of the invention which recognizes the target or portions thereof. Typically the antigen recognition domain comprises the variable region of the antibody or a portion thereof, e.g., one, two, three, four, five, six, or more hypervariable regions. The terms "V.sub.H" or "VH" refer to the variable region of an immunoglobulin heavy chain, including an Fv, scFv, dsFv or Fab. The terms "V.sub.L" or "VL" refer to the variable region of an immunoglobulin light chain, including an Fv, scFv, dsFv or Fab.

[0199] Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies.

[0200] As used herein, "fragment" is defined as at least a portion of the variable region of the immunoglobulin molecule, which binds to its target, i.e. the antigen recognition domain or the antigen binding region. Some of the constant region of the immunoglobulin may be included. Examples of antibody functional fragments include, but are not limited to, complete antibody molecules, humanized antibodies, antibody fragments, such as Fv, single chain Fv (scFv), hypervariable regions ro complementarity determining regions (CDRs), V.sub.L (light chain variable region), V.sub.H (heavy chain variable region), Fab, F(ab)2' and any combination of those or any other portion of an immunoglobulin peptide capable of binding to target antigen (see, e.g., Fundamental Immunology (Paul ed., 4th. 1999). As appreciated by one of skill in the art, various antibody fragments can be obtained by a variety of methods, for example, digestion of an intact antibody with an enzyme, such as pepsin; or de novo synthesis. Antibody fragments are often synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., (1990) Nature 348:552). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al., J. Immunol. 148: 1547 (1992), Pack and Pluckthun, Biochemistry 31: 1579 (1992), Zhu et al. Protein Sci. 6: 781 (1997), Hu et al. Cancer Res. 56: 3055 (1996), Adams et al., Cancer Res. 53: 4026 (1993), and McCartney, et al., Protein Eng. 8: 301 (1995).

[0201] A "humanized antibody" refers to an antibody in which the antigen binding loops, i.e., complementarity determining regions (CDRs), comprised by the V.sub.H and V.sub.L regions are grafted to a human framework sequence. Typically, the humanized antibodies have the same binding specificity as the non-humanized antibodies described herein. Techniques for humanizing antibodies are well known in the art and are described in e.g., U.S. Pat. Nos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87/02671; EP Patent Application 0173494; Jones et al., Nature 321: 522 (1986); and Verhoyen et al., Science 239: 1534 (1988). Humanized antibodies are further described in, e.g., Winter and Milstein, Nature 349: 293 (1991).

[0202] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985)).

[0203] Methods of producing of polyclonal antibodies are known to those of skill in the art. In an exemplary method, an inbred strain of mice (e.g., BALB/C mice) or rabbits is immunized with the chelate or a close structural analogue using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. Alternatively, or in addition to the use of an adjuvant, the chelate is coupled to a carrier that is itself immunogenic (e.g., keyhole limpit hemocyanin ("KLH"). The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the beta subunits. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired.

[0204] Monoclonal antibodies are obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, for example, Kohler & Milstein, Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246: 1275-1281 (1989).

[0205] Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Typically, polyclonal antisera with a titer of 10.sup.4 or greater are selected and tested for cross reactivity against different chelates, using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a K.sub.d of at least about 0.1 mM, more usually at least about 1 .mu.M, preferably, at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M or better.

[0206] Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to reactive chelates and other diagnostic, analytical and therapeutic agents. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to produce and identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348: 552-554 (1990); Marks et al., Biotechnology 10: 779-783 (1992)).

[0207] In an exemplary embodiment, an animal, such as a rabbit or mouse is immunized with a chelate, or an immunogenic construct. The antibodies produced as a result of the immunization are preferably isolated using standard methods.

[0208] In a still further preferred embodiment, the antibody is a humanized antibody. "Humanized" refers to a non-human polypeptide sequence that has been modified to minimize immunoreactivity in humans, typically by altering the amino acid sequence to mimic existing human sequences, without substantially altering the function of the polypeptide sequence (see, e.g., Jones et al., Nature 321: 522-525 (1986), and published UK patent application No. 8707252).

[0209] In another preferred embodiment, the present invention provides an antibody, as described above, further comprising a member selected from detectable labels, biologically active agents and combinations thereof attached to the antibody.

[0210] When the antibody is conjugated to a detectable label, the label is preferably a member selected from the group consisting of radioactive isotopes, fluorescent agents, fluorescent agent precursors, chromophores, enzymes and combinations thereof. Methods for conjugating various groups to antibodies are well known in the art. For example, a detectable label that is frequently conjugated to an antibody is an enzyme, such as horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, and glucose oxidase.

[0211] Methods of producing antibodies labeled with small molecules, for example, fluorescent agents, are well known in the art. Fluorescent labeled antibodies can be used in immunohistochemical staining (Osborn et al., Methods Cell Biol. 24: 97-132 (1990); in flow cytometry or cell sorting techniques (Ormerod, M. G. (ed.), FLOW CYTOMETRY. A PRACTICAL APPROACH, IRL Press, New York, 1990); for tracking and localization of antigens, and in various double-staining methods (Kawamura, A., Jr., FLUORESCENT ANTIBODY TECHNIQUES AND THEIR APPLICATION, Univ. Tokyo Press, Baltimore, 1977).

[0212] Many reactive fluorescent labels are available commercially (e.g., Molecular Probes, Eugene, Oreg.) or they can be synthesized using art-recognized techniques. In an exemplary embodiment, an antibody of the invention is labeled with an amine-reactive fluorescent agent, such as fluorescein isothiocyanate under mildly basic conditions. For other examples of antibody labeling techniques, see, Goding, J. Immunol. Methods 13: 215-226 (1976); and in, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, pp. 6-58, Academic Press, Orlando (1988).

[0213] Monoclonal antibodies specific for the antigenic peptide of interest may be prepared, for example, using the technique of Kohler and Milstein (1976) and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the peptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the peptide. Hybridomas having high reactivity and specificity are preferred.

[0214] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The peptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

[0215] Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on Protein A bead columns.

[0216] Monoclonal antibodies and fragments thereof may be coupled to one or more therapeutic agents. Suitable agents in this regard include radioactive tracers and chemotherapeutic agents, which may be used, for example, to purge autologous bone marrow in vitro). Representative therapeutic agents include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. For diagnostic purposes, coupling of radioactive agents may be used to facilitate tracing of metastases or to determine the location of hematological malignancy related-positive tumors.

[0217] A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

[0218] Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.

[0219] It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as the linker group. Coupling may be affected, for example, through amino groups, carboxyl groups, and sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958.

[0220] Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group that is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (U.S. Pat. No. 4,489,710), by irradiation of a photolabile bond (U.S. Pat. No. 4,625,014), by hydrolysis of derivatized amino acid side chains (U.S. Pat. No. 4,638,045), by serum complement-mediated hydrolysis (U.S. Pat. No. 4,671,958), and acid-catalyzed hydrolysis (U.S. Pat. No. 4,569,789).

[0221] It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used. A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (U.S. Pat. No. 4,507,234), peptides and polysaccharides such as aminodextran (U.S. Pat. No. 4,699,784). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (U.S. Pat. No. 4,429,008 and U.S. Pat. No. 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562 discloses representative chelating compounds and their synthesis.

[0222] A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.

[0223] Also provided herein are anti-idiotypic antibodies that mimic an immunogenic portion of hematological malignancy related. Such antibodies may be raised against an antibody, or an antigen-binding fragment thereof, that specifically binds to an immunogenic portion of hematological malignancy related, using well-known techniques. Anti-idiotypic antibodies that mimic an immunogenic portion of hematological malignancy related are those antibodies that bind to an antibody, or antigen-binding fragment thereof, that specifically binds to an immunogenic portion of hematological malignancy related, as described herein.

[0224] Irrespective of the source of the original hematological malignancy related peptide-specific antibody, the intact antibody, antibody multimers, or any one of a variety of functional, antigen-binding regions of the antibody may be used in the present invention. Exemplary functional regions include scFv, Fv, Fab', Fab and F(ab').sub.2 fragments of the hematological malignancy related peptide-specific antibodies. Techniques for preparing such constructs are well known to those in the art and are further exemplified herein.

[0225] The choice of antibody construct may be influenced by various factors. For example, prolonged half-life can result from the active readsorption of intact antibodies within the kidney, a property of the Fc piece of immunoglobulin. IgG based antibodies, therefore, are expected to exhibit slower blood clearance than their Fab' counterparts. However, Fab' fragment-based compositions will generally exhibit better tissue penetrating capability.

[0226] Antibody fragments can be obtained by proteolysis of the whole immunoglobulin by the non-specific thiol protease, papain. Papain digestion yields two identical antigen-binding fragments, termed "Fab fragments," each with a single antigen-binding site, and a residual "Fc fragment."

[0227] Papain should first be activated by reducing the sulfhydryl group in the active site with cysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in the stock enzyme should be removed by chelation with EDTA (2 mM) to ensure maximum enzyme activity. Enzyme and substrate are normally mixed together in the ratio of 1:100 by weight. After incubation, the reaction can be stopped by irreversible alkylation of the thiol group with iodoacetamide or simply by dialysis. The completeness of the digestion should be monitored by SDS-PAGE and the various fractions separated by Protein A-Sepharose or ion exchange chromatography.

[0228] The usual procedure for preparation of F(ab).sub.2 fragments from IgG of rabbit and human origin is limited proteolysis by the enzyme pepsin. The conditions, 100.times. antibody excess wt./wt. in acetate buffer at pH 4.5, 37.degree. C., suggest that antibody is cleaved at the C-terminal side of the inter-heavy-chain disulfide bond. Rates of digestion of mouse IgG may vary with subclass and it may be difficult to obtain high yields of active F(ab')2 fragments without some undigested or completely degraded IgG. In particular, IgG.sub.2b is highly susceptible to complete degradation. The other subclasses require different incubation conditions to produce optimal results, all of which is known in the art.

[0229] Pepsin treatment of intact antibodies yields an F(ab).sub.2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. Digestion of rat IgG by pepsin requires conditions including dialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for four hrs with 1% wt./wt. pepsin; IgG.sub.1 and IgG.sub.2a digestion is improved if first dialyzed against 0.1 M formate buffer, pH 2.8, at 4.degree. C., for 16 hrs followed by acetate buffer. IgG.sub.2b gives more consistent results with incubation in staphylococcal V8 protease (3% wt./wt.) in 0.1 M sodium phosphate buffer, pH 7.8, for four hrs at 37.degree. C.

[0230] A Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteine(s) from the antibody hinge region. F(ab').sub.2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0231] The term "variable," as used herein in reference to antibodies, means that certain portions of the variable domains differ extensively in sequence among antibodies, and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments termed "hypervariable regions," both in the light chain and the heavy chain variable domains.

[0232] The more highly conserved portions of variable domains are called the framework region (FR). The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a R-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases, forming part of, the P-sheet structure.

[0233] The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (Kabat et al., 1991, specifically incorporated herein by reference). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0234] The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat et al., 1991, specifically incorporated herein by reference) and/or those residues from a "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0235] An "Fv" fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, con-covalent association. It is in this configuration that three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V.sub.H-V.sub.L dimer. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0236] "Single-chain Fv" or "sFv" antibody fragments comprise the V.sub.H and V.sub.L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains that enables the sFv to form the desired structure for antigen binding.

[0237] "Diabodies" are small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described in European Pat. Appl. No. EP 404,097 and Intl. Pat. Appl. Publ. No. WO 93/11161, each specifically incorporated herein by reference. "Linear antibodies", which can be bispecific or monospecific, comprise a pair of tandem Fd segments (V.sub.H--C.sub.H1-V.sub.H-C.sub.H1) that form a pair of antigen binding regions, as described in Zapata et al. (1995), specifically incorporated herein by reference.

[0238] Other types of variants are antibodies with improved biological properties relative to the parent antibody from which they are generated. Such variants, or second-generation compounds, are typically substitutional variants involving one or more substituted hypervariable region residues of a parent antibody. A convenient way for generating such substitutional variants is affinity maturation using phage display.

[0239] In affinity maturation using phage display, several hypervariable region sites (e.g., 6 to 7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine-scanning mutagenesis can be performed on hypervariable region residues identified as contributing significantly to antigen binding.

[0240] Alternatively, or in addition, the crystal structure of the antigen-antibody complex be delineated and analyzed to identify contact points between the antibody and target. Such contact residues and neighboring residues are candidates for substitution. Once such variants are generated, the panel of variants is subjected to screening, and antibodies with analogues but different or even superior properties in one or more relevant assays are selected for further development.

[0241] In using a Fab' or antigen binding fragment of an antibody, with the attendant benefits on tissue penetration, one may derive additional advantages from modifying the fragment to increase its half-life. A variety of techniques may be employed, such as manipulation or modification of the antibody molecule itself, and also conjugation to inert carriers. Any conjugation for the sole purpose of increasing half-life, rather than to deliver an agent to a target, should be approached carefully in that Fab' and other fragments are chosen to penetrate tissues. Nonetheless, conjugation to non-protein polymers, such PEG and the like, is contemplated.

[0242] Modifications other than conjugation are therefore based upon modifying the structure of the antibody fragment to render it more stable, and/or to reduce the rate of catabolism in the body. One mechanism for such modifications is the use of D-amino acids in place of L-amino acids. Those of ordinary skill in the art will understand that the introduction of such modifications needs to be followed by rigorous testing of the resultant molecule to ensure that it still retains the desired biological properties. Further stabilizing modifications include the use of the addition of stabilizing moieties to either the N-terminal or the C-terminal, or both, which is generally used to prolong the half-life of biological molecules. By way of example only, one may wish to modify the termini by acylation or amination.

[0243] Moderate conjugation-type modifications for use with the present invention include incorporating a salvage receptor binding epitope into the antibody fragment. Techniques for achieving this include mutation of the appropriate region of the antibody fragment or incorporating the epitope as a peptide tag that is attached to the antibody fragment. Intl. Pat. Appl. Publ. No. WO 96/32478 is specifically incorporated herein by reference for the purposes of further exemplifying such technology. Salvage receptor binding epitopes are typically regions of three or more amino acids from one or two lops of the Fc domain that are transferred to the analogous position on the antibody fragment. The salvage receptor-binding epitopes disclosed in Intl. Pat. Appl. Publ. No. WO 98/45331 are incorporated herein by reference for use with the present invention.

[0244] 4.3 T Cell Compositions Specific for Hematological Malignancy-Related Peptides

[0245] Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for hematological malignancy related. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be present within (or isolated from) bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell separation system, such as the Isolex.TM. System, available from Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; Intl. Pat. Appl. Publ. No. WO 89/06280; Intl. Pat. Appl. Publ. No. WO 91/16116 and Intl. Pat. Appl. Publ. No. WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.

[0246] T cells may be stimulated with hematological malignancy related peptide, polynucleotide encoding a hematological malignancy related peptide and/or an antigen-presenting cell (APC) that expresses a hematological malignancy related peptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the hematological malignancy related peptide. Preferably, a hematological malignancy related peptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of antigen-specific T cells. Briefly, T cells, which may be isolated from a patient or a related or unrelated donor by routine techniques (such as by Ficoll/Hypaque.RTM. density gradient centrifugation of peripheral blood lymphocytes), are incubated with hematological malignancy related peptide. For example, T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37.degree. C. with hematological malignancy related peptide (e.g., 5 to 25 .mu.g/ml) or cells synthesizing a comparable amount of hematological malignancy related peptide. It may be desirable to incubate a separate aliquot of a T cell sample in the absence of hematological malignancy related peptide to serve as a control.

[0247] T cells are considered to be specific for a hematological malignancy related peptide if the T cells kill target cells coated with a hematological malignancy related peptide or expressing a gene encoding such a peptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al. (1994). Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Other ways to detect T cell proliferation include measuring increases in interleukin-2 (IL-2) production, Ca.sup.2+ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively, synthesis of lymphokines (such as interferon-gamma) can be measured or the relative number of T cells that can respond to a hematological malignancy related peptide may be quantified. Contact with a hematological malignancy related peptide (200 ng/ml -100 .mu.g/ml, preferably 100 ng/ml -25 .mu.g/ml) for 3-7 days should result in at least a two-fold increase in proliferation of the T cells and/or contact as described above for 2-3 hrs should result in activation of the T cells, as measured using standard cytokine assays in which a two-fold increase in the level of cytokine release (e.g., TNF or IFN-.gamma.) is indicative of T cell activation (Coligan et al., 1998). hematological malignancy related specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient or a related or unrelated donor and are administered to the patient following stimulation and expansion.

[0248] T cells that have been activated in response to a hematological malignancy related peptide, polynucleotide or hematological malignancy related-expressing APC may be CD4.sup.+ and/or CD8.sup.+. Specific activation of CD4.sup.+ or CD8.sup.+ T cells may be detected in a variety of ways. Methods for detecting specific T cell activation include detecting the proliferation of T cells, the production of cytokines (e.g., lymphokines), or the generation of cytolytic activity (i.e., generation of cytotoxic T cells specific for hematological malignancy related). For CD4.sup.+ T cells, a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells. For CD8.sup.+ T cells, a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity.

[0249] For therapeutic purposes, CD4.sup.+ or CD8.sup.+ T cells that proliferate in response to the hematological malignancy related peptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to hematological malignancy related peptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a hematological malignancy related peptide. The addition of stimulator cells is preferred where generating CD8.sup.+ T cell responses. T cells can be grown to large numbers in vitro with retention of specificity in response to intermittent restimulation with hematological malignancy related peptide. Briefly, for the primary in vitro stimulation (IVS), large numbers of lymphocytes (e.g., greater than 4.times.10.sup.7) may be placed in flasks with media containing human serum. hematological malignancy related peptide (e.g., peptide at 10 .mu.g/ml) may be added directly, along with tetanus Stoxoid (e.g., 5 .mu.g/ml). The flasks may then be incubated (e.g., 37.degree. C. for 7 days). For a second IVS, T cells are then harvested and placed in new flasks with 2-3.times.10.sup.7 irradiated peripheral blood mononuclear cells. hematological malignancy related peptide (e.g., 10 .mu.g/ml) is added directly. The flasks are incubated at 37.degree. C. for 7 days. On day 2 and day 4 after the second IVS, 2-5 units of interleukin-2 (IL-2) may be added. For a third IVS, the T cells may be placed in wells and stimulated with the individual's own EBV transformed B cells coated with the peptide. IL-2 may be added on days 2 and 4 of each cycle. As soon as the cells are shown to be specific cytotoxic T cells, they may be expanded using a 10-day stimulation cycle with higher IL-2 (20 units) on days 2, 4 and 6.

[0250] Alternatively, one or more T cells that proliferate in the presence of hematological malignancy related peptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution. Responder T cells may be purified from the peripheral blood of sensitized patients by density gradient centrifugation and sheep red cell rosetting and established in culture by stimulating with the nominal antigen in the presence of irradiated autologous filler cells. In order to generate CD4.sup.+ T cell lines, hematological malignancy related peptide is used as the antigenic stimulus and autologous peripheral blood lymphocytes (PBL) or lymphoblastoid cell lines (LCL) immortalized by infection with Epstein Barr virus are used as antigen-presenting cells. In order to generate CD8.sup.+ T cell lines, autologous antigen-presenting cells transfected with an expression vector that produces hematological malignancy related peptide may be used as stimulator cells. Established T cell lines may be cloned 2-4 days following antigen stimulation by plating stimulated T cells at a frequency of 0.5 cells per well in 96-well flat-bottom plates with 1.times.10.sup.6 irradiated PBL or LCL cells and recombinant interleukin-2 (rIL2) (50 U/ml). Wells with established clonal growth may be identified at approximately 2-3 weeks after initial plating and restimulated with appropriate antigen in the presence of autologous antigen-presenting cells, then subsequently expanded by the addition of low doses of rIL2 (10 U/ml) 2-3 days following antigen stimulation. T cell clones may be maintained in 24-well plates by periodic restimulation with antigen and rIL2 approximately every two weeks. Cloned and/or expanded cells may be administered back to the patient as described, for example, by Chang et al., (1996).

[0251] Within certain embodiments, allogeneic T-cells may be primed (i.e., sensitized to hematological malignancy related) in vivo and/or in vitro. Such priming may be achieved by contacting T cells with a hematological malignancy related peptide, a polynucleotide encoding such a peptide or a cell producing such a peptide under conditions and for a time sufficient to permit the priming of T cells. In general, T cells are considered to be primed if, for example, contact with a hematological malignancy related peptide results in proliferation and/or activation of the T cells, as measured by standard proliferation, chromium release and/or cytokine release assays as described herein. A stimulation index of more than two fold increase in proliferation or lysis, and more than three fold increase in the level of cytokine, compared to negative controls indicates T-cell specificity. Cells primed in vitro may be employed, for example, within bone marrow transplantation or as donor lymphocyte infusion.

[0252] T cells specific for hematological malignancy related can kill cells that express hematological malignancy related protein. Introduction of genes encoding T-cell receptor (TCR) chains for hematological malignancy related are used as a means to quantitatively and qualitatively improve responses to hematological malignancy related bearing leukemia and cancer cells. Vaccines to increase the number of T cells that can react to hematological malignancy related positive cells are one method of targeting hematological malignancy related bearing cells. T cell therapy with T cells specific for hematological malignancy related is another method. An alternative method is to introduce the TCR chains specific for hematological malignancy related into T cells or other cells with lytic potential. In a suitable embodiment, the TCR alpha and beta chains are cloned out from a hematological malignancy related specific T cell line and used for adoptive T cell therapy, such as described in WO 96/30516, incorporated herein by reference.

[0253] 4.4 Pharmaceutical Compositions and Vaccine Formulations

[0254] Within certain aspects, peptides, polynucleotides, antibodies and/or T cells may be incorporated into pharmaceutical compositions or immunogenic compositions (i.e., vaccines). Alternatively, a pharmaceutical composition may comprise an antigen-presenting cell (e.g., a dendritic cell) transfected with a hematological malignancy related polynucleotide such that the antigen-presenting cell expresses a hematological malignancy related peptide. Pharmaceutical compositions comprise one or more such compounds or cells and a physiologically acceptable carrier or excipient. Vaccines may comprise one or more such compounds or cells and an immunostimulant, such as an adjuvant or a liposome (into which the compound is incorporated). An immunostimulant may be any substance that enhances or potentiates an immune response (antibody- and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated) (U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in, for example, Powell and Newman (1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion peptide or as a separate compound, within the composition or vaccine.

[0255] Within certain embodiments, pharmaceutical compositions and vaccines are designed to elicit T cell responses specific for a hematological malignancy related peptide in a patient, such as a human. In general, T cell responses may be favored through the use of relatively short peptides (e.g., comprising less than 23 consecutive amino acid residues of a native hematological malignancy related peptide, preferably 4-16 consecutive residues, more preferably 8-16 consecutive residues and still more preferably 8-10 consecutive residues). Alternatively, or in addition, a vaccine may comprise an immunostimulant that preferentially enhances a T cell response. In other words, the immunostimulant may enhance the level of a T cell response to a hematological malignancy related peptide by an amount that is proportionally greater than the amount by which an antibody response is enhanced. For example, when compared to a standard oil based adjuvant, such as CFA, an immunostimulant that preferentially enhances a T cell response may enhance a proliferative T cell response by at least two fold, a lytic response by at least 10%, and/or T cell activation by at least two fold compared to hematological malignancy related-negative control cell lines, while not detectably enhancing an antibody response. The amount by which a T cell or antibody response to a hematological malignancy related peptide is enhanced may generally be determined using any representative technique known in the art, such as the techniques provided herein.

[0256] A pharmaceutical composition or vaccine may contain DNA encoding one or more of the peptides as described above, such that the peptide is generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems and mammalian expression systems. Numerous gene delivery techniques are well known in the art (Rolland, 1998, and references cited therein). Appropriate nucleic acid expression systems contain the necessary DNA, cDNA or RNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the peptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus (Fisher-Hoch et al., 1989; Flexner et al., 1989; Flexner et al., 1990; U.S. Pat. No. 4,603,112, U.S. Pat. No. 4,769,330, U.S. Pat. No. 5,017,487; Intl. Pat. Appl. Publ. No. WO 89/01973; U.S. Pat. No. 4,777,127; Great Britain Patent No. GB 2,200,651; European Patent No. EP 0,345,242; Intl. Pat. Appl. Publ. No. WO 91/02805; Berkner, 1988; Rosenfeld et al., 1991; Kolls et al., 1994; Kass-Eisler et al., 1993; Guzman et al., 1993a; and Guzman et al., 1993). Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al. (1993) and reviewed by Cohen (1993). The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a peptide component. Such vaccines may provide for an enhanced immune response.

[0257] As noted above, a pharmaceutical composition or vaccine may comprise an antigen-presenting cell that expresses a hematological malignancy related peptide. For therapeutic purposes, as described herein, the antigen-presenting cell is preferably an autologous dendritic cell. Such cells may beprepared and transfected using standard techniques (Reeves et al., 1996; Tuting et al., 1998; and Nair et al., 1998). Expression of a hematological malignancy related peptide on the surface of an antigen-presenting cell may be confirmed by in vitro stimulation and standard proliferation as well as chromium release assays, as described herein.

[0258] It will be apparent to those of ordinary skill in the art having the benefit of the present teachings that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and peptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts). The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other significant untoward reaction when administered to an animal, or a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the Food and Drug Administration Office of Biologics standards. Supplementary active ingredients can also be incorporated into the compositions.

[0259] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. For certain topical applications, formulation as a cream or lotion, using well-known components, is preferred.

[0260] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, peptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate, or formulated with one or more liposomes, microspheres, nanoparticles, or micronized delivery systems using well-known technology.

[0261] Any of a variety of immunostimulants, such as adjuvants, may be employed in the preparation of vaccine compositions of this invention. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, alum-based adjuvants (e.g., Alhydrogel, Rehydragel, aluminum phosphate, Algammulin, aluminum hydroxide); oil based adjuvants (Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.), Specol, RIBI, TiterMax, Montanide ISA50 or Seppic MONTANIDE ISA 720); nonionic block copolymer-based adjuvants, cytokines (e.g., GM-CSF or Flat3-ligand); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and Quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0262] Hemocyanins and hemoerythrins may also be used in the invention. The use of hemocyanin from keyhole limpet (KLH) is particularly preferred, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed. Various polysaccharide adjuvants may also be used. Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.

[0263] A further preferred group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-a-isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.

[0264] U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide that is proposed for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is said to be effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses. The compounds of U.S. Pat. No. 4,950,645, and Intl. Pat. Appl. Publ. No. WO 91/16347 are also proposed for use in achieving particular aspects of the present invention.

[0265] BCG and BCG-cell wall skeleton (CWS) may also be used as adjuvants in the invention, with or without trehalose dimycolate. Trehalose dimycolate may be used itself. Azuma et al. (1988) show that trehalose dimycolate administration correlates with augmented resistance to influenza virus infection in mice. Trehalose dimycolate may be prepared as described in U.S. Pat. No. 4,579,945.

[0266] Amphipathic and surface-active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of preferred adjuvants for use with the immunogens of the present invention. Nonionic block copolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) may also be employed. Oligonucleotides, as described by Yamamoto et al. (1988) are another useful group of adjuvants. Quil A and lentinen are also preferred adjuvants.

[0267] Superantigens are also contemplated for use as adjuvants in the present invention. "Superantigens" are generally bacterial products that stimulate a greater proportion of T lymphocytes than peptide antigens without a requirement for antigen processing (Mooney et. al., 1994). Superantigens include Staphylococcus exoproteins, such as the .alpha., .beta., .gamma. and .delta. enterotoxins from S. aureus and S. epidermidis, and the .alpha., .beta., .gamma. and .delta. E. coli exotoxins.

[0268] Common Staphylococcus enterotoxins are known as staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB), with enterotoxins through E (SEE) being described (Rott et. al., 1992). Streptococcus pyogenes B (SEB), Clostridium perfringens enterotoxin (Bowness et. al., 1992), cytoplasmic membrane-associated protein (CAP) from S. pyogenes (Sato et. al., 1994) and toxic shock syndrome toxin-1 (TSST-1) from S. aureus (Schwab et. al., 1993) are further useful superantigens.

[0269] One group of adjuvants particularly preferred for use in the invention are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals.

[0270] The detoxified endotoxins may be combined with other adjuvants. Combination of detoxified endotoxins with trehalose dimycolate is contemplated, as described in U.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins are also envisioned to be useful, as described in U.S. Pat. No. 4,520,019.

[0271] MPL is currently one preferred immunopotentiating agent for use herein. References that concern the uses of MPL include Tomai et al. (1987), Chen et al. (1991) and Garg and Subbarao (1992), that each concern certain roles of MPL in the reactions of aging mice; Elliott et al. (1991), that concerns the D-galactosamine loaded mouse and its enhanced sensitivity to lipopolysaccharide and MPL; Chase et al. (1986), that relates to bacterial infections; and Masihi et al. (1988), that describes the effects of MPL and endotoxin on resistance of mice to Toxoplasma gondii. Fitzgerald (1991) also reported on the use of MPL to up-regulate the immunogenicty of a syphilis vaccine and to confer significant protection against challenge infection in rabbits.

[0272] Thus MPL is known to be safe for use, as shown in the above model systems. Phase-I clinical trials have also shown MPL to be safe for use (Vosika et al., 1984). Indeed, 100 .mu.g/m.sup.2 is known to be safe for human use, even on an outpatient basis (Vosika et al., 1984).

[0273] MPL generally induces polyclonal B cell activation (Baker et al., 1994), and has been shown to augment antibody production in many systems, for example, in immunologically immature mice (Baker et al., 1988); in aging mice (Tomai and Johnson, 1989); and in nude and Xid mice (Madonna and Vogel, 1986; Myers et al., 1995). Antibody production has been shown against erythrocytes (Hraba et al., 1993); T cell dependent and independent antigens; Pnu-immune vaccine (Garg and Subbarao, 1992); isolated tumor-associated antigens (U.S. Pat. No. 4,877,611); against syngeneic tumor cells (Livingston et al., 1985; Ravindranath et al., 1994a; b); and against tumor-associated gangliosides (Ravindranath et al., 1994a; b).

[0274] Another useful attribute of MPL is that is augments IgM responses, as shown by Baker et al. (1988a), who describe the ability of MPL to increase antibody responses in young mice. This is a particularly useful feature of an adjuvant for use in certain embodiments of the present invention. Myers et al. (1995) recently reported on the ability of MPL to induce IgM antibodies, by virtue T cell-independent antibody production.

[0275] In the Myers et al. (1995) studies, MPL was conjugated to the hapten, TNP. MPL was proposed for use as a carrier for other haptens, such as peptides.

[0276] MPL also activates and recruits macrophages (Verma et al., 1992). Tomai and Johnson (1989) showed that MPL-stimulated T cells enhance IL-1 secretion by macrophages. MPL is also known to activate superoxide production, lysozyme activity, phagocytosis, and killing of Candida in murine peritoneal macrophages (Chen et al., 1991).

[0277] The effects of MPL on T cells include the endogenous production of cytotoxic factors, such as TNF, in serum of BCG-primed mice by MPL (Bennett et al., 1988). Kovach et al. (1990) and Elliot et al. (1991) also show that MPL induces TNF activity. MPL is known to act with TNF-.alpha. to induce release of IFN-.gamma. by NK cells. IFN-.gamma. production by T cells in response to MPL was also documented by Tomai and Johnson (1989), and Odean et al. (1990).

[0278] MPL is also known to be a potent T cell adjuvant. For example, MPL stimulates proliferation of melanoma-antigen specific CTLs (Mitchell et al., 1988, 1993). Further, Baker et al. (1988b) showed that nontoxic MPL inactivated suppressor T cell activity. Naturally, in the physiological environment, the inactivation of T suppressor cells allows for increased benefit for the animal, as realized by, e.g., increased antibody production. Johnson and Tomai (1988) have reported on the possible cellular and molecular mediators of the adjuvant action of MPL.

[0279] MPL is also known to induce aggregation of platelets and to phosphorylate a platelet protein prior to induction of serotonin secretion (Grabarek et al., 1990). This study shows that MPL is involved in protein kinase C activation and signal transduction.

[0280] Many articles concern the structure and function of MPL include. These include Johnson et al. (1990), that describes the structural characterization of MPL homologs obtained from Salmonella minnesota Re595 lipopolysaccharide. The work of Johnson et al. (1990), in common with Grabarek et al. (1990), shows that the fatty acid moieties of MPL can vary, even in commercial species. In separating MPL into eight fractions by thin layer chromatography, Johnson et al. (1990) found that three were particularly active, as assessed using human platelet responses. The chemical components of the various MPL species were characterized by Johnson et al. (1990).

[0281] Baker et al. (1992) further analyzed the structural features that influence the ability of lipid A and its analogs to abolish expression of suppressor T cell activity. They reported that decreasing the number of phosphate groups in lipid A from two to one (i.e., creating monophosphoryl lipid A, MPL) as well as decreasing the fatty acyl content, primarily by removing the residue at the 3 position, resulted in a progressive reduction in toxicity; however, these structural modifications did not influence its ability to abolish the expression of Ts function (Baker et al., 1992). These types of MPL are ideal for use in the present invention.

[0282] Baker et al. (1992) also showed that reducing the fatty acyl content from five to four (lipid A precursor IV.sub.A or I.sub.a) eliminated the capacity to influence Ts function but not to induce polyclonal activation of B cells. These studies show that in order to be able to abolish the expression of Ts function, lipid A must be a glucosamine disaccharide; may have either one or two phosphate groups; and must have at least five fatty acyl groups. Also, the chain length of the nonhydroxylated fatty acid, as well as the location of acyloxyacyl groups (2' versus 3' position), may play an important role (Baker et al., 1992).

[0283] In examining the relationship between chain length and position of fatty acyl groups on the ability of lipid A to abolish the expression of suppressor T-cell (Ts) activity, Baker et al. (1994) found that fatty acyl chain lengths of C.sub.12 to C.sub.14 appeared to be optimal for bioactivity. Therefore, although their use is still possible, lipid A preparations with fatty acyl groups of relatively short chain length (C.sub.10 to C.sub.12 from Pseudomonas aeruginosa and Chromobacterium violaceum) or predominantly long chain length (C.sub.18 from Helicobacter pylori) are less preferred for use in this invention.

[0284] Baker et al. (1994) also showed that the lipid A proximal inner core region oligosaccharides of some bacterial lipopolysaccharides increase the expression of Ts activity; due mainly to the capacity of such oligosaccharides, which are relatively conserved in structure among gram-negative bacterial, to enlarge or expand upon the population of CD8.sup.+ Ts generated during the course of a normal antibody response to unrelated microbial antigens. The minimal structure required for the expression of the added immunosuppression observed was reported to be a hexasaccharide containing one 2-keto-3-deoxyoctonate residue, two glucose residues, and three heptose residues to which are attached two pyrophosphorylethanolamine groups (Baker et al., 1994). This information may be considered in utilizing or even designing further adjuvants for use in the invention.

[0285] In a generally related line of work, Tanamoto et al. (1994a; b; 1995) described the dissociation of endotoxic activities in a chemically synthesized Lipid A precursor after acetylation or succinylation. Thus, compounds such as "acetyl 406" and "succinyl 516" (Tanamoto et al., 1994a; b; 1995) are also contemplated for use in the invention.

[0286] Synthetic MPLs form a particularly preferred group of antigens. For example, Brade et al. (1993) described an artificial glycoconjugate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A that binds to anti-Lipid A MAbs. This is one candidate for use in certain aspects of the invention.

[0287] The MPL derivatives described in U.S. Pat. No. 4,987,237 are particularly contemplated for use in the present invention. U.S. Pat. No. 4,987,237 describes MPL derivatives that contain one or more free groups, such as amines, on a side chain attached to the primary hydroxyl groups of the monophosphoryl lipid A nucleus through an ester group. The derivatives provide a convenient method for coupling the lipid A through coupling agents to various biologically active materials. The immunostimulant properties of lipid A are maintained. All MPL derivatives in accordance with U.S. Pat. No. 4,987,237 are envisioned for use in the MPL adjuvant-incorporated cells of this invention.

[0288] Various adjuvants, even those that are not commonly used in humans, may still be employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.

[0289] Within the vaccines provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-.gamma., TNF.alpha., IL-2 and IL-12) tend to favor the induction of cell-mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines see e.g., Mosmann and Coffman (1989).

[0290] Preferred adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, Wash.; see e.g., U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094, each of which is specifically incorporated herein by reference in its entirety). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in Intl. Pat. Appl. Publ. No. WO 96/02555 and Intl. Pat. Appl. Publ. No. WO 99/33488. Immunostimulatory DNA sequences are also described, for example, by Sato et al. (1996). Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL (see e.g., Intl. Pat. Appl. Publ. No. WO 94/00153), or a less reactogenic composition where the QS21 is quenched with cholesterol (see e.g., Intl. Pat. Appl. Publ. No. WO 96/33739). Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion has also been described (see e.g., Intl. Pat. Appl. Publ. No. WO 95/17210).

[0291] Other preferred adjuvants include Montanide ISA 720 (Seppic), SAF (Chiron), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa Corporation), RC-529 (Corixa Corporation) and aminoalkyl glucosaminide 4-phosphates (AGPs).

[0292] Any vaccine provided herein may be prepared using well-known methods that result in a combination of one or more antigens, one or more immunostimulants or adjuvants and one or more suitable carriers, excipients, or pharmaceutically acceptable buffers. The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel [composed of polysaccharides, for example] that effects a slow release of compound following administration). Such formulations may generally be prepared using well-known technology (Coombes et al., 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a peptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate-controlling membrane.

[0293] Carriers for use within such formulations are preferably biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose and dextran. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (U.S. Pat. No. 5,151,254; Intl. Pat. Appl. Publ. No. WO 94/20078; Intl. Pat. Appl. Publ. No. WO/94/23701; and Intl. Pat. Appl. Publ. No. WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0294] Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0295] Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (Timmerman and Levy, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (Zitvogel et al., 1998).

[0296] Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF.alpha. to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

[0297] Dendritic cells are conveniently categorized as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fc.gamma. receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0298] APCs may generally be transfected with a polynucleotide encoding a hematological malignancy related peptide, such that the peptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen-presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in Intl. Pat. Appl. Publ. No. WO 97/24447, or the gene gun approach described by Mahvi et al. (1997). Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the hematological malignancy related peptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the peptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the peptide.

[0299] Combined therapeutics is also contemplated, and the same type of underlying pharmaceutical compositions may be employed for both single and combined medicaments. Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

[0300] 4.5 Diagnostic and Prognostic Methods for Hematological Malignancy Diseases

[0301] The present invention further provides methods for detecting a malignant disease associated with one or more of the polypeptide or polynucleotide compositions disclosed herein, and for monitoring the effectiveness of an immunization or therapy for such a disease. To determine the presence or absence of a malignant disease associated with one or more of the polypeptide or polynucleotide compositions disclosed herein, a patient may be tested for the level of T cells specific for one or more of such compositions. Within certain methods, a biological sample comprising CD4.sup.+ and/or CD8.sup.+ T cells isolated from a patient is incubated with one or more of the polypeptide or polynucleotide compositions disclosed herein, and/or an APC that expresses one or more of such peptides or polypeptides, and the presence or absence of specific activation of the T cells is detected, as described herein. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37.degree. C. with one or more of the disclosed peptide, polypeptide or polynucleotide compositions (e.g., 5-25 .mu.g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of the composition to serve as a control. For CD4.sup.+ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8.sup.+ T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a malignant disease associated with expression or one or more of the disclosed polypeptide or polynucleotide compositions. Further correlation may be made, using methods well known in the art, between the level of proliferation and/or cytolytic activity and the predicted response to therapy. In particular, patients that display a higher antibody, proliferative and/or lytic response may be expected to show a greater response to therapy.

[0302] Within other methods, a biological sample obtained from a patient is tested for the level of antibody specific for one or more of the hematological malignancy-related peptides or polypeptide s disclosed herein. The biological sample is incubated with hematological malignancy-related peptide or polypeptide, or a polynucleotide encoding such a peptide or polypeptide, and/or an APC that expresses such a peptide or polypeptide under conditions and for a time sufficient to allow immunocomplexes to form. Immunocomplexes formed between the selected peptide or polypeptide and antibodies in the biological sample that specifically bind to the selected peptide or polypeptide are then detected. A biological sample for use within such methods may be any sample obtained from a patient that would be expected to contain antibodies. Suitable biological samples include blood, sera, ascites, bone marrow, pleural effusion, and cerebrospinal fluid.

[0303] The biological sample is incubated with the selected peptide or polypeptide in a reaction mixture under conditions and for a time sufficient to permit immunocomplexes to form between the selected peptide or polypeptide and antibodies that are immunospecific for such a peptide or polypeptide. For example, a biological sample and a selected peptide or polypeptide peptide may be incubated at 4.degree. C. for 24-48 hrs.

[0304] Following the incubation, the reaction mixture is tested for the presence of immunocomplexes. Detection of immunocomplexes formed between the selected peptide or polypeptide and antibodies present in the biological sample may be accomplished by a variety of known techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISA). Suitable assays are well known in the art and are amply described in the scientific and patent literature (Harlow and Lane, 1988). Assays that may be used include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique (U.S. Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., 1970); the "western blot" method (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., 1980); enzyme-linked immunosorbent assays (Raines and Ross, 1982); immunocytochemical techniques, including the use of fluorochromes (Brooks et al., 1980); and neutralization of activity (Bowen-Pope et al., 1984). Other immunoassays include, but are not limited to, those described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876.

[0305] For detection purposes, the selected peptide or polypeptide may either be labeled or unlabeled. Unlabeled polypeptide peptide may be used in agglutination assays or in combination with labeled detection reagents that bind to the immunocomplexes (e.g., anti-immunoglobulin, protein G, Protein A or a lectin and secondary antibodies, or antigen-binding fragments thereof, capable of binding to the antibodies that specifically bind to the selected hematological malignancy-related peptide or polypeptide). If the selected peptide or polypeptide is labeled, the reporter group may be any suitable reporter group known in the art, including radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.

[0306] Within certain assays, unlabeled peptide or polypeptide is immobilized on a solid support. The solid support may be any material known to those of ordinary skill in the art to which the peptide may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The peptide may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the selected peptide or polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of peptide ranging from about 10 ng to about 10 .mu.g, and preferably about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate amount of peptide.

[0307] Following immobilization, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin, Tween.TM. 20.TM. (Sigma Chemical Co., St. Louis, Mo.), heat-inactivated normal goat serum (NGS), or BLOTTO (buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent) may be used. The support is then incubated with a biological sample suspected of containing specific antibody. The sample can be applied neat, or, more often, it can be diluted, usually in a buffered solution which contains a small amount (0.1%-5.0% by weight) of protein, such as BSA, NGS, or BLOTTO. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of antibody or an antigen binding fragment that is immunospecific for the selected peptide or polypeptide within a sample containing such an antibody or binding fragment thereof. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound antibody or antibody fragment. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 min is generally sufficient.

[0308] Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween.TM. 20. A detection reagent that binds to the immunocomplexes and that comprises at least a first detectable label or "reporter" molecule may then be added. The detection reagent is incubated with the immunocomplex for an amount of time sufficient to detect the bound antibody or antigen binding fragment thereof. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound label or detection reagent is then removed and bound label or detection reagent is detected using a suitable assay or analytical instrument. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive labels, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent or chemiluminescent moieties and various chromogens, fluorescent labels and such like. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups (e.g., horseradish peroxidase, .beta.-galactosidase, alkaline phosphatase and glucose oxidase) may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products. Regardless of the specific method employed, a level of bound detection reagent that is at least two fold greater than background (i.e., the level observed for a biological sample obtained from a disease-free individual) indicates the presence of a malignant disease associated with expression of the selected peptide or polypeptide.

[0309] In general, methods for monitoring the effectiveness of an immunization or therapy involve monitoring changes in the level of antibodies or T cells specific for the selected peptide or polypeptide in a sample, or in an animal such as a human patient. Methods in which antibody levels are monitored may comprise the steps of: (a) incubating a first biological sample, obtained from a patient prior to a therapy or immunization, with a selected peptide or polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow immunocomplexes to form; (b) detecting immunocomplexes formed between the selected peptide or polypeptide and antibodies or antigen binding fragments in the biological sample that specifically bind to the selected peptide or polypeptide; (c) repeating steps (a) and (b) using a second biological sample taken from the patient at later time, such as for example, following a given therapy or immunization; and (d) comparing the number of immunocomplexes detected in the first and second biological samples. Alternatively, a polynucleotide encoding the selected peptide or polypeptide, or an APC expressing the selected peptide or polypeptide may be employed in place of the selected peptide or polypeptide itself. Within such methods, immunocomplexes between the selected peptide or polypeptide encoded by a polynucleotide, or expressed by the APC, and antibodies and/or antigen binding fragments in the biological sample are detected.

[0310] Methods in which T cell activation and/or the number of hematological malignancy polypeptide-specific precursors are monitored may comprise the steps of: (a) incubating a first biological sample comprising CD4.sup.+ and/or CD8.sup.+ cells (e.g., bone marrow, peripheral blood or a fraction thereof), obtained from a patient prior to a therapy or immunization, with a hematological malignancy peptide or polypeptide, wherein the incubation is performed under conditions and for a time sufficient to allow specific activation, proliferation and/or lysis of T cells; (b) detecting an amount of activation, proliferation and/or lysis of the T cells; (c) repeating steps (a) and (b) using a second biological sample comprising CD4.sup.+ and/or CD8.sup.+ T cells, and taken from the same patient following therapy or immunization; and (d) comparing the amount of activation, proliferation and/or lysis of T cells in the first and second biological samples. Alternatively, a polynucleotide encoding a hematological malignancy related peptide, or an APC expressing such a peptide may be employed in place of the hematological malignancy peptide itself.

[0311] A biological sample for use within such methods may be any sample obtained from a patient that would be expected to contain antibodies, CD4.sup.+ T cells and/or CD8.sup.+ T cells. Suitable biological samples include blood, sera, ascites, bone marrow, pleural effusion and cerebrospinal fluid. A first biological sample may be obtained prior to initiation of therapy or immunization or part way through a therapy or vaccination regime. The second biological sample should be obtained in a similar manner, but at a time following additional therapy or immunization. The second biological sample may be obtained at the completion of, or part way through, therapy or immunization, provided that at least a portion of therapy or immunization takes place between the isolation of the first and second biological samples.

[0312] Incubation and detection steps for both samples may generally be performed as described above. A statistically significant increase in the number of immunocomplexes in the second sample relative to the first sample reflects successful therapy or immunization.

[0313] 4.6 Administration of Pharmaceutical Compositions and Formulations

[0314] In certain embodiments, the present invention concerns formulation of one or more of the polynucleotide, polypeptide, peptide, antibody, or antigen binding fragment compositions disclosed herein in pharmaceutically acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of anti-cancer therapy, or in combination with one or more diagnostic or therapeutic agents.

[0315] It will also be understood that, if desired, the nucleic acid segment, RNA, or DNA compositions disclosed herein may be administered in combination with other agents as well, such as, e.g., proteins or peptides or various pharmaceutically-active agents. As long as the composition comprises at least one of the genetic expression constructs disclosed herein, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The RNA- or DNA-derived compositions may thus be delivered along with various other agents as required in the particular instance. Such RNA or DNA compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may comprise substituted or derivatized RNA or DNA compositions. Such compositions may include one or more therapeutic gene constructs, either alone, or in combination with one or more modified peptide or nucleic acid substituent derivatives, and/or other anticancer therapeutics.

[0316] The formulation of pharmaceutically-acceptable excipients and carrier solutions are well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, intravenous, intranasal, transdermal, intraprostatic, intratumoral, and/or intramuscular administration and formulation.

[0317] 4.6.1 Injectable Delivery

[0318] For example, the pharmaceutical compositions disclosed herein may be administered parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U.S. Pat. No. 5,543,158, U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free-base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0319] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0320] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Hoover, 1975). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.

[0321] Sterile injectable solutions may be prepared by incorporating the gene therapy constructs in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0322] The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

[0323] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0324] 4.6.2 Intranasal Delivery

[0325] One may use nasal solutions or sprays, aerosols or even inhalants for the treatment of hematological malignancies with one of more of the disclosed peptides and polynucleotides. Nasal solutions are usually aqueous solutions designed for administration to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of from about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known.

[0326] Inhalations and inhalants are pharmaceutical preparations designed for delivering a drug or compound into the respiratory tree of a patient. A vapor or mist is administered and reaches the affected area, often to give relief from symptoms of bronchial and nasal congestion. However, this route can also be employed to deliver agents into the systemic circulation. Inhalations may be administered by the nasal or oral respiratory routes. The administration of inhalation solutions is only effective if the droplets are sufficiently fine and uniform in size so that the mist reaches the bronchioles.

[0327] Another group of products, also known as inhalations, and sometimes called insufflations, consists of finely powdered or liquid drugs that are carried into the respiratory passages by the use of special delivery systems, such as pharmaceutical aerosols, that hold a solution or suspension of the drug in a liquefied gas propellant. When released through a suitable valve and oral adapter, a metered does of the inhalation is propelled into the respiratory tract of the patient.

[0328] Particle size is of importance in the administration of this type of preparation. It has been reported that the optimum particle size for penetration into the pulmonary cavity is of the order of about 0.5 to about 7 .mu.m. Fine mists are produced by pressurized aerosols and hence their use in considered advantageous.

[0329] 4.6.3 Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

[0330] In certain embodiments, the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the polynucleotide compositions of the present invention into suitable host cells. In particular, the polynucleotide compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0331] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-lives (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516, specifically incorporated herein by reference in its entirety). Further, various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporated herein by reference in its entirety).

[0332] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC12 cells (Renneisen et al., 1990; Muller et al., 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs (Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses (Faller and Baltimore, 1984), transcription factors and allosteric effectors (Nicolau and Gersonde, 1979) into a variety of cultured cell lines and animals. In addition, several successful clinical trails examining the effectiveness of liposome-mediated drug delivery have been completed (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al., 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery (Mori and Fukatsu, 1992).

[0333] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 .ANG., containing an aqueous solution in the core.

[0334] Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.

[0335] In addition to the teachings of Couvreur et al. (1977; 1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars, and drugs.

[0336] Alternatively, the invention provides for pharmaceutically acceptable nanocapsule formulations of the polynucleotide compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 .mu.m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated herein by reference in its entirety). In particular, methods of polynucleotide delivery to a target cell using either nanoparticles or nanospheres (Schwab et al., 1994; Truong-Le et al., 1998) are also particularly contemplated to be useful in formulating the disclosed compositions for administration to an animal, and to a human in particular.

[0337] 4.7 Therapeutic Agents and Kits

[0338] The invention also provides one or more of the hematological malignancy-related compositions formulated with one or more pharmaceutically acceptable excipients, carriers, diluents, adjuvants, and/or other components for use in the preparation of medicaments, or diagnostic reagents, as well as various kits comprising one or more of such compositions, medicaments, or formulations intended for administration to an animal in need thereof, or for use in one or more diagnostic assays for identifying polynucleotides, polypeptides, and/or antibodies that are specific for one or more hematological malignancy-related compounds as described herein. In addition to the disclosed epitopes, antibodies and antigen binding fragments, antibody- or antigen binding fragment-encoding polynucleotides or additional anticancer agents, polynucleotides, peptides, antigens, or other therapeutic compounds as may be employed in the formulation of particular compositions and formulations disclosed herein, and particularly in the preparation of anticancer agents or anti-hematological malignancies therapies for administration to the affected mammal:

[0339] As such, preferred animals for administration of the pharmaceutical compositions disclosed herein include mammals, and particularly humans. Other preferred animals include primates, sheep, goats, bovines, equines, porcines, lupines, canines, and felines, as well as any other mammalian species commonly considered pets, livestock, or commercially relevant animal species. The compositions and formulations may include partially or significantly purified polypeptide, polynucleotide, or antibody or antigen binding fragment compositions, either alone, or in combination with one or more additional active ingredients, anticancer agents, vaccines, adjuvants, or other therapeutics which may be obtained from natural or recombinant sources, or which may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing one or more nucleic acid segments that encode one or more such additional active ingredients, carriers, adjuvants, cofactors, or other therapeutic compound.

[0340] 4.8 Diagnostic Reagents and Kits

[0341] The invention further provides diagnostic reagents and kits comprising one or more such reagents for use in a variety of diagnostic assays, including for example, immunoassays such as ELISA and "sandwich"-type immunoassays. Such kits may preferably include at least a first peptide, or a first antibody or antigen binding fragment of the invention, a functional fragment thereof, or a cocktail thereof, and means for signal generation. The kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used. The signal generating means may come pre-associated with an antibody of the invention or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of microtiter plates, microspheres, or other materials suitable for immobilizing proteins, peptides, or polypeptides. Preferably, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art.

[0342] Such kits are useful in the detection, monitoring and diagnosis of conditions characterized by over-expression or inappropriate expression of hematological malignancy-related peptides, polypeptides, antibodies, and/or polynucleotides, as well as hybridomas, host cells, and vectors comprising one or more such compositions as disclosed herein.

[0343] The therapeutic and diagnostic kits of the present invention may also be prepared that comprise at least one of the antibody, peptide, antigen binding fragment, hybridoma, vector, vaccine, polynucleotide, or cellular compositions disclosed herein and instructions for using the composition as a diagnostic reagent or therapeutic agent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the diagnostic and/or therapeutic composition(s) may be placed, and preferably suitably aliquoted. Where a second therapeutic agent is also provided, the kit may also contain a second distinct container into which this second diagnostic and/or therapeutic composition may be placed. Alternatively, a plurality of compounds may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container. The kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorigenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the diagnostic or therapeutic composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.

[0344] 4.9 Polynucleotide Compositions

[0345] As used herein, the terms "DNA segment" and "polynucleotide" refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms "DNA segment" and "polynucleotide" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

[0346] As will be understood by those skilled in the art, the DNA segments of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.

[0347] "Isolated," as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the DNA segment does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

[0348] As will be recognized by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

[0349] Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a hematological malignancy-related tumor protein or a portion thereof) or may comprise a variant, or a biological or antigenic functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native tumor protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. The term "variants" also encompasses homologous genes of xenogenic origin.

[0350] When comparing polynucleotide or polypeptide sequences, two sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0351] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0352] Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0353] One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2:0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

[0354] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0355] Therefore, the present invention encompasses polynucleotide and polypeptide sequences having substantial identity to the sequences disclosed herein, for example those comprising at least 50% sequence identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide or polypeptide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0356] In additional embodiments, the present invention provides isolated polynucleotides and polypeptides comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.

[0357] The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative DNA segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.

[0358] In other embodiments, the present invention is directed to polynucleotides that are capable of hybridizing under moderately stringent conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight; followed by washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS.

[0359] Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

[0360] 4.10 Probes and Primers

[0361] In other embodiments of the present invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. As such, it is contemplated that nucleic acid segments that comprise a sequence region of at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 nucleotide long contiguous sequence the disclosed polynucleotides will find particular utility in a variety of hybridization embodiments. Longer contiguous identical or complementary sequences, e.g., those of about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 525, 550, 575, 600, 650, 700, 750, 800, 850, 900, 950, or even 1000 or so nucleotides (including all intermediate lengths) and all full-length sequences as the disclosed polynucleotides will also be of use in certain embodiments as probes, primers, or amplification targets and such like.

[0362] The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers, for use in preparing other genetic constructions, and for identifying and characterizing full-length polynucleotides and full, or substantially full-length cDNAs, mRNAs, and such like.

[0363] Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches identical or complementary to one or more polynucleotide sequences as disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern hybridization analyses and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or so and up to and including larger contiguous complementary sequences, including those of about 70, 80, 90, 100, 120, 140, 160, 180, or 200 or so nucleotides in length may also be used, according to the given desired goal, and the particular length of the complementary sequences one wishes to detect by hybridization analysis.

[0364] The use of a hybridization probe of about between about 20 and about 500 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than about 20 or so bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of between about 25 and 300 or so contiguous nucleotides, or even longer where desired.

[0365] Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the disclosed sequences, or to any contiguous portion of such a sequence, from about 15 to 30 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence.

[0366] Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR.TM. technology of U.S. Pat. No. 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

[0367] The nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest. Depending on the application envisioned, one will typically desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50.degree. C. to about 70.degree. C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.

[0368] Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20.degree. C. to about 55.degree. C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

[0369] 4.11 Polynucleotide Identification and Characterization

[0370] Polynucleotides may be identified, prepared and/or manipulated using any of a variety of well established techniques. For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs for tumor-associated expression (i.e., expression that is at least two fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using a Synteni microarray (Palo Alto, Calif.) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as hematological malignancy-related tumor cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized.

[0371] An amplified portion of a polynucleotide of the present invention may be used to isolate a full length gene from a suitable library (e.g., a hematological malignancy-related tumor cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.

[0372] For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with .sup.32P) using well known techniques. A bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.

[0373] Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software or algorithms or formulas well known in the art.

[0374] One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.

[0375] In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments.

[0376] 4.12 Polynucleotide Expression in Host Cells

[0377] In other embodiments of the invention, polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

[0378] As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

[0379] Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.

[0380] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.

[0381] Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).

[0382] A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

[0383] In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

[0384] A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0385] The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector--enhancers, promoters, 5' and 3' untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

[0386] In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of .beta.-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0387] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0388] In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0389] An insect system may also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91:3224-3227).

[0390] In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

[0391] Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

[0392] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

[0393] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.

[0394] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk.sup.- or aprt.sup.-cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0395] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

[0396] Alternatively, host cells which contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.

[0397] A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J Exp. Med. 158:1211-1216).

[0398] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a-variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0399] Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

[0400] In addition to recombinant production methods, polypeptides of the invention, and fragments thereof, may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.

[0401] 4.13 Site-Specific Mutagenesis

[0402] Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent polypeptides, through specific mutagenesis of the underlying polynucleotides that encode them. The technique, well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

[0403] In certain embodiments of the present invention, the inventors contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the antigenicity of a polypeptide vaccine. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter a specific portion of a DNA molecule. In such embodiments, a primer comprising typically about 14 to about 25 nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

[0404] As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.

[0405] In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

[0406] The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

[0407] As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987). Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U.S. Pat. No. 4,237,224, specifically incorporated herein by reference in its entirety.

[0408] 4.14 Polynucleotide Amplification Techniques

[0409] A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR.TM.) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCR.TM., two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse transcription and PCR.TM. amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.

[0410] Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specifically incorporated herein by reference in its entirety). In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR.TM., bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.

[0411] Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880, incorporated herein by reference in its entirety, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

[0412] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[.alpha.-thio]triphosphates in one strand of a restriction site (Walker et al., 1992, incorporated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.

[0413] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA.

[0414] Sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 3' and 5' sequences of non-target DNA and an internal or "middle" sequence of the target protein specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe are identified as distinctive products by generating a signal that is released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to a target gene specific expressed nucleic acid.

[0415] Still other amplification methods described in Great Britain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR-like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes is added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

[0416] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer that has sequences specific to the target sequence. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat-denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target-specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into DNA, and transcribed once again with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target-specific sequences.

[0417] Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting as a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

[0418] PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" (Frohman, 1990), and "one-sided PCR" (Ohara, 1989) which are well-known to those of skill in the art.

[0419] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu and Dean, 1996, incorporated herein by reference in its entirety), may also be used in the amplification of DNA sequences of the present invention.

[0420] 4.15 In Vivo Polynucleotide Delivery Techniques

[0421] In additional embodiments, genetic constructs comprising one or more of the polynucleotides of the invention are introduced into cells in vivo. This may be achieved using any of a variety or well known approaches, several of which are outlined below for the purpose of illustration.

[0422] 4.15.1 Adenovirus

[0423] One of the preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized.

[0424] The expression vector comprises a genetically engineered form of an adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

[0425] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

[0426] In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.

[0427] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions (Graham and Preve., 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately 5.5 kB of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1-deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).

[0428] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the currently preferred helper cell line is 293.

[0429] Recently, Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

[0430] Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain a conditional replication-defective adenovirus vector for use in the present invention, since Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

[0431] As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.

[0432] Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10.sup.9-10.sup.11 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

[0433] Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

[0434] 4.15.2 Retroviruses

[0435] The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-Stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

[0436] In order to construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0437] A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.

[0438] A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).

[0439] 4.15.3 Adeno-Associated Viruses

[0440] AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replications is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka and McLaughlin, 1988).

[0441] The AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs. There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three viral promoters have been identified and named p5, p19, and p40, according to their map position. Transcription from p5 and p19 results in production of rep proteins, and transcription from p40 produces the capsid proteins (Hermonat and Muzyczka, 1984).

[0442] There are several factors that prompted researchers to study the possibility of using rAAV as an expression vector. One is that the requirements for delivering a gene to integrate into the host chromosome are surprisingly few. It is necessary to have the 145-bp ITRs, which are only 6% of the AAV genome. This leaves room in the vector to assemble a 4.5-kb DNA insertion. While this carrying capacity may prevent the AAV from delivering large genes, it is amply suited for delivering the antisense constructs of the present invention.

[0443] AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response.

[0444] 4.15.4 Other Viral Vectors as Expression Constructs

[0445] Other viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio viruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al., 1990).

[0446] With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al. (1991) introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991).

[0447] 4.15.5 Non-Viral Vectors

[0448] In order to effect expression of the oligonucleotide or polynucleotide sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, one preferred mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.

[0449] Once the expression construct has been delivered into the cell the nucleic acid encoding the desired oligonucleotide or polynucleotide sequences may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the construct may be stably integrated into the genome of the cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

[0450] In certain embodiments of the invention, the expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Reshef (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0451] Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

[0452] Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e. ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention.

[0453] 4.16 Antisense Oligonucleotides

[0454] The end result of the flow of genetic information is the synthesis of protein. DNA is transcribed by polymerases into messenger RNA and translated on the ribosome to yield a folded, functional protein. Thus there are several steps along the route where protein synthesis can be inhibited. The native DNA segment coding for a polypeptide described herein, as all such mammalian DNA strands, has two strands: a sense strand and an antisense strand held together by hydrogen bonding. The messenger RNA coding for polypeptide has the same nucleotide sequence as the sense DNA strand except that the DNA thymidine is replaced by uridine. Thus, synthetic antisense nucleotide sequences will bind to a mRNA and inhibit expression of the protein encoded by that mRNA.

[0455] The targeting of antisense oligonucleotides to mRNA is thus one mechanism to shut down protein synthesis, and, consequently, represents a powerful and targeted therapeutic approach. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829, each specifically incorporated herein by reference in its entirety). Further, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABA.sub.A receptor and human EGF (Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al., 1998; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288, each specifically incorporated herein by reference in its entirety). Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683, each specifically incorporated herein by reference in its entirety).

[0456] Therefore, in exemplary embodiments, the invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof. In one embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNAs comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein.

[0457] Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence (i.e. in these illustrative examples the rat and human sequences) and determination of secondary structure, T.sub.m, binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.

[0458] Highly preferred target regions of the mRNA, are those which are at or near the AUG translation initiation codon, and those sequences which were substantially complementary to 5' regions of the mRNA. These secondary structure analyses and target site selection considerations were performed using v.4 of the OLIGO primer analysis software (Rychlik, 1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

[0459] The use of an antisense delivery method employing a short peptide vector, termed MPG (27 residues), is also contemplated. The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of SV40 T-antigen (Morris et al., 1997). It has been demonstrated that several molecules of the MPG peptide coat the antisense oligonucleotides and can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%). Further, the interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane (Morris et al., 1997).

[0460] 4.17 Ribozymes

[0461] Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

[0462] Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855 (specifically incorporated herein by reference) reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990). Recently, it was reported that ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.

[0463] Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0464] The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.

[0465] The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis .delta. virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al. (1992). Examples of hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al. (1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein by reference). An example of the hepatitis .delta. virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive, 1993); and an example of the Group I intron is described in (U.S. Pat. No. 4,987,071, specifically incorporated herein by reference). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.

[0466] In certain embodiments, it may be important to produce enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target, such as one of the sequences disclosed herein. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. Alternatively, the ribozymes can be expressed from DNA or RNA vectors that are delivered to specific cells.

[0467] Small enzymatic nucleic acid motifs (e.g., of the hammerhead or the hairpin structure) may also be used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. Alternatively, catalytic RNA molecules can be expressed within cells from eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet et al., 1992; Dropulic et al., 1992; Weerasinghe et al, 1991; Ojwang et al, 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in the art realize that any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector. The activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl. Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa et al., 1992; Taira et al., 1991; and Ventura et al., 1993).

[0468] Ribozymes may be added directly, or can be complexed with cationic lipids, lipid complexes, packaged within liposomes, or otherwise delivered to target cells. The RNA or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their incorporation in biopolymers.

[0469] Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be utilized when necessary.

[0470] Hammerhead or hairpin ribozymes may be individually analyzed by computer folding (Jaeger et al., 1989) to assess whether the ribozyme sequences fold into the appropriate secondary structure. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 or so bases on each arm are able to bind to, or otherwise interact with, the target RNA.

[0471] Ribozymes of the hammerhead or hairpin motif may be designed to anneal to various sites in the mRNA message, and can be chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al (1987) and in Scaringe et al. (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. Average stepwise coupling yields are typically >98%. Hairpin ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992). Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high pressure liquid chromatography and resuspended in water.

[0472] Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990; Pieken et al, 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

[0473] Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles. Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions of ribozyme delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.

[0474] Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Saber et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yu et al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors).

[0475] Ribozymes may be used as diagnostic tools to examine genetic drift and mutations within diseased cells. They can also be used to assess levels of the target RNA molecule. The close relationship between ribozyme activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA. By using multiple ribozymes, one may map nucleotide changes which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with ribozymes may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease. These studies will lead to better treatment of the disease progression by affording the possibility of combinational therapies (e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules). Other in vitro uses of ribozymes are well known in the art, and include detection of the presence of mRNA associated with an IL-5 related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a ribozyme using standard methodology.

[0476] 4.18 Peptide Nucleic Acids

[0477] In certain embodiments, the inventors contemplate the use of peptide nucleic acids (PNAs) in the practice of the methods of the invention. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, 1997). PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (1997) and is incorporated herein by reference. As such, in certain embodiments, one may prepare PNA sequences that are complementary to one or more portions of the ACE mRNA sequence, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of ACE-specific mRNA, and thereby alter the level of ACE activity in a host cell to which such PNA compositions have been administered.

[0478] PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., 1991; Hanvey et al., 1992; Hyrup and Nielsen, 1996; Neilsen, 1996). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc (Dueholm et al., 1994) or Fmoc (Thomson et al., 1995) protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used (Christensen et al., 1995).

[0479] PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., 1995). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.

[0480] As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse-phase high-pressure liquid chromatography (Norton et al., 1995) providing yields and purity of product similar to those observed during the synthesis of peptides.

[0481] Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (Norton et al., 1995; Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al., 1995; Ulmann et al., 1996; Koch et al., 1995; Orum et al., 1995; Footer et al., 1996; Griffith et al., 1995; Kremsky et al., 1996; Pardridge et al., 1995; Boffa et al, 1995; Landsdorp et al., 1996; Gambacorti-Passerini et al., 1996; Armitage et al., 1997; Seeger et al., 1997; Ruskowski et al., 1997). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.

[0482] In contrast to DNA and RNA, which contain negatively charged linkages, the PNA backbone is neutral. In spite of this dramatic alteration, PNAs recognize complementary DNA and RNA by Watson-Crick pairing (Egholm et al., 1993), validating the initial modeling by Nielsen et al. (1991). PNAs lack 3' to 5' polarity and can bind in either parallel or anti-parallel fashion, with the anti-parallel mode being preferred (Egholm et al., 1993).

[0483] Hybridization of DNA oligonucleotides to DNA and RNA is destabilized by electrostatic repulsion between the negatively charged phosphate backbones of the complementary strands. By contrast, the absence of charge repulsion in PNA-DNA or PNA-RNA duplexes increases the melting temperature (T.sub.m) and reduces the dependence of T.sub.m on the concentration of mono- or divalent cations (Nielsen et al., 1991). The enhanced rate and affinity of hybridization are significant because they are responsible for the surprising ability of PNAs to perform strand invasion of complementary sequences within relaxed double-stranded DNA. In addition, the efficient hybridization at inverted repeats suggests that PNAs can recognize secondary structure effectively within double-stranded DNA. Enhanced recognition also occurs with PNAs immobilized on surfaces, and Wang et al. have shown that support-bound PNAs can be used to detect hybridization events (Wang et al., 1996).

[0484] One might expect that tight binding of PNAs to complementary sequences would also increase binding to similar (but not identical) sequences, reducing the sequence specificity of PNA recognition. As with DNA hybridization, however, selective recognition can be achieved by balancing oligomer length and incubation temperature. Moreover, selective hybridization of PNAs is encouraged by PNA-DNA hybridization being less tolerant of base mismatches than DNA-DNA hybridization. For example, a single mismatch within a 16 bp PNA-DNA duplex can reduce the T.sub.m by up to 15.degree. C. (Egholm et al., 1993). This high level of discrimination has allowed the development of several PNA-based strategies for the analysis of point mutations (Wang et al., 1996; Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996; Perry-O'Keefe et al., 1996).

[0485] High-affinity binding provides clear advantages for molecular recognition and the development of new applications for PNAs. For example, 11-13 nucleotide PNAs inhibit the activity of telomerase, a ribonucleo-protein that extends telomere ends using an essential RNA template, while the analogous DNA oligomers do not (Norton et al., 1996).

[0486] Neutral PNAs are more hydrophobic than analogous DNA oligomers, and this can lead to difficulty solubilizing them at neutral pH, especially if the PNAs have a high purine content or if they have the potential to form secondary structures. Their solubility can be enhanced by attaching one or more positive charges to the PNA termini (Nielsen et al., 1991).

[0487] Findings by Allfrey and colleagues suggest that strand invasion will occur spontaneously at sequences within chromosomal DNA (Boffa et al., 1995; Boffa et al., 1996). These studies targeted PNAs to triplet repeats of the nucleotides CAG and used this recognition to purify transcriptionally active DNA (Boffa et al., 1995) and to inhibit transcription (Boffa et al., 1996). This result suggests that if PNAs can be delivered within cells then they will have the potential to be general sequence-specific regulators of gene expression. Studies and reviews concerning the use of PNAs as antisense and anti-gene agents include Nielsen et al. (1993b), Hanvey et al. (1992), and Good and Nielsen (1997). Koppelhus et al. (1997) have used PNAs to inhibit HIV-1 inverse transcription, showing that PNAs may be used for antiviral therapies.

[0488] Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (1993) and Jensen et al (1997). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al. using BIAcore.TM. technology.

[0489] Other applications of PNAs include use in DNA strand invasion (Nielsen et al., 1991), antisense inhibition (Hanvey et al., 1992), mutational analysis (Orum et al., 1993), enhancers of transcription (Mollegaard et al., 1994), nucleic acid purification (Orum et al., 1995), isolation of transcriptionally active genes (Boffa et al., 1995), blocking of transcription factor binding (Vickers et al., 1995), genome cleavage (Veselkov et al., 1996), biosensors (Wang et al., 1996), in situ hybridization (Thisted et al., 1996), and in a alternative to Southern blotting (Perry-O'Keefe, 1996).

[0490] 4.19 Polypeptide, Peptides and Peptide Variants

[0491] The present invention, in other aspects, provides polypeptide compositions. Generally, a polypeptide of the invention will be an isolated polypeptide (or an epitope, variant, or active fragment thereof) derived from a mammalian species. Preferably, the polypeptide is encoded by a polynucleotide sequence disclosed herein or a sequence which hybridizes under moderately stringent conditions to a polynucleotide sequence disclosed herein. Alternatively, the polypeptide may be defined as a polypeptide which comprises a contiguous amino acid sequence from an amino acid sequence disclosed herein, or which polypeptide comprises an entire amino acid sequence disclosed herein.

[0492] In the present invention, a polypeptide composition is also understood to comprise one or more polypeptides that are immunologically reactive with antibodies generated against a polypeptide of the invention, particularly a polypeptide having the amino acid sequence encoded by the disclosed polynucleotides, or to active fragments, or to variants or biological functional equivalents thereof.

[0493] Likewise, a polypeptide composition of the present invention is understood to comprise one or more polypeptides that are capable of eliciting antibodies that are immunologically reactive with one or more polypeptides encoded by one or more contiguous nucleic acid sequences disclosed in this application, or to active fragments, or to variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.

[0494] As used herein, an active fragment of a polypeptide includes a whole or a portion of a polypeptide which is modified by conventional techniques, e.g., mutagenesis, or by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure function, antigenicity, etc., as a polypeptide as described herein.

[0495] In certain illustrative embodiments, the polypeptides of the invention will comprise at least an immunogenic portion of a hematological malignancy-related tumor protein or a variant thereof, as described herein. As noted above, a "hematological malignancy-related tumor protein" is a protein that is expressed by hematological malignancy-related tumor cells. Proteins that are hematological malignancy-related tumor proteins also react detectably within an immunoassay (such as an ELISA) with antisera from a patient with hematological malignancy. Polypeptides as described herein may be of any length. Additional sequences derived from the native protein and/or heterologous sequences may be present, and such sequences may (but need not) possess further immunogenic or antigenic properties.

[0496] An "immunogenic portion," as used herein is a portion of a protein that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Such immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of a hematological malignancy-related tumor protein or a variant thereof. Certain preferred immunogenic portions include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted. Other preferred immunogenic portions may contain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.

[0497] Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen-specific" if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well known techniques. An immunogenic portion of a native hematological malignancy-related tumor protein is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, .sup.125I-labeled Protein A.

[0498] As noted above, a composition may comprise a variant of a native hematological malignancy-related tumor protein. A polypeptide "variant," as used herein, is a polypeptide that differs from a native hematological malignancy-related tumor protein in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished. In other words, the ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein. Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein.

[0499] Polypeptide variants encompassed by the present invention include those exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described above) to the polypeptides disclosed herein.

[0500] Preferably, a variant contains conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr, (2) cys, ser, tyr, thr, (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

[0501] As noted above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0502] Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

[0503] Portions and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

[0504] Within certain specific embodiments, a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

[0505] Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

[0506] A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0507] The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

[0508] Fusion proteins are also provided. Such proteins comprise a polypeptide as described herein together with an unrelated immunogenic protein. Preferably the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

[0509] Within preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.

[0510] In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

[0511] In general, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

[0512] 4.20 Binding Agents

[0513] The present invention further employs agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a hematological malignancy-related antigen. As used herein, an antibody, or antigen-binding fragment thereof, is said to "specifically bind" to a hematological malignancy-related antigen if it reacts at a detectable level (within, for example, an ELISA) with, and does not react detectably with unrelated proteins under similar conditions. As used herein, "binding" refers to a noncovalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to "bind," in the context of the present invention, when the binding constant for complex formation exceeds about 10.sup.3 L/mol. The binding constant maybe determined using methods well known in the art.

[0514] Binding agents may be further capable of differentiating between patients with and without a hematological malignancy. Such binding agents generate a signal indicating the presence of a hematological malignancy in at least about 20% of patients with the disease, and will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the disease. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, urine and/or tumor biopsies) from patients with and without a hematological malignancy (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent. It will be apparent that a statistically significant number of samples with and without the disease should be assayed. Each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.

[0515] Any agent that satisfies the above requirements may be a binding agent. For example, a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

[0516] Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

[0517] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

[0518] Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

[0519] Monoclonal antibodies, and fragments thereof, of the present invention may be coupled to one or more therapeutic agents, such as radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi. Preferred drugs include methotrexate, and pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein. For certain in vivo and ex vivo therapies, an antibody or fragment thereof is preferably coupled to a cytotoxic agent, such as a radioactive or chemotherapeutic moiety.

[0520] A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

[0521] Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.

[0522] It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Pat. No. 4,671,958.

[0523] Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No. 4,569,789).

[0524] It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used.

[0525] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Pat. No. 4,507,234), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide. For example, U.S. Pat. No. 4,673,562 discloses representative chelating compounds and their synthesis.

[0526] A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.

[0527] 4.21 Vaccines

[0528] In certain preferred embodiments of the present invention, vaccines are provided. The vaccines will generally comprise one or more pharmaceutical compositions, such as those discussed above, in combination with an immunostimulant. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in, for example, M. F. Powell and M. J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine.

[0529] Illustrative vaccines may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component. Such vaccines may provide for an enhanced immune response.

[0530] It will be apparent that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).

[0531] While any suitable carrier known to those of ordinary skill in the art may be employed in the vaccine compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. One may also employ a carrier comprising the particulate-protein complexes described in U.S. Pat. No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.

[0532] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

[0533] Any of a variety of immunostimulants may be employed in the vaccines of this invention. For example, an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0534] Within the vaccines provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Th1 type. High levels of Th1-type cytokines (e.g., IFN-.gamma., TNF.alpha., IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

[0535] Preferred adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa Corporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

[0536] Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties.

[0537] Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient. The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 14:1429-1438, 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.

[0538] Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0539] Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0540] Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).

[0541] Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF.alpha. to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

[0542] Dendritic cells are conveniently categorized as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0543] APCs may generally be transfected with a polynucleotide encoding a hematological malignancy-related tumor protein (or portion or other variant thereof) such that the hematological malignancy-related tumor polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the hematological malignancy-related tumor polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

[0544] Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

[0545] 4.22 Cancer Therapy

[0546] In further aspects of the present invention, the compositions described herein may be used for immunotherapy of cancer, such as hematological malignancy. Within such methods, pharmaceutical compositions and vaccines are typically administered to a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer. A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor. Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. Administration may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.

[0547] Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein).

[0548] Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8.sup.+ cytotoxic T lymphocytes and CD4.sup.+ T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Pat. No. 4,918,164) for passive immunotherapy.

[0549] Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).

[0550] Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.

[0551] Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 .mu.g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

[0552] In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to a hematological malignancy-related tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

[0553] 4.23 Cancer Detection and Diagnosis

[0554] In general, a cancer may be detected in a patient based on the presence of one or more hematological malignancy-related tumor proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a cancer such as hematological malignancy. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of a cancer. In general, a hematological malignancy-related tumor sequence should be present at a level that is at least three fold higher in tumor tissue than in normal tissue

[0555] There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.

[0556] In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length hematological malignancy-related tumor proteins and portions thereof to which the binding agent binds, as described above.

[0557] The solid support may be any material known to those of ordinary skill in the art to which the tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 0.10 .mu.g, and preferably about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate amount of binding agent.

[0558] Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

[0559] In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

[0560] More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20.TM. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with hematological malignancy. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

[0561] Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM.. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include those groups recited above.

[0562] The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

[0563] To determine the presence or absence of a cancer, such as hematological malignancy, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.

[0564] In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

[0565] Of course, numerous other assay protocols exist that are suitable for use with the tumor proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use hematological malignancy-related tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such hematological malignancy-related tumor protein specific antibodies may correlate with the presence of a cancer.

[0566] A cancer may also, or alternatively, be detected based on the presence of T cells that specifically react with a hematological malignancy-related tumor protein in a biological sample. Within certain methods, a biological sample comprising CD4.sup.+ and/or CD8.sup.+ T cells isolated from a patient is incubated with a hematological malignancy-related tumor polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37.degree. C. with polypeptide (e.g., 5-25 .mu.g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of hematological malignancy-related tumor polypeptide to serve as a control. For CD4.sup.+ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8.sup.+ T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a cancer in the patient.

[0567] As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a hematological malignancy-related tumor protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a hematological malignancy-related tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the hematological malignancy-related tumor protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a hematological malignancy-related tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.

[0568] To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a hematological malignancy-related tumor protein that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence disclosed in this application. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, Stockton Press, N Y, 1989).

[0569] One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.

[0570] In another embodiment, the compositions described herein may be used as markers for the progression of cancer. In this embodiment, assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.

[0571] Certain in vivo diagnostic assays may be performed directly on a tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.

[0572] As noted above, to improve sensitivity, multiple hematological malignancy-related tumor protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.

[0573] 4.24 Preparation of DNA Sequences

[0574] Certain nucleic acid sequences of cDNA molecules encoding portions of hematological malignancy-related antigens were isolated by PCR.TM.-based subtraction. This technique serves to normalize differentially expressed cDNAs, facilitating the recovery of rare transcripts, and also has the advantage of permitting enrichment of cDNAs with small amounts of polyA RNA material and without multiple rounds of hybridization. To obtain antigens overexpressed in non-Hodgkin's lymphomas, two subtractions were performed with a tester library prepared from a pool of three T cell non-Hodgkin's lymphoma mRNAs. The two libraries were independently subtracted with different pools of driver cDNAs. Driver #1 contained cDNA prepared from specific normal tissues (lymph node, bone marrow, T cells, heart and brain), and this subtraction generated the library TCS-D1 (T cell non-Hodgkin's lymphoma subtracted library with driver #1). Driver #2 contained non-specific normal tissues (colon, large intestine, lung, pancreas, spinal cord, skeletal muscle, liver, kidney, skin and brain), and this subtraction generated the library TCS-D2 (T cell non-Hodgkin's lymphoma subtraction library with driver #2). Two other subtractions were performed with a tester library prepared from a pool of three B cell non-Hodgkin's lymphoma mRNAs. The two libraries were independently subtracted with different pools of driver cDNAs. Driver #1 contained cDNA prepared from specific normal tissues (lymph node, bone marrow, B cells, heart and brain), and this subtraction generated the library BCNHL/D1 (B cell non-Hodgkin's lymphoma subtracted library with driver #1). Driver #2 contained non-specific normal tissues (brain, lung, pancreas, spinal cord, skeletal muscle, colon, spleen, large intestine and PBMC), and this subtraction generated the library BCNHL/D2 (B cell non-Hodgkin's lymphoma subtraction library with driver #2). PCR.TM.-amplified pools were generated from the subtracted libraries and clones were sequenced.

[0575] Hematological malignancy-related antigen sequences may be further characterized using any of a variety of well known techniques. For example, PCR.TM. amplified clones may be arrayed onto glass slides for microarray analysis. To determine tissue distribution, the arrayed clones may be used as targets to be hybridized with different first strand cDNA probes, including lymphoma probes, leukemia probes and probes from different normal tissues. Leukemia and lymphoma probes may be generated from cryopreserved samples obtained at the time of diagnosis from NHL, Hodgkin's disease, AML, CML, CLL, ALL, MDS and myeloma patients with poor outcome (patients who failed to achieve complete remission following conventional chemotherapy or relapsed) or good outcome (patients who achieved long term remission). To analyze gene expression during hematopoetic differentiation, probes may be generated from >95% pure fractions of CD34+, CD2+, CD14+, CD15+ and CD19+ cells derived from healthy individuals.

[0576] Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding a hematological malignancy-related antigen, and administering the transfected cells to the patient).

[0577] A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate hematological malignancy-related antigen expression. cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells or tissues to facilitate the production of antisense RNA. An antisense polynucleotide may be used, as described herein, to inhibit expression of a hematological malignancy-related antigen. Antisense technology can be used to control gene expression through triple-helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules (see Gee et al., In Huber and Canrr, Molecular and Immunologic Approaches, Futura Publishing Co. (Mt. Kisco, N.Y.; 1994)). Alternatively, an antisense molecule may be designed to hybridize with a control region of a gene (e.g., promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes.

[0578] A portion of a coding sequence or of a complementary sequence may also be designed as a probe or primer to detect gene expression. Probes may be labeled with a variety of reporter groups, such as radionuclides and enzymes, and are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides in length and still more preferably at least 30 nucleotides in length. Primers, as noted above, are preferably 22-30 nucleotides in length.

[0579] Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl-methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0580] Hematological malignancy-related antigen polynucleotides may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

[0581] Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

[0582] Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0583] 4.25 Therapeutic Methods

[0584] In further aspects of the present invention, the compositions described herein may be used for immunotherapy of hematological malignancies including adult and pediatric AML, CML, ALL, CLL, myelodysplastic syndromes (MDS), myeloproliferative syndromes (MPS), secondary leukemia, multiple myeloma, Hodgkin's lymphoma and Non-Hodgkin's lymphomas. In addition, compositions described herein may be used for therapy of diseases associated with an autoimmune response against hematopoetic precursor cells, such as severe aplastic anemia.

[0585] Immunotherapy may be performed using any of a variety of techniques, in which compounds or cells provided herein function to remove hematological malignancy-related antigen-expressing cells from a patient. Such removal may take place as a result of enhancing or inducing an immune response in a patient specific for hematological malignancy-related antigen or a cell expressing hematological malignancy-related antigen. Alternatively, hematological malignancy-related antigen-expressing cells may be removed ex vivo (e.g., by treatment of autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood). Fractions of bone marrow or peripheral blood may be obtained using any standard technique in the art.

[0586] Within such methods, pharmaceutical compositions and vaccines are typically administered to a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with a hematological malignancy. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the development of a malignancy or to treat a patient afflicted with a malignancy. A hematological malignancy may be diagnosed using criteria generally accepted in the art. Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs, or bone marrow transplantation (autologous, allogeneic or syngeneic).

[0587] Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein).

[0588] Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8.sup.+ cytotoxic T lymphocytes and CD4.sup.+ T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Pat. No. 4,918,164) for passive immunotherapy.

[0589] Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).

[0590] Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.

[0591] The compositions provided herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated). As discussed in greater detail below, binding agents and T cells as provided herein may be used for purging of autologous stem cells. Such purging may be beneficial prior to, for example, bone marrow transplantation or transfusion of blood or components thereof. Binding agents, T cells, antigen presenting cells (APC) and compositions provided herein may further be used for expanding and stimulating (or priming) autologous, allogeneic, syngeneic or unrelated hematological malignancy-related antigen-specific T-cells in vitro and/or in vivo. Such hematological malignancy-related antigen-specific T cells may be used, for example, within donor lymphocyte infusions.

[0592] Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 100 .mu.g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

[0593] In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to a hematological malignancy-related antigen generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

[0594] Within further aspects, methods for inhibiting the development of a malignant disease associated with hematological malignancy-related antigen expression involve the administration of autologous T cells that have been activated in response to a hematological malignancy-related antigen polypeptide or hematological malignancy-related antigen-expressing APC, as described above. Such T cells may be CD4.sup.+ and/or CD8.sup.+, and may be proliferated as described above. The T cells may be administered to the individual in an amount effective to inhibit the development of a malignant disease. Typically, about 1.times.10.sup.9 to 1.times.10.sup.11 T cells/M.sup.2 are administered intravenously, intracavitary or in the bed of a resected tumor. It will be evident to those skilled in the art that the number of cells and the frequency of administration will be dependent upon the response of the patient.

[0595] Within certain embodiments, T cells may be stimulated prior to an autologous bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a patient may be contacted with a hematological malignancy-related antigen polypeptide, a polynucleotide encoding a hematological malignancy-related antigen polypeptide and/or an APC that expresses a hematological malignancy-related antigen polypeptide under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or hematological malignancy-related antigen-specific T cells may then be administered to a patient using standard techniques.

[0596] Within related embodiments, T cells of a related or unrelated donor may be stimulated prior to a syngeneic or allogeneic (related or unrelated) bone marrow transplantation. Such stimulation may take place in vivo or in vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow or peripheral blood) obtained from a related or unrelated donor may be contacted with a-hematological malignancy-related antigen polypeptide, hematological malignancy-related antigen polynucleotide and/or APC that expresses a hematological malignancy-related antigen polypeptide under conditions and for a time sufficient to permit the stimulation of T cells as described above. Bone marrow, peripheral blood stem cells and/or hematological malignancy-related antigen-specific T cells may then be administered to a patient using standard techniques.

[0597] Within other embodiments, hematological malignancy-related antigen-specific T cells, antibodies or antigen-binding fragments thereof as described herein may be used to remove cells expressing hematological malignancy-related antigen from a biological sample, such as autologous bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood (e.g., CD34.sup.+ enriched peripheral blood (PB) prior to administration to a patient). Such methods may be performed by contacting the biological sample with such T cells, antibodies or antibody fragments under conditions and for a time sufficient to permit the reduction of hematological malignancy-related antigen expressing cells to less than 10%, preferably less than 5% and more preferably less than 1%, of the total number of myeloid or lymphatic cells in the bone marrow or peripheral blood. Such contact may be achieved, for example, using a column to which antibodies are attached using standard techniques. Antigen-expressing cells are retained on the column. The extent to which such cells have been removed may be readily determined by standard methods such as, for example, qualitative and quantitative PCR analysis, morphology, immunohistochemistry and FACS analysis. Bone marrow or PB (or a fraction thereof) may then be administered to a patient using standard techniques.

[0598] 4.26 Diagnostic Methods

[0599] In general, a hematological malignancy may be detected in a patient based on the presence of hematological malignancy-related antigen and/or polynucleotide in a biological sample (such as blood, sera, urine and/or tumor biopsies) obtained from the patient. In other words, hematological malignancy-related antigens may be used as a marker to indicate the presence or absence of such a malignancy. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding hematological malignancy-related antigen, which is also indicative of the presence or absence of a hematological malignancy. In general, hematological malignancy-related antigen should be present at a level that is at least three fold higher in a sample obtained from a patient afflicted with a hematological malignancy than in the sample obtained from an individual not so afflicted.

[0600] There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a hematological malignancy in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.

[0601] In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length hematological malignancy-related antigens and portions thereof to which the binding agent binds, as described above.

[0602] The solid support may be any material known to those of ordinary skill in the art to which the hematological malignancy-related antigen polypeptide may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 .mu.g, and preferably about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate amount of binding agent.

[0603] Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

[0604] In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

[0605] More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20.TM. (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with a hematological malignancy. Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

[0606] Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM.. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include those groups recited above.

[0607] The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

[0608] To determine the presence or absence of a hematological malignancy, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a hematological malignancy is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the malignancy. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the malignancy. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a malignancy.

[0609] In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of a hematological malignancy. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

[0610] Of course, numerous other assay protocols exist that are suitable for use with the hematological malignancy-related antigen sequences or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use hematological malignancy-related antigen polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of hematological malignancy-related antigen-specific antibodies may correlate with the presence of a hematological.

[0611] A malignancy may also, or alternatively, be detected based on the presence of T cells that specifically react with hematological malignancy-related antigen in a biological sample. Within certain methods, a biological sample comprising CD4.sup.+ and/or CD8.sup.+ T cells isolated from a patient is incubated with a hematological malignancy-related antigen polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses such a polypeptide, and the presence or absence of specific activation of the T cells is detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37.degree. C. with Mtb-81 or Mtb-67.2 polypeptide (e.g., 5-25 .mu.g/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of hematological malignancy-related antigen polypeptide to serve as a control. For CD4.sup.+T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8.sup.+ T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a hematological malignancy in the patient.

[0612] As noted above, a hematological malignancy may also, or alternatively, be detected based on the level of mRNA encoding hematological malignancy-related antigen in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of hematological malignancy-related antigen cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the hematological malignancy-related antigen protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding hematological malignancy-related antigen may be used in a hybridization assay to detect the presence of polynucleotide encoding hematological malignancy-related antigen in a biological sample.

[0613] To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding hematological malignancy-related antigen that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, Stockton Press, N Y, 1989).

[0614] One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample such as a biopsy tissue and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a hematological malignancy. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the sample from a normal individual is typically considered positive.

[0615] In preferred embodiments, such assays may be performed using samples enriched for cells expressing the hematological malignancy-related antigen(s) of interest. Such enrichment may be achieved, for example, using a binding agent as provided herein to remove the cells from the remainder of the biological sample. The removed cells may then be assayed as described above for biological samples.

[0616] In further embodiments, hematological malignancy-related antigens may be used as markers for monitoring disease progression or the response to therapy of a hematological malignancy. In this embodiment, assays as described above for the diagnosis of a hematological malignancy may be performed over time, and the change in the level of reactive polypeptide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a malignancy is progressing in those patients in whom the level of polypeptide detected by the binding agent increases over time. In contrast, the malignancy is not progressing when the level of reactive polypeptide either remains constant or decreases with time.

[0617] Certain in vivo diagnostic assays may be performed directly on a tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.

[0618] As noted above, to improve sensitivity, multiple markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of markers may be based on routine experiments to determine combinations that results in optimal sensitivity.

[0619] Further diagnostic applications include the detection of extramedullary disease (e.g., cerebral infiltration of blasts in leukemias). Within such methods, a binding agent may be coupled to a tracer substance, and the diagnosis is performed in vivo using well known techniques. Coupled binding agent may be administered as described above, and extramedullary disease may be detected based on assaying the presence of tracer substance. Alternatively, a tracer substance may be associated with a T cell specific for hematological malignancy-related antigen, permitting detection of extramedullary disease based on assays to detect the location of the tracer substance.

[0620] 4.27 Exemplary Definitions

[0621] In accordance with the present invention, nucleic acid sequences include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from native sources, chemically synthesized, modified, or otherwise prepared in whole or in part by the hand of man.

[0622] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein. For purposes of the present invention, the following terms are defined below:

[0623] A, an: In accordance with long standing patent law convention, the words "a" and "an" when used in this application, including the claims, denotes "one or more".

[0624] Expression: The combination of intracellular processes, including transcription and translation undergone by a polynucleotide such as a structural gene to synthesize the encoded peptide or polypeptide.

[0625] Promoter: a term used to generally describe the region or regions of a nucleic acid sequence that regulates transcription.

[0626] Regulatory Element: a term used to generally describe the region or regions of a nucleic acid sequence that regulates transcription.

[0627] Structural gene: A gene or sequence region that is expressed to produce an encoded peptide or polypeptide.

[0628] Transformation: A process of introducing an exogenous polynucleotide sequence (e.g., a vector, a recombinant DNA or RNA molecule) into a host cell or protoplast in which that exogenous nucleic acid segment is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and naked nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.

[0629] Transformed cell: A host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.

[0630] Transgenic cell: Any cell derived or regenerated from a transformed cell or derived from a transgenic cell, or from the progeny or offspring of any generation of such a transformed host cell.

[0631] Transgenic animal: An animal or a progeny or an offspring of any generation thereof that is derived from a transformed animal cell, wherein the animal's DNA contains an introduced exogenous nucleic acid molecule not originally present in a native, wild type, non-transgenic animal of the same species. The terms "transgenic animal" and "transformed animal" have sometimes been used in the art as synonymous terms to define an animal, the genetic contents of which has been modified to contain one or more exogenous nucleic acid segments.

[0632] Vector: A nucleic acid molecule, typically comprised of DNA, capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment. A plasmid, cosmid, or a virus is an exemplary vector.

[0633] The terms "substantially corresponds to", "substantially homologous", or "substantial identity" as used herein denotes a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared. The percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, in the case of sequence homology of two or more polynucleotide sequences, the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides. Desirably, which highly homologous fragments are desired, the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).

[0634] The term "naturally occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring. As used herein, laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally occurring animals.

[0635] As used herein, a "heterologous" is defined in relation to a predetermined referenced gene sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation. Likewise, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.

[0636] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.

[0637] As used herein, a "transcription factor recognition site" and a "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s) which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted on the basis of known consensus sequence motifs, or by other methods known to those of skill in the art.

[0638] As used herein, the term "operably linked" refers to a linkage of two or more polynucleotides or two or more nucleic acid sequences in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not contiguous.

[0639] "Transcriptional unit" refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first cis-acting promoter sequence and optionally linked operably to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.

[0640] As noted above, the present invention is generally directed to compositions and methods for using the compositions, for example in the therapy and diagnosis of cancer, such as hematological malignancy. Certain illustrative compositions described herein include hematological malignancy-related tumor polypeptides, polynucleotides encoding such polypeptides, binding agents such as antibodies, antigen presenting cells (APCs) and/or immune system cells (e.g., T cells). A "hematological malignancy-related tumor protein," as the term is used herein, refers generally to a protein that is expressed in hematological malignancy-related tumor cells at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in a normal tissue, as determined using a representative assay provided herein. Certain hematological malignancy-related tumor proteins are tumor proteins that react detectably (within an immunoassay, such as an ELISA or Western blot) with antisera of a patient afflicted with hematological malignancy.

[0641] 4.28 Biological Functional Equivalents

[0642] Modification and changes may be made in the structure of the polynucleotides and peptides of the present invention and still obtain a functional molecule that encodes a peptide with desirable characteristics, or still obtain a genetic construct with the desirable expression specificity and/or properties. As it is often desirable to introduce one or more mutations into a specific polynucleotide sequence, various means of introducing mutations into a polynucleotide or peptide sequence known to those of skill in the art may be employed for the preparation of heterologous sequences that may be introduced into the selected cell or animal species. In certain circumstances, the resulting encoded peptide sequence is altered by this mutation, or in other cases, the sequence of the peptide is unchanged by one or more mutations in the encoding polynucleotide. In other circumstances, one or more changes are introduced into the promoter and/or enhancer regions of the polynucleotide constructs to alter the activity, or specificity of the expression elements and thus alter the expression of the heterologous therapeutic nucleic acid segment operably positioned under the control of the elements.

[0643] When it is desirable to alter the amino acid sequence of one or more of the heterologous peptides encoded by the expression construct to create an equivalent, or even an improved, second-generation molecules, the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 1.

[0644] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC CCC GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA CCC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0645] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0646] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within .+-.2 is preferred, those that are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0647] As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those that are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

[0648] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

5. EXAMPLES

[0649] The following examples are included to demonstrate preferred embodiments of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention described in the appended claims.

5.1 Example 1

Identification of Hematological Malignancy-Related Antigen Polynucleotides

[0650] This Example illustrates the identification of hematological malignancy-related antigen polynucleotides from non-Hodgkin's lymphomas.

[0651] Hematological malignancy-related antigen polynucleotides were isolated by PCR-based subtraction. PolyA mRNA was prepared from T cell non-Hodgkin's lymphomas, B cell non-Hodgkin's lymphomas and normal tissues. Six cDNA libraries were constructed, PCR-subtracted and analyzed. Two libraries were constructed using pools of three T cell non-Hodgkin's lymphoma mRNAs (referred to herein as TCS libraries). Two others were constructed using pools of three B cell non-Hodgkin's lymphoma mRNAs (referred to herein as BCNHL libraries). Two other libraries were constructed using a pool of 2 Hodgkin's lymphoma mRNAs (referred to herein as HLS libraries. cDNA synthesis, hybridization and PCR amplification were performed according to Clontech's user manual (PCR-Select cDNA Subtraction), with the following changes: 1) cDNA was restricted with a mixture of enzymes, including MscI, PvuII, StuI and DraI, instead of the single enzyme RsaI; and 2) the ratio of driver to tester cDNA was increased in the hybridization steps (to 76:1) to give a more stringent subtraction.

[0652] The two TCS libraries were independently subtracted with different pools of driver cDNAs. Driver #1 contained cDNA prepared from specific normal tissues (lymph node, bone marrow, T cells, heart and brain), and this subtraction generated the library TCS-D1 (T cell non-Hodgkin's lymphoma subtracted library with driver #1). Driver #2 contained non-specific normal tissues (colon, large intestine, lung, pancreas, spinal cord, skeletal muscle, liver, kidney, skin and brain), and this subtraction generated the library TCS-D2 (T cell non-Hodgkin's lymphoma subtraction library with driver #2).

[0653] Similarly, the two BCNHL libraries were independently subtracted with different pools of driver cDNAs. Driver #1 contained cDNA prepared from specific normal tissues (lymph node, bone marrow, B cells, heart and brain), and this subtraction generated the library BCNHL/D1 (B cell non-Hodgkin's lymphoma subtracted library with driver #1). Driver #2 contained non-specific normal tissues (brain, lung, pancreas, spinal cord, skeletal muscle, colon, spleen, large intestine and PBMC), and this subtraction generated the library BCNHL/D2 (B cell non-Hodgkin's lymphoma subtraction library with driver #2).

[0654] The two HLS libraries were independently subtracted with different pools of driver cDNAs. Driver #1 contained cDNA prepared from specific normal tissues (lymph node, bone marrow, B cells and lung) and this subtraction generated HLS-D1 (Hodgkin's lymphoma subtraction library with driver #1). Driver #2 contained non-specific normal tissues (colon, large intestine, lung, pancreas, spinal cord, skeletal muscle, liver, kidney, skin and brain) and this generated the library HLS-D2 (Hodgkin's lymphoma subtraction library with driver #2).

[0655] To analyze the efficiency of the subtraction, actin (a housekeeping gene) was PCR amplified from dilutions of subtracted as well as unsubtracted PCR samples. Furthermore, the complexity and redundancy of each library was characterized by sequencing 96 clones from each of the PCR subtraction libraries (TCS-D1, TCS-D2, BCNHL/D1, BCNHL/D2, HLS-D1 and HLS-D2). These analyses indicated that the libraries are enriched for genes overexpressed in leukemia tissues and specifically T cell and B cell non-Hodgkin's lymphoma and M. Hodgkin's lymphoma samples.

[0656] Following PCR amplification, the cDNAs were cloned into the pCR2.1-TOPO plasmid vector (Invitrogen).

[0657] Sequences obtained from these analyses were searched against known sequences in the publicly available databases using the BLAST 2.0 release. The default BLAST parameters used were as follows: GAP PARAMETERS: Open Gap=0, Extended Gap=0; OUTPUT PARAMETERS: Expect=10.0, Threshold=0, Number of Alignments=250; For BLASTN, the search parameters were as follows: Mismatch=-3, Reward=1, Word size=0. The alignments were presented pair-wise, with a window percent identity=22. All available protein and nucleotide databases were searched, including, PIR, SwissPROT, GenBank, Mouse EST, Human EST, Other EST, Human repeat and high throughput sequences, and published patents and patent application database.

[0658] From these, a number of unique sequences were identified that represented novel polynucleotide sequences that had not previously been described in the GenBank and other sequence databases. A number of other sequences were identified that appeared to contain significant homology with one or more sequences previously identified in the databases, although they were described only as genomic or cDNA clones, and had no known function. The remaining sequences corresponded to known genes. The clones obtained from this analysis are summarized in Tables 2-6 in co-pending application U.S. Ser. No. 09/796,692.

5.2 Example 2

Analysis of Subtracted cDNA Sequences by Microarray Analysis

[0659] Subtracted cDNA sequences were analyzed by microarray analysis to evaluate their expression in hematological malignancies and normal tissues. Using this approach, cDNA sequences were PCR amplified and their mRNA expression profiles in hematological malignancies and normal tissues are examined using cDNA microarray technology essentially as described (Shena et al., 1995).

[0660] In brief, the clones identified from the subtracted cDNA libraries analyses were immobilized and arrayed onto glass slides as multiple replicas on microarray slides and the slides were hybridized with two different sets of probes, with each location on the microarray slide corresponding to a unique cDNA clone (as many as 5500 clones can be arrayed on a single slide, or chip). Each chip is hybridized with a pair of cDNA probes that are fluorescence-labeled with Cy3 and Cy5, respectively. The set of probes derived from the hematological malignancies was labeled with cy3 while the other set of probes derived from a pool of normal tissues was labeled with cy5. Typically, 1 .mu.g of polyA.sup.+ RNA was used to generate each cDNA probe. After hybridization, the chips were scanned and the fluorescence intensity recorded for both Cy3 and Cy5 channels. The difference in intensities (i.e., cy3/cy5 ratios) following hybridization with both probe sets provided the information on the relative expression level of each cDNA sequences immobilized on the slide in tumor versus normal tissues. There are multiple built-in quality control steps. First, the probe quality is monitored using a panel of ubiquitously expressed genes. Secondly, the control plate also can include yeast DNA fragments of which complementary RNA may be spiked into the probe synthesis for measuring the quality of the probe and the sensitivity of the analysis. This methodology provides a sensitivity of 1 in 100,000 copies of mRNA, and the reproducibility of the technology may be ensured by including duplicated control cDNA elements at different locations.

[0661] Analysis of hematological malignancy subtracted clones by microarray analyses on a variety of microarray chips identified the sequences set forth in SEQ ID NO: 1 through SEQ ID NO:668 of co-pending application U.S. Ser. No. 09/796,692 as being at least two-fold overexpressed in hematological malignancies versus normal tissues.

5.3 Example 3

Polynucleotide and Polypeptide Compositions: Brief Description of the cDNA Clones and Open Reading Frames Identified by Subtractive Hybridization and Microarray Analysis

[0662] Table 7 in co-pending application U.S. Ser. No. 09/796,692 lists the sequences of the polynucleotides obtained during the analyses of the present invention. Shown are the 668 polynucleotide sequences, along with their clone name identifiers, as well as the serial number and filing date of the priority provisional patent application in which the clone was first identified.

[0663] Table 8 in co-pending application U.S. Ser. No. 09/796,692 identifies the putative open reading frames obtained from analyses of the cDNA sequences obtained in SEQ ID NO:1-SEQ ID NO:668 in the co-pending application. Shown are the sequence identifiers, the clone name and translation frame, and the start and stop nucleotides in the corresponding DNA sequence used to generate the polypeptide sequence of the open reading frame.

[0664] Table 9 in co-pending application U.S. Ser. No. 09/796,692 identifies an additional set of particular hematological malignancy-related cDNA sequences that were obtained using the subtractive library and microarray methods as described above. These sequences, designated SEQ ID NO:2533-SEQ ID NO:9597 in the co-pending application, are shown in the Table along with the original clone name, and the serial number and filing date of the priority provisional application in which the clone was first described.

5.4 Example 4

Additional Analysis of cDNA Clones and Orfs Identified by Subtractive Hybridization and Microarray Analysis

[0665] This example describes microarray analysis of leukemia tumor- and tissue-specific cDNAs.

[0666] Microarray analysis identifies many potential genes that are overexpressed in specific tissues/tumors. However, these genes often represent known genes or genes that subsequently are found by RealTime PCR analysis to have a broader expression profile. This disclosure describes analyses which combine microarray analysis (CorixArray) and comparisons to public databases to identify and prioritize candidate sequences for RealTime analysis, thus allowing the identification of sequences with favorable expression profiles in a more efficient manner.

[0667] Clones are tested for overexpression in lymphoma tumor samples as compared to normal tissues using Corixa Leukemia/Lymphoma Chip#3 (LyC3). The analyzed clones are originally randomly picked from lymphoma PCR subtracted libraries: B-cell non-Hodgkin's lymphoma libraries (BCNHL/D1 and BCNHL/D2; CID000153); T-cell non-Hodgkin's lymphoma libraries (TCS-D1 and TCS-D2; CID000166); Hodgkin's lymphoma libraries (HLS-D1 and HLS-D2; CID000204 and CID000275) and a Clontech-generated T8 leukemia PCR subtracted library. A total of 5184 clones were arrayed: 2304 from BCNHL libraries, 288 from TCS libraries, 1344 from HLS libraries, and 960 from a Clontech-T8 library. In addition, a selection of 288 clones from the above libraries that had been identified from prior leukemia/lymphoma chips were re-analyzed on LyC3.

[0668] cDNA inserts for arraying are amplified by PCR using vector-specific primers. The arrays are probed with 43 probe pairs. Analysis is performed using CorixArray computational analysis. Analysis consisted of determining the ratio of the mean or median hybridization signal for a particular element (cDNA) using two sets of probes. The ratio is a reflection of the over- or under-expression of the element (cDNA) within the probe population. Probe groups are set up to identify elements (cDNAs) with high differential expression in probe group #1. Probe group #1 typically consists of 20 tumor RNAs, each probe representing a subset of lymphoma (e.g., B-cell non-Hodgkins lymphoma, T-cell non-Hodgkins lymphoma and Hodgkins lymphoma). Probes in group #2 include 16 essential and non-essential normal tissues (see, FIG. 4). A threshold (fold-overexpression in probe group #1) is set at 3.0. This threshold is set to identify elements with overexpression that could be reproducibly detected based on the quality of the chip. The sequences are sorted initially based on their CorixArray analysis, specifically on the basis of their mean signal 2 values.

[0669] Sequences having a mean signal 2<0.1 can be considered as sequences with low/no expression in normal tissues. Sequences having a mean signal 2 between 0.1 and 0.2 can be considered as clones with a potential for some expression in normal tissues. Sequences having a mean signal 2>0.2 and can be considered as clones that have the potential to have expression in some normal tissues.

5.5 Example 5

Identification of Candidate Genes with the Same Tissue Expression Profile as CD20 and CD52

[0670] This example identifies leukemia tumor and tissue-specific genes that have similar tissue expression profiles as CD20 and CD52 (FIG. 3). Antibodies against these two markers have been used for the therapy of hematological malignancies and other diseases associated with expression of these markers, i.e., FDA-approved Rituximab (anti-CD20 Ab) and Campath (anti-CD52 Ab). The similarity in gene expression between our candidate genes and CD20 and CD52 suggests that the described genes will also be useful as compounds for the diagnosis and therapy of hematological malignancies and other cancerous and non-cancerous diseases associated with expression of one or more of the described antigens.

[0671] RealTime PCR was used to compare the expression profiles of the candidate genes with the expression profiles of CD20 and CD52. FIG. 8 illustrates the expression of the candidate genes in hematopoietic subsets and hematological malignancies. Data summarized in this sheets shows that using a combination of the PCR subtracted cDNA libraries, microarray analyses, and RealTime PCR, it is possible to identify genes differentially expressed in normal B-cells, lymphomas, myeloma, chronic lymphocytic leukemia, and acute myeloid leukemia.

5.6 Example 6

Analysis of Ly1484 (SEQ ID NOS:16-18 and 120-121), One of the Genes with a Similar Expression Profile as CD20 and CD52

[0672] This example illustrates the typical procedure used to identify antigens for use as therapeutics, diagnostics, etc. and preferred methods for developing therapeutics and diagnostics for leukemia/lymphoma diseases. First, candidate genes highly enriched in leukemia/lymphoma cells are identified using PCR subtraction library cloning. Next, subtracted cDNA sequences are analyzed by microarray analysis to evaluate their expression in hematological malignancies and normal tissues. Since microarray analysis often identifies genes that represent known genes or genes that subsequently are found by RealTime analysis to have broader expression profiles, the microarray analysis is combined with comparisons to public databases to identify and prioritize candidate sequences for analysis. Next, RealTime PCR is used to analyze the expression profiles in various hematological subsets. In some cases, further analysis is focused on antigens with expression profiles similar to known therapeutics. For these genes, structural prediction programs are used to identify transmembrane domains, antigen-specific CTL are generated using human in vitro priming, and humanized monoclonal and polyclonal antibodies are generated as reagents for the diagnosis and therapy of malignancies and autoimmune disorders associated with antigen expression.

[0673] Ly1484P PCR subtraction library clone sequences matched the Genbank clone KIAA1607 (acc. no. XM033378, XM033379 and AB046827) and FJL00111 (acc. no. AK024502). Overexpression of Ly1484 was documented by microarray analyses and RealTime PCR. Ly1484P is overexpressed in B cell neoplasms, while expression in normal tissues is restricted to normal B-cells.

[0674] A full length sequence of candidate Ly1484P was obtained using the Genbank database. Ly1484P was mapped to human chromosome 10. There is both a long and short version of Ly1484P (long version--SEQ ID NO: 120; short version--SEQ ID NO:121). TMpred analysis of Ly1484P indicates that this protein contains a transmembrane domain. Using the TSITES program, T-helper epitopes have also been identified (FIGS. 7 & 8). Polypeptides have been generated and are being used to generate antibodies that are specific for Ly1484P. These humanized monoclonal antibodies may be used (conjugated or unconjugated) for the diagnosis and therapy of malignancies and autoimmune disorders associated with Ly1484 expression.

5.7 Example 7

Identification of Transmembrane Domains

[0675] Structural prediction programs known to those of skill in the art were used to identify transmembrane domains in the candidate antigens described herein.

[0676] For Ly1728P, amino acid residues 6-22, 47-65, 227-243, and 228-245 of SEQ ID NO:2 were identified as putative transmembrane domains.

[0677] For Ly1732P, amino acid residues 60-76 and 55-76 of SEQ ID NO:4 were identified as putative transmembrane domains.

[0678] For Ly1888P, amino acids 1-22, 3-21, 52-68, 254-273, 252-273, and 256-272 of SEQ ID NO: 6 were identified as putative transmembrane domains.

[0679] For Ly1452P amino acids 354-375, 355-377 and 425-441 of SEQ ID NO: 10 (splice variant 1) were identified as putative transmembrane domains and amino acids 354-375, 355-377 and 425-441 of SEQ ID NO: 12 (splice variant 2) were identified as putative transmembrane domains.

[0680] Ly1462P, amino acids 2-23, 3-21, 369-385, 976-999, 977-993, and 979-1000 of SEQ ID NO: 15 were identified as putative transmembrane domains.

[0681] For Ly1484P, amino acids 51-67, 322-338, 666-682, 736-752, 1078-1094, 24-43, 53-69, 118-136, 319-335, 730-752, 1586-1602, 48-69, 88-109, 114-135, 196-217, 300-321, 323-344, 389-410, 502-523, 659-680, 714-735, 1076-1097, 1158-1179, and 1321-1342 of SEQ ID NO: 18 were identified as putative transmembrane domains, amino acids 10-86, 63-84, 118-139, 480-501, 562-583, 725-746, 70-86, 113-129, 134-156, 280-296, 481-497, 560-577, 653-674, 721-738, 734-752, 833-869, 879-895, 990-1006, 1023-1048, 1070-1087, 1112-1138, 1135-1170, and 1146-1170 of SEQ ID NO: 120 were identified as putative transmembrane domains, amino acids 94-129, 102-123, 367-383, 394-423, 447-464, and 493-513, of SEQ ID NO: 121 were identified as putative transmembrane domains.

[0682] For Ly1486P, amino acids 24-40 and 24-45 of SEQ ID NO: 21 were identified as putative transmembrane domains.

[0683] For Ly1693P, amino acids 47-63, 80-96, 114-130, 158-174, 207-223, 237-253, 289-305, 117-134, 144-160, 167-184, 516-535, 44-65, 85-106, 111-132, 150-171, 204-225, 242-263, and 290-311 of SEQ ID NO: 26 were identified as putative transmembrane domains.

[0684] For Ly1715P, amino acids 38-54, 38-56, and 34-55 of SEQ ID NO: 29 were identified as putative transmembrane domains.

[0685] For Ly1727P, amino acids 68-100, 214-234, 304-320, 84-105, 217-238, and 302-323 of SEQ ID NO: 32 were identified as putative transmembrane domains.

[0686] For Ly1905P, amino acids 68-100, 214-234, 84-105, and 217-238 of SEQ ID NO: 40 were identified as putative transmembrane domains.

[0687] For Ly1885P, amino acids 218-234, 219-235, 626-654, and 219-240 of SEQ ID NO: 35 were identified as putative transmembrane domains.

[0688] For Ly663S, amino acids 22-38, 41-57, 60-76, 92-108, 242-258, 20-38, 23-57, 60-77, 86-102, 241-257, 14-35, 89-110, and 248-269 of SEQ ID NO: 43 were identified as putative transmembrane domains.

[0689] For Ly664S, amino acids 11-27, 15-31, 74-93, 209-227, 8-29, and 67-88 of SEQ ID NO: 45 were identified as putative transmembrane domains.

[0690] For Ly667S, amino acids 13-31, 139-157, 184-202, 231-247, 329-351, 435-451, 473-490, 609-626, 685-705, 688-704, 7-28, 233-254, and 685-706 of SEQ ID NO: 48 were identified as putative transmembrane domains.

[0691] For Ly677S, amino acids 149-165, 10-30, 144-165, 7-28, and 144-165 of SEQ ID NO: 54 were identified as putative transmembrane domains.

[0692] For Ly1891P, amino acids 31-47, 66-82, 93-109, 128-144, 171-187, 205-221, 242-258, 31-48, 64-82, 94-111, 120-145, 170-187, 205-221, 244-266, and 29-50, 63-84, 93-114, 124-145, 168-189, 206-227, and 241-262 of SEQ ID NO: 56 were identified as putative transmembrane domains.

[0693] For CD138, amino acids 255-271, 4-21, 258-276, 6-27, and 256-277 of SEQ ID NO: 58 were identified as putative transmembrane domains.

[0694] For CD22, amino acids 688-704, 3-19, 29-46, 157-174, 185-201, 349-366, 386-406, 479-505, 688-709, 1-22, 159-180, and 689-710 of SEQ ID NO: 61 were identified as putative transmembrane domains.

[0695] For CD79beta, amino acids 161-177, 4-29, 59-78, 160-181, 4-25, and 161-180 of SEQ ID NO: 66 were identified as putative transmembrane domains.

[0696] For Ly1454P, amino acids 5-22, 231-249, 7-28, and 425-446 of SEQ ID NO: 74 were identified as putative transmembrane domains. 0.16561 For Ly1485P, amino acid residues 2-19 of SEQ ID NO:76 were identified as a putative transmembrane domain.

[0697] Ly1500P, amino acid residues 10-31 and 327-344 of SEQ ID NO: 80 (splice variant 1) were identified as putative transmembrane domains, amino acid residues 13-38, 71-92, and 388-405 of SEQ ID NO: 82 (splice variant 2) were identified as putative transmembrane domains, and amino acids 25-46 and 341-359 of SEQ ID NO: 84 (splice variant 3) were identified as putative transmembrane domains.

[0698] For Ly1516P, amino acids 142-158, 177-193, 207-223, 238-254, 271-287, 142-158, 177-193, 207-223, 238-254, and 271-287 of SEQ ID NO:87 were identified as putative transmembrane domains.

[0699] For Ly1729P, amino acids 420-436 431-437, and 412-433 of SEQ ID NO:101 were identified as putative transmembrane domains.

[0700] For Ly1859P, amino acid residues 128-144, 293-311, 408-425, 435-454, 465-483, 516-533, 290-311; 435-456, and 507-528 of SEQ ID NO 107 were identified as putative transmembrane domains.

[0701] For Ly1866P, amino acids 47-65 and 50-71 of SEQ ID NO: 109 were identified as putative transmembrane domains.

[0702] For Ly669S, amino acids 489-505, 13-29, 38-57, 73-89, 94-114, 252-268, 307-324, 329-346, 489-509, 4-25, and 486-507 of SEQ ID NO:114 were identified as putative transmembrane domains.

[0703] For Ly672S, amino acids 11-27, 284-300, 325-341, 345-361, 407-423, 7-28, 102-118, 174-198, 283-299, 325-341, 347-383, 403-423, 431-454, 473-492, 11-32, 286-307, 322-343, 345-366, 404-425, 430-451, and 469-490 of SEQ ID NO: 117 were identified as putative transmembrane domains.

[0704] For Ly675S, amino acids 154-170, 187-203, 428-444, 518-534, 846-862, 81-97, 155-172, 235-251, 374-391, 428-444, 477-195, 520-542, 539-573, 694-714, 807-823, 843-862, 50-71, 77-98, 145-166, 518-539, 802-823, and 845-866 of SEQ ID NO:119 were identified as putative transmembrane domains.

5.8 Example 8

Realtime PCR Analysis to Identify Antigens Overexpressed in Chronic Lymphocytic Leukemia and Multiple Myeloma

[0705] Overexpression of candidate antigens in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) was confirmed by RealTime PCR.

[0706] Real-time PCR evaluates the level of PCR product accumulation during amplification (see, e.g., Gibson et al., Genome Research 6:995-1001 (1996); Heid et al., Genome Research 6:986-994 (1996)). RealTime PCR permits quantitative evaluation of mRNA levels in multiple samples. Briefly, mRNA is extracted from tumor and normal tissue and cDNA is prepared using standard techniques. Real-time PCR is performed, for example, using a Perkin Elmer/Applied Biosystems (Foster City, Calif.) 9700 Prism instrument. Matching primers are designed for genes of interest using, for example, the primer express program provided by Perkin Elmer/Applied Biosystems (Foster City, Calif.). Optimal concentrations of primers and probes are initially determined by those of ordinary skill in the art, and control (e.g., ft-actin) primers and probes are obtained commercially from, for example, Perkin Elmer/Applied Biosystems (Foster City, Calif.). To quantitate the amount of specific RNA in a sample, a standard curve is generated using a plasmid containing the gene of interest. Standard curves are generated using the Ct values determined in the real-time PCR, which are related to the initial cDNA concentration used in the assay. Standard dilutions ranging from 10-10.sup.6 copies of the gene of interest are generally sufficient. In addition, a standard curve is generated for the control sequence. This permits standardization of initial RNA content of a tissue sample to the amount of control for comparison purposes.

Summary of Results

TABLE-US-00002 [0707] Antigen CLL MM Ly1728P no yes Ly1732P yes yes Ly1888P yes no Ly 1452P yes no Ly1462P yes no Ly1484P not determined not determined Ly1486P yes no Ly1677P yes no Ly1682P yes no Ly1693P yes yes Ly1697P yes no Ly1715P yes yes Ly1727P yes yes Ly1905P yes yes Ly1885P yes yes Ly663S yes yes Ly664S yes yes Ly667S yes yes Ly677S yes yes Ly1891P not determined not determined CD138 no yes CD22 yes no CD79beta yes yes Ly1450P not determined not determined Ly1451P yes yes Ly1454P yes not determined Ly1485P not determined not determined Ly1500P yes no Ly1516P not determined not determined Ly1678P yes yes Ly1680P yes no Ly1686P yes no Ly1687P yes no Ly1706P yes yes Ly1712P yes yes Ly1729P yes yes Ly1848P yes yes Ly1859P yes no Ly1866P yes yes Ly1867P yes no Ly1868P yes no Ly1886P yes no Ly669S yes yes Ly672S yes yes Ly675S yes yes

[0708] These sequences can conveniently be used to diagnose, treat, and prevent malignant diseases that overexpress these genes, including multiple myeloma, B-cell lymphomas, and B-CLL. For example, monoclonal antibodies, including humanized monoclonal antibodies can be used for diagnosis and therapy of disorders associated with expression of antigens overexpressed in hematological malignancies.

5.9 Example 9

Sequence Analyses, Expression Analyses, and Structure Analyses of Other Antigens with Similar Expression Profiles as CD20 & CD52

Summary of Results

TABLE-US-00003 [0709] Antigen Sequence Analysis Ly1728P FOAP-2 ("novel gene over-expressed in macrophages") Ly1732P B-cell maturation factor (BCM), tumor necrosis factor receptor superfamily, member 17. BCM bins to TALL-1, a member of the TNF family. Ly1888P anti-Fas-induced apoptosis protein (TOSO); experimentally shown to be expressed on the cell surface. Ly 1452P anti-Fas-induced apoptosis protein (TOSO); experimentally shown to be expressed on the cell surface. Ly1462P Human Epstein-Barr virus complement receptor type II Ly1484P KIAA1607 (cDNA sequence present in GenBank). Ly1486P Fc fragment of IgE, low affinity II receptor. Ly1677P novel Ly1682P novel Ly1693P Chemokine receptor CXCR4 Ly1697P novel Ly1715P lectin-like NK cell receptor Ly1727P Splice variants of the hpim-2 gene (homologs of the mouse pim-2 oncogene). Predicted to be a serine threonine kinase with a role in cell proliferation. Ly1905P Splice variants of the hpim-2 gene (homologs of the mouse pim-2 oncogene). Predicted to be a serine threonine kinase with a role in cell proliferation. Ly1885P An apparent splice form of the cell cycle progression 8 protein: one of a family of proteins involved in restoration of cell cycle progression (by blocking arrest in G1 phase). Ly663S leukocyte surface antigen CD37 Ly664S protein with unknown function that shares 30-40% identity with various thioredoxins Ly667S Semaphorin B (semaphorins are a large family of secreted and transmembrane proteins; setna domains occur in the hepatocyte growth factor receptor) Ly677S leukocyte surface recptor CD79A Ly1891P orphan G-protein coupled receptor (GPRC5D) CD138 CD138 CD22 CD22 CD79beta CD79beta Ly1450P novel (matches GenBank seq FLJ23202; no ORF) Ly1451P novel (matches GenBank seq FLJ39358 no ORF) Ly1454P novel (matches GenBank seq FLJ40597; ORF) Ly1485P novel (matches genomic DNA only) Ly1500P BANK protein: B-cell scaffold protein with ankyrin repeats; a substrate for tyrosine phosphorylation upon B-cell antigen receptor stimulation. Ly1516P Rh-related antigen CD47, a signal transducer integrin- associated protein Ly1678P novel (matches genomic DNA only) Ly1680P novel (matches genomic DNA only) Ly1686P novel (matches genornic DNA only) Ly1687P novel (matches genomic DNA only) Ly1706P novel (matches genomic DNA only) Ly1712P novel Ly1729P Hematopoietic cell-specific Lyn substrate 1 (HCLS1) protein; t N-erminal half hs repeats with significant identity to a helix-turn-helix DNA binding motif; the C-terminal half is similar to domains that act as substrates for protein tyrosine kinases suggesting that this protein may be involved in signal transduction and regulation of gene expression. Ly1848P novel (matches genomic DNA only) Ly1859P novel (matches GenBank seq FLJ00140; ORF) Ly1866P Matches a gene in GenBank referred to as "similar to hypothetical protein PR01722." Ly1867P novel (matches genomic DNA only) Ly1868P novel (matches genomic DNA only) Ly1886P novel (matches genomic DNA only) Ly669S intercellular adhesion molecule 3 (ICAM3) Ly672S cisplatin resistance related protein CRR9p Ly675S KIAA0906 (partial cDNA/protein sequences present in GenBank).

5.10 Example 10

Sequence Analysis of Ly1451

[0710] The Ly1451 (SEQ ID NOS:69-71 and 124) sequence derived from a lymphoma PCR subtraction library clone was used to query several public databases, including GenBank and GenSeq. No matches (>90 identity) were detected for the 5'-proximal 51 bp suggesting that this sequence may contain a repeat element. A BLASTN search of the LifeSeq database (Incyte) identified a 980 bp template (template #1076101.8; SEQ ID NO:124 that contained all 240 bp of Ly1451. This template consisted of sequences from 6 clones, of which 2 (33%) were derived from hematologic/immune tissue libraries. Template #1076101.8 was part of a bin containing 11 templates derived from a total of 104 clones, of which 12 (9%) were derived from hematologic/immune tissue libraries.

[0711] This sequence (SEQ ID NO: 124) was used to search further public databases but no additional sequences were obtained. However, these searches indicate this sequence is a human endogenous retroviral sequence (HERV) encoding polypeptides corresponding to portions of the integrase and envelope genes. A single ORF with an ATG translational start site is contained in the forward read of LS1076101.8

[0712] The polypeptide encoded by this ORF (SEQ ID NO:124) is not predicted to have a transmembrane domain.

5.11 Example 11

Expression of Ly1452 Lymphoma Antigens Encoded by a Specific Gene, Ly1452, Associated with B Cell Leukemias, Lymphomas and Multiple Myelomas

[0713] Recombinantly expressed Ly1452 antigens were constructed to allow for quick and easy purification of the protein.

[0714] The open reading frame for Ly1452 was PCR amplified and subcloned into a modified pET28 vector with a His tag in-frame and recombinantly expressed in E. coli (His-Ly1452: SEQ ID NO:10,482 (nt), SEQ ID NO:10,483 (protein).

Ly1452P expression in E. coli

[0715] The open reading frame of the LS coding region was PCR amplified with the following primers:

TABLE-US-00004 PDM-797 (SEQ ID NO: 10,975) 5'gtgtcacaatctacagtcaggcaggattctcc 3' Tin 64.degree. C. PDM-799 (SEQ ID NO: 10,976) 5'gttatgtagcggccgcttatcatgttgctgcagag 3' Tm 67.degree. C.

[0716] Using the following conditions:

10 .mu.l 10.times. Herculase buffer 1 .mu.l 10 mM dNTPs 2 .mu.l 10 .mu.M each oligo 83 .mu.l sterile water 1.0 .mu.l Herculase DNA polymerase (Stratagene, La Jolla, Calif.)

50 .eta.g DNA

TABLE-US-00005 [0717] 98.degree. C. 3 minutes 98.degree. C. 40 seconds 60.degree. C. 30 seconds 72.degree. C. 2 minute .times. 10 cycles 98.degree. C. 40 seconds 60.degree. C. 30 seconds 72.degree. C. 2 minutes 30 seconds .times. 10 cycles 98.degree. C. 40 seconds 60.degree. C. 30 seconds 72.degree. C. 3 minutes .times. 10 cycles 98.degree. C. 40 seconds 60.degree. C. 30 seconds 72.degree. C. 3 minutes 30 seconds .times. 10 cycles 72.degree. C. 4 minutes

[0718] The PCR product was digested with Xho I and cloned into pPDM His (a modified pET28 vector with a His tag in frame on the 5' end) that had been digested with Eco72I and XhoI. Construct was confirmed through sequence analysis and transformed into BLR (DE3) pLysS and HMS 174 pLys S cells.

[0719] Recombinant proteins are also expressed without a histidine tag or with other lymphoma antigens. They are also expressed in other vectors, including other E. coli constructs, Baculovirus, yeast, and mammalian expression vectors. This recombinant antigen can be used to make polyclonal and monoclonal antibodies or used in immunological assays.

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[1075] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Accordingly, the exclusive rights sought to be patented are as described in the claims below:

Sequence CWU 1

1

12412672DNAHomo sapiens 1cttccagaga gcaatatggc tggttcccca acatgcctca ccctcatcta tatcctttgg 60cagctcacag ggtcagcagc ctctggaccc gtgaaagagc tggtcggttc cgttggtggg 120gccgtgactt tccccctgaa gtccaaagta aagcaagttg actctattgt ctggaccttc 180aacacaaccc ctcttgtcac catacagcca gaagggggca ctatcatagt gacccaaaat 240cgtaataggg agagagtaga cttcccagat ggaggctact ccctgaagct cagcaaactg 300aagaagaatg actcagggat ctactatgtg gggatataca gctcatcact ccagcagccc 360tccacccagg agtacgtgct gcatgtctac gagcacctgt caaagcctaa agtcaccatg 420ggtctgcaga gcaataagaa tggcacctgt gtgaccaatc tgacatgctg catggaacat 480ggggaagagg atgtgattta tacctggaag gccctggggc aagcagccaa tgagtcccat 540aatgggtcca tcctccccat ctcctggaga tggggagaaa gtgatatgac cttcatctgc 600gttgccagga accctgtcag cagaaacttc tcaagcccca tccttgccag gaagctctgt 660gaaggtgctg ctgatgaccc agattcctcc atggtcctcc tgtgtctcct gttggtgccc 720ctcctgctca gtctctttgt actggggcta tttctttggt ttctgaagag agagagacaa 780gaagagtaca ttgaagagaa gaagagagtg gacatttgtc gggaaactcc taacatatgc 840ccccattctg gagagaacac agagtacgac acaatccctc acactaatag aacaatccta 900aaggaagatc cagcaaatac ggtttactcc actgtggaaa taccgaaaaa gatggaaaat 960ccccactcac tgctcacgat gccagacaca ccaaggctat ttgcctatga gaatgttatc 1020tagacagcag tgcactcccc taagtctctg ctcaaaaaaa aaacaattct cggcccaaag 1080aaaacaatca gaagaattca ctgatttgac tagaaacatc aaggaagaat gaagaacgtt 1140gacttttttc caggataaat tatctctgat gcttctttag atttaagagt tcataattcc 1200atccactgct gagaaatctc ctcaaaccca gaaggtttaa tcacttcatc ccaaaaatgg 1260gattgtgaat gtcagcaaac cataaaaaaa gtgcttagaa gtattcctat agaaatgtaa 1320atgcaaggtc acacatatta atgacagcct gttgtattaa tgatggctcc aggtcagtgt 1380ctggagtttc attccatccc agggcttgga tgtaaggatt ataccaagag tcttgctacc 1440aggagggcaa gaagaccaaa acagacagac aagtccagca gaagcagatg cacctgacaa 1500aaatggatgt attaattggc tctataaact atgtgcccag cactatgctg agcttacact 1560aattggtcag acgtgctgtc tgccctcatg aaattggctc caaatgaatg aactactttc 1620atgagcagtt gtagcaggcc tgaccacaga ttcccagagg gccaggtgtg gatccacagg 1680acttgaaggt caaagttcac aaagatgaag aatcagggta gctgaccatg tttggcagat 1740actataatgg agacacagaa gtgtgcatgg cccaaggaca aggacctcca gccaggcttc 1800atttatgcac ttgtgctgca aaagaaaagt ctaggtttta aggctgtgcc agaacccatc 1860ccaataaaga gaccgagtct gaagtcacat tgtaaatcta gtgtaggaga cttggagtca 1920ggcagtgaga ctggtggggc acggggggca gtgggtactt gtaaaccttt aaagatggtt 1980aattcattca atagatattt attaagaacc tatgcggccc ggcatggtgg ctcacacctg 2040taatcccagc actttgggag gccaaggtgg gtgggtcatc tgaggtcagg agttcaagac 2100cagcctggcc aacatggtga aaccccatct ctactaaaga tacaaaaatt tgctgagcgt 2160ggtggtgtgc acctgtaatc ccagctactc gagaggccaa ggcatgagaa tcgcttgaac 2220ctgggaggtg gaggttgcag tgagctgaga tggcaccact gcactccggc ctaggcaacg 2280agagcaaaac tccaatacaa acaaacaaac aaacacctgt gctaggtcag tctggcacgt 2340aagatgaaca tccctaccaa cacagagctc accatctctt atacttaagt gaaaaacatg 2400gggaagggga aaggggaatg gctgcttttg atatgttccc tgacacatat cttgaatgga 2460gacctcccta ccaagtgatg aaagtgttga aaaacttaat aacaaatgct tgttgggcaa 2520gaatgggatt gaggattatc ttctctcaga aaggcattgt gaaggaattg agccagatct 2580ctctccctac tgcaaaaccc tattgtagta aaaaagtctt ctttactatc ttaataaaac 2640agatattgtg agattcaaaa aaaaaaaaaa aa 26722335PRTHomo sapiens 2Met Ala Gly Ser Pro Thr Cys Leu Thr Leu Ile Tyr Ile Leu Trp Gln1 5 10 15Leu Thr Gly Ser Ala Ala Ser Gly Pro Val Lys Glu Leu Val Gly Ser 20 25 30Val Gly Gly Ala Val Thr Phe Pro Leu Lys Ser Lys Val Lys Gln Val 35 40 45Asp Ser Ile Val Trp Thr Phe Asn Thr Thr Pro Leu Val Thr Ile Gln 50 55 60Pro Glu Gly Gly Thr Ile Ile Val Thr Gln Asn Arg Asn Arg Glu Arg65 70 75 80Val Asp Phe Pro Asp Gly Gly Tyr Ser Leu Lys Leu Ser Lys Leu Lys 85 90 95Lys Asn Asp Ser Gly Ile Tyr Tyr Val Gly Ile Tyr Ser Ser Ser Leu 100 105 110Gln Gln Pro Ser Thr Gln Glu Tyr Val Leu His Val Tyr Glu His Leu 115 120 125Ser Lys Pro Lys Val Thr Met Gly Leu Gln Ser Asn Lys Asn Gly Thr 130 135 140Cys Val Thr Asn Leu Thr Cys Cys Met Glu His Gly Glu Glu Asp Val145 150 155 160Ile Tyr Thr Trp Lys Ala Leu Gly Gln Ala Ala Asn Glu Ser His Asn 165 170 175Gly Ser Ile Leu Pro Ile Ser Trp Arg Trp Gly Glu Ser Asp Met Thr 180 185 190Phe Ile Cys Val Ala Arg Asn Pro Val Ser Arg Asn Phe Ser Ser Pro 195 200 205Ile Leu Ala Arg Lys Leu Cys Glu Gly Ala Ala Asp Asp Pro Asp Ser 210 215 220Ser Met Val Leu Leu Cys Leu Leu Leu Val Pro Leu Leu Leu Ser Leu225 230 235 240Phe Val Leu Gly Leu Phe Leu Trp Phe Leu Lys Arg Glu Arg Gln Glu 245 250 255Glu Tyr Ile Glu Glu Lys Lys Arg Val Asp Ile Cys Arg Glu Thr Pro 260 265 270Asn Ile Cys Pro His Ser Gly Glu Asn Thr Glu Tyr Asp Thr Ile Pro 275 280 285His Thr Asn Arg Thr Ile Leu Lys Glu Asp Pro Ala Asn Thr Val Tyr 290 295 300Ser Thr Val Glu Ile Pro Lys Lys Met Glu Asn Pro His Ser Leu Leu305 310 315 320Thr Met Pro Asp Thr Pro Arg Leu Phe Ala Tyr Glu Asn Val Ile 325 330 3353834DNAHomo sapiens 3ttgtaagata ttacttgtcc ttccaggctg ttctttctgt agctcccttg ttttcttttt 60gtgatcatgt tgcagatggc tgggcagtgc tcccaaaatg aatattttga cagtttgttg 120catgcttgca taccttgtca acttcgatgt tcttctaata ctcctcctct aacatgtcag 180cgttattgta atgcaagtgt gaccaattca gtgaaaggaa cgaatgcgat tctctggacc 240tgtttgggac tgagcttaat aatttctttg gcagttttcg tgctaatgtt tttgctaagg 300aagataagct ctgaaccatt aaaggacgag tttaaaaaca caggatcagg tctcctgggc 360atggctaaca ttgacctgga aaagagcagg actggtgatg aaattattct tccgagaggc 420ctcgagtaca cggtggaaga atgcacctgt gaagactgca tcaagagcaa accgaaggtc 480gactctgacc attgctttcc actcccagct atggaggaag gcgcaaccat tcttgtcacc 540acgaaaacga atgactattg caagagcctg ccagctgctt tgagtgctac ggagatagag 600aaatcaattt ctgctaggta attaaccatt tcgactcgag cagtgccact ttaaaaatct 660tttgtcagaa tagatgatgt gtcagatctc tttaggatga ctgtattttt cagttgccga 720tacagctttt tgtcctctaa ctgtggaaac tctttatgtt agatatattt ctctaggtta 780ctgttgggag cttaatggta gaaacttcct tggtttctat gattaaagtc tttt 8344184PRTHomo sapiens 4Met Leu Gln Met Ala Gly Gln Cys Ser Gln Asn Glu Tyr Phe Asp Ser1 5 10 15Leu Leu His Ala Cys Ile Pro Cys Gln Leu Arg Cys Ser Ser Asn Thr 20 25 30Pro Pro Leu Thr Cys Gln Arg Tyr Cys Asn Ala Ser Val Thr Asn Ser 35 40 45Val Lys Gly Thr Asn Ala Ile Leu Trp Thr Cys Leu Gly Leu Ser Leu 50 55 60Ile Ile Ser Leu Ala Val Phe Val Leu Met Phe Leu Leu Arg Lys Ile65 70 75 80Ser Ser Glu Pro Leu Lys Asp Glu Phe Lys Asn Thr Gly Ser Gly Leu 85 90 95Leu Gly Met Ala Asn Ile Asp Leu Glu Lys Ser Arg Thr Gly Asp Glu 100 105 110Ile Ile Leu Pro Arg Gly Leu Glu Tyr Thr Val Glu Glu Cys Thr Cys 115 120 125Glu Asp Cys Ile Lys Ser Lys Pro Lys Val Asp Ser Asp His Cys Phe 130 135 140Pro Leu Pro Ala Met Glu Glu Gly Ala Thr Ile Leu Val Thr Thr Lys145 150 155 160Thr Asn Asp Tyr Cys Lys Ser Leu Pro Ala Ala Leu Ser Ala Thr Glu 165 170 175Ile Glu Lys Ser Ile Ser Ala Arg 18051339DNAHomo sapiens 5ttgcactcta gaagggacaa tggacttctg gctttggcca ctttacttcc tgccagtatc 60gggggccctg aggatcctcc cagaagtaaa ggtagagggg gagctgggcg gatcagttac 120catcaagtgc ccacttcctg aaatgcatgt gaggatatat ctgtgccggg agatggctgg 180atctggaaca tgtggtaccg tggtatccac caccaacttc atcaaggcag aatacaaggg 240ccgagttact ctgaagcaat acccacgcaa gaatctgttc ctagtggagg taacacagct 300gacagaaagt gacagcggag tctatgcctg cggagcgggc atgaacacag accggggaaa 360gacccagaaa gtcaccctga atgtccacag tgaatacgag ccatcatggg aagagcagcc 420aatgcctgag actccaaaat ggtttcatct gccctatttg ttccagatgc ctgcatatgc 480cagttcttcc aaattcgtaa ccagagttac cacaccagct caaaggggca aggtccctcc 540agttcaccac tcctccccca ccacccaaat cacccaccgc cctcgagtgt ccagagcatc 600ttcagtagca ggtgacaagc cccgaacctt cctgccatcc actacagcct caaaaatctc 660agctctggag gggctgctca agccccagac gcccagctac aaccaccaca ccaggctgca 720caggcagaga gcactggact atggctcaca gtctgggagg gaaggccaag gatttcacat 780cctgatcccg accatcctgg gccttttcct gctggcactt ctggggctgg tggtgaaaag 840ggccgttgaa aggaggaaag ccctctccag gcgggcccgc cgactggccg tgaggatgcg 900cgccctggag agctcccaga ggccccgcgg gtcgccgcga ccgcgctccc aaaacaacat 960ctacagcgcc tgcccgcggc gcgctcgtgg agcggacgct gcaggcacag gggaagcccc 1020cgttcccggc cccggagcgc cgttgccccc cgccccgctg caggtgtctg aatctccctg 1080gctccatgcc ccatctctga agaccagctg tgaatacgtg agcctctacc accagcctgc 1140cgccatgatg gaggacagtg attcagatga ctacatcaat gttcctgcct gacaactccc 1200cagctatccc ccaaccccag gctcggactg tggtgccaag gagtctcatc tatctgctga 1260tgtccaatac ctgcttcatg tgttctcaga gccctcatca ttcccatgcc ccatctcgat 1320cccatcccca tctatctgt 13396390PRTHomo sapiens 6Met Asp Phe Trp Leu Trp Pro Leu Tyr Phe Leu Pro Val Ser Gly Ala1 5 10 15Leu Arg Ile Leu Pro Glu Val Lys Val Glu Gly Glu Leu Gly Gly Ser 20 25 30Val Thr Ile Lys Cys Pro Leu Pro Glu Met His Val Arg Ile Tyr Leu 35 40 45Cys Arg Glu Met Ala Gly Ser Gly Thr Cys Gly Thr Val Val Ser Thr 50 55 60Thr Asn Phe Ile Lys Ala Glu Tyr Lys Gly Arg Val Thr Leu Lys Gln65 70 75 80Tyr Pro Arg Lys Asn Leu Phe Leu Val Glu Val Thr Gln Leu Thr Glu 85 90 95Ser Asp Ser Gly Val Tyr Ala Cys Gly Ala Gly Met Asn Thr Asp Arg 100 105 110Gly Lys Thr Gln Lys Val Thr Leu Asn Val His Ser Glu Tyr Glu Pro 115 120 125Ser Trp Glu Glu Gln Pro Met Pro Glu Thr Pro Lys Trp Phe His Leu 130 135 140Pro Tyr Leu Phe Gln Met Pro Ala Tyr Ala Ser Ser Ser Lys Phe Val145 150 155 160Thr Arg Val Thr Thr Pro Ala Gln Arg Gly Lys Val Pro Pro Val His 165 170 175His Ser Ser Pro Thr Thr Gln Ile Thr His Arg Pro Arg Val Ser Arg 180 185 190Ala Ser Ser Val Ala Gly Asp Lys Pro Arg Thr Phe Leu Pro Ser Thr 195 200 205Thr Ala Ser Lys Ile Ser Ala Leu Glu Gly Leu Leu Lys Pro Gln Thr 210 215 220Pro Ser Tyr Asn His His Thr Arg Leu His Arg Gln Arg Ala Leu Asp225 230 235 240Tyr Gly Ser Gln Ser Gly Arg Glu Gly Gln Gly Phe His Ile Leu Ile 245 250 255Pro Thr Ile Leu Gly Leu Phe Leu Leu Ala Leu Leu Gly Leu Val Val 260 265 270Lys Arg Ala Val Glu Arg Arg Lys Ala Leu Ser Arg Arg Ala Arg Arg 275 280 285Leu Ala Val Arg Met Arg Ala Leu Glu Ser Ser Gln Arg Pro Arg Gly 290 295 300Ser Pro Arg Pro Arg Ser Gln Asn Asn Ile Tyr Ser Ala Cys Pro Arg305 310 315 320Arg Ala Arg Gly Ala Asp Ala Ala Gly Thr Gly Glu Ala Pro Val Pro 325 330 335Gly Pro Gly Ala Pro Leu Pro Pro Ala Pro Leu Gln Val Ser Glu Ser 340 345 350Pro Trp Leu His Ala Pro Ser Leu Lys Thr Ser Cys Glu Tyr Val Ser 355 360 365Leu Tyr His Gln Pro Ala Ala Met Met Glu Asp Ser Asp Ser Asp Asp 370 375 380Tyr Ile Asn Val Pro Ala385 39072007DNAArtificial SequenceDescription of Artificial SequenceLy1452 open reading frame His tag fusion 7atgcagcatc accaccatca ccacgtgtca caatctacag tcaggcagga ttctcctgtg 60gagccctggg aagggatcag cgatcactct ggcattattg atggttcgcc cagactcctg 120aacactgacc atcctccttg ccaattagac atcaggctca tgaggcacaa agctgtctgg 180attaaccccc aggatgtgca gcaacagccg caggacttgc aatctcaggt gccagcagca 240gggaacagtg ggacccattt tgtgacagat gctgcctctc cctcaggccc ttcaccttcg 300tgcctcgggg actccctggc agagacaacg ttgtctgagg ataccacaga ctccgttggc 360agcgcttctc cccatggctc gagtgaaaag agtagcagct tctctctgtc ctcaacagag 420gtacacatgg tccgcccagg atactctcat cgggtgtctc tgcccacaag ccctgggatt 480ttggccacct ccccatatcc tgagactgac agtgcttttt ttgagccttc ccatctgaca 540tctgctgctg atgaaggtgc tgttcaagtc agtagaagaa ccatttcttc gaattccttc 600tcaccagagg tatttgtgct gcctgttgat gtagaaaagg aaaatgccca cttttatgtt 660gcagatatga ttatatcagc aatggagaaa atgaagtgta acattctgag tcaacagcag 720acagagagct ggagtaaaga agtcagtggg ttacttggga gtgatcagcc tgactctgaa 780atgacttttg ataccaacat aaagcaagag tctgggtctt ctacttcttc atacagtggc 840tatgaaggtt gtgctgtgtt acaggtcagc ccagtgactg aaacacgtac ttaccatgat 900gtgaaagaga tttgcaaatg cgatgttgat gaatttgtta ttttagagct tggagatttt 960aatgatatca cagaaacctg tagctgttcc tgcagctcct ctaagagtgt cacttatgag 1020ccagacttca attctgcaga actattagcc aaagagctgt accgcgtgtt ccagaagtgc 1080tggatactgt cagtagttaa ttctcagctg gcaggttccc tgagtgcagc tggctcgata 1140gtcgtaaatg aagagtgtgt ccgaaaagac tttgaatcca gtatgaatgt agtacaggaa 1200attaaattta agtctaggat cagagggact gaagactggg ctcctcctag atttcaaatc 1260atatttaata ttcatccacc actcaagagg gaccttgtgg tggcagccca gaattttttc 1320tgtgccggct gtggaactcc agtagagcct aagtttgtga agcggctccg gtactgcgaa 1380tacctaggga agtatttctg tgactgctgc cactcatatg cagagtcgtg catccctgcc 1440cgaatcctga tgatgtggga cttcaagaag tactacgtca gcaatttctc caaacagctg 1500ctcgacagca tatggcacca gcccattttc aatttgctga gcatcggcca aagcctgtat 1560gcgaaagcca aggagctgga cagagtgaag gaaattcagg agcagctctt ccatatcaag 1620aagctgttga agacctgtag gtttgctaac agtgcattaa aggagttcga gcaggtgccg 1680ggacacttga ctgatgagct ccacctgttc tcccttgagg acctggtcag gatcaagaaa 1740gggctgctgg cacccttact caaggacatt ctgaaagctt cccttgcaca tgtggctggc 1800tgtgagctgt gtcaaggaaa gggctttatt tgtgaatttt gccagaatac gactgtcatc 1860ttcccatttc agacagcaac atgtagaaga tgttcagcgt gcagggcttg ctttcacaaa 1920cagtgcttcc agtcctccga gtgcccccgg tgtgcgagga tcacagcgag gagaaaactt 1980ctggaaagtg tggcctctgc agcaaca 20078669PRTArtificial SequenceDescription of Artificial SequenceLy1452 open reading frame His tag fusion 8Met Gln His His His His His His Val Ser Gln Ser Thr Val Arg Gln1 5 10 15Asp Ser Pro Val Glu Pro Trp Glu Gly Ile Ser Asp His Ser Gly Ile 20 25 30Ile Asp Gly Ser Pro Arg Leu Leu Asn Thr Asp His Pro Pro Cys Gln 35 40 45Leu Asp Ile Arg Leu Met Arg His Lys Ala Val Trp Ile Asn Pro Gln 50 55 60Asp Val Gln Gln Gln Pro Gln Asp Leu Gln Ser Gln Val Pro Ala Ala65 70 75 80Gly Asn Ser Gly Thr His Phe Val Thr Asp Ala Ala Ser Pro Ser Gly 85 90 95Pro Ser Pro Ser Cys Leu Gly Asp Ser Leu Ala Glu Thr Thr Leu Ser 100 105 110Glu Asp Thr Thr Asp Ser Val Gly Ser Ala Ser Pro His Gly Ser Ser 115 120 125Glu Lys Ser Ser Ser Phe Ser Leu Ser Ser Thr Glu Val His Met Val 130 135 140Arg Pro Gly Tyr Ser His Arg Val Ser Leu Pro Thr Ser Pro Gly Ile145 150 155 160Leu Ala Thr Ser Pro Tyr Pro Glu Thr Asp Ser Ala Phe Phe Glu Pro 165 170 175Ser His Leu Thr Ser Ala Ala Asp Glu Gly Ala Val Gln Val Ser Arg 180 185 190Arg Thr Ile Ser Ser Asn Ser Phe Ser Pro Glu Val Phe Val Leu Pro 195 200 205Val Asp Val Glu Lys Glu Asn Ala His Phe Tyr Val Ala Asp Met Ile 210 215 220Ile Ser Ala Met Glu Lys Met Lys Cys Asn Ile Leu Ser Gln Gln Gln225 230 235 240Thr Glu Ser Trp Ser Lys Glu Val Ser Gly Leu Leu Gly Ser Asp Gln 245 250 255Pro Asp Ser Glu Met Thr Phe Asp Thr Asn Ile Lys Gln Glu Ser Gly 260 265 270Ser Ser Thr Ser Ser Tyr Ser Gly Tyr Glu Gly Cys Ala Val Leu Gln 275 280 285Val Ser Pro Val Thr Glu Thr Arg Thr Tyr His Asp Val Lys Glu Ile 290 295 300Cys Lys Cys Asp Val Asp Glu Phe Val Ile Leu Glu Leu Gly Asp Phe305 310 315 320Asn Asp Ile Thr Glu Thr Cys Ser Cys Ser Cys Ser Ser Ser Lys Ser 325 330 335Val Thr Tyr Glu Pro Asp Phe Asn Ser Ala Glu Leu Leu Ala Lys Glu 340 345 350Leu Tyr Arg Val Phe Gln Lys Cys Trp Ile Leu Ser Val Val Asn Ser 355 360 365Gln Leu Ala Gly Ser Leu Ser Ala Ala

Gly Ser Ile Val Val Asn Glu 370 375 380Glu Cys Val Arg Lys Asp Phe Glu Ser Ser Met Asn Val Val Gln Glu385 390 395 400Ile Lys Phe Lys Ser Arg Ile Arg Gly Thr Glu Asp Trp Ala Pro Pro 405 410 415Arg Phe Gln Ile Ile Phe Asn Ile His Pro Pro Leu Lys Arg Asp Leu 420 425 430Val Val Ala Ala Gln Asn Phe Phe Cys Ala Gly Cys Gly Thr Pro Val 435 440 445Glu Pro Lys Phe Val Lys Arg Leu Arg Tyr Cys Glu Tyr Leu Gly Lys 450 455 460Tyr Phe Cys Asp Cys Cys His Ser Tyr Ala Glu Ser Cys Ile Pro Ala465 470 475 480Arg Ile Leu Met Met Trp Asp Phe Lys Lys Tyr Tyr Val Ser Asn Phe 485 490 495Ser Lys Gln Leu Leu Asp Ser Ile Trp His Gln Pro Ile Phe Asn Leu 500 505 510Leu Ser Ile Gly Gln Ser Leu Tyr Ala Lys Ala Lys Glu Leu Asp Arg 515 520 525Val Lys Glu Ile Gln Glu Gln Leu Phe His Ile Lys Lys Leu Leu Lys 530 535 540Thr Cys Arg Phe Ala Asn Ser Ala Leu Lys Glu Phe Glu Gln Val Pro545 550 555 560Gly His Leu Thr Asp Glu Leu His Leu Phe Ser Leu Glu Asp Leu Val 565 570 575Arg Ile Lys Lys Gly Leu Leu Ala Pro Leu Leu Lys Asp Ile Leu Lys 580 585 590Ala Ser Leu Ala His Val Ala Gly Cys Glu Leu Cys Gln Gly Lys Gly 595 600 605Phe Ile Cys Glu Phe Cys Gln Asn Thr Thr Val Ile Phe Pro Phe Gln 610 615 620Thr Ala Thr Cys Arg Arg Cys Ser Ala Cys Arg Ala Cys Phe His Lys625 630 635 640Gln Cys Phe Gln Ser Ser Glu Cys Pro Arg Cys Ala Arg Ile Thr Ala 645 650 655Arg Arg Lys Leu Leu Glu Ser Val Ala Ser Ala Ala Thr 660 66593604DNAHomo sapiens 9gggagcctat agggttcaat aacttgcagt gtggttgggg cttatggatg ctggatgaag 60ataagtgagg gaggtttaca gggacagcat catgtcaggc cttgagggca agaatagctc 120tccagacccc cagctggcca tgtggtgagt tcagggccca aatcaagtag taccagcaat 180cagggaactc ctatctgttt tgaatggatt cacaccagcc acaagcctgg aaagatggtg 240tcacaatcta cagtcaggca ggattctcct gtggagccct gggaagggat cagcgatcac 300tctggcatta ttgatggttc gcccagactc ctgaacactg accatcctcc ttgccaatta 360gacatcaggc tcatgaggca caaagctgtc tggattaacc cccaggatgt gcagcaacag 420ccgcaggact tgcaatctca ggtgccagca gcagggaaca gtgggaccca ttttgtgaca 480gatgctgcct ctccctcagg cccttcacct tcgtgcctcg gggactccct ggcagagaca 540acgttgtctg aggataccac agactccgtt ggcagcgctt ctccccatgg ctcgagtgaa 600aagagtagca gcttctctct gtcctcaaca gaggtacaca tggtccgccc aggatactct 660catcgggtgt ctctgcccac aagccctggg attttggcca cctccccata tcctgagact 720gacagtgctt tttttgagcc ttcccatctg acatctgctg ctgatgaagg tgctgttcaa 780gtcagtagaa gaaccatttc ttcgaattcc ttctcaccag aggtatttgt gctgcctgtt 840gatgtagaaa aggaaaatgc ccacttttat gttgcagata tgattatatc agcaatggag 900aaaatgaagt gtaacattct gagtcaacag cagacagaga gctggagtaa agaagtcagt 960gggttacttg ggagtgatca gcctgactct gaaatgactt ttgataccaa cataaagcaa 1020gagtctgggt cttctacttc ttcatacagt ggctatgaag gttgtgctgt gttacaggtc 1080agcccagtga ctgaaacacg tacttaccat gatgtgaaag agatttgcaa atgcgatgtt 1140gatgaatttg ttattttaga gcttggagat tttaatgata tcacagaaac ctgtagctgt 1200tcctgcagct cctctaagag tgtcacttat gagccagact tcaattctgc agaactatta 1260gccaaagagc tgtaccgcgt gttccagaag tgctggatac tgtcagtagt taattctcag 1320ctggcaggtt ccctgagtgc agctggctcg atagtcgtaa atgaagagtg tgtccgaaaa 1380gactttgaat ccagtatgaa tgtagtacag gaaattaaat ttaagtctag gatcagaggg 1440actgaagact gggctcctcc tagatttcaa atcatattta atattcatcc accactcaag 1500agggaccttg tggtggcagc ccagaatttt ttctgtgccg gctgtggaac tccagtagag 1560cctaagtttg tgaagcggct ccggtactgc gaatacctag ggaagtattt ctgtgactgc 1620tgccactcat atgcagagtc gtgcatccct gcccgaatcc tgatgatgtg ggacttcaag 1680aagtactacg tcagcaattt ctccaaacag ctgctcgaca gcatatggca ccagcccatt 1740ttcaatttgc tgagcatcgg ccaaagcctg tatgcgaaag ccaaggagct ggacagagtg 1800aaggaaattc aggagcagct cttccatatc aagaagctgt tgaagacctg taggtttgct 1860aacagtgcat taaaggagtt cgagcaggtg ccgggacact tgactgatga gctccacctg 1920ttctcccttg aggacctggt caggatcaag aaagggctgc tggcaccctt actcaaggac 1980attctgaaag cttcccttgc acatgtggct ggctgtgagc tgtgtcaagg aaagggcttt 2040atttgtgaat tttgccagaa tacgactgtc atcttcccat ttcagacagc aacatgtaga 2100agatgttcag cgtgcagggc ttgctttcac aaacagtgct tccagtcctc cgagtgcccc 2160cggtgtgcga ggatcacagc gaggagaaaa cttctggaaa gtgtggcctc tgcagcaaca 2220tgatgcccct gagtactgtg aaaaagactg ttcaacatgc cttatgataa caccgatttg 2280tgtctattat tggtgacatt gttttagata ttgggtattg tatattaagg aaaaagatgg 2340tctatattct ctttattgca tatacttaat gtttcaaaag aatgcagatt ctgtgtttaa 2400gcacagggct gatagttgtg gttttgttta caaatgttct gttttggctg ctattggttt 2460tttaaagagg ttttttatac ttttgtattt gaatagttat gtttcactga tgctgagcca 2520gtttgtatgt gtgtgcatat atgtgaactg taactgacaa gatgaattac tcagtttctc 2580tttctctaaa gcttgtttga tgaaactggt tggtcctttc agtgaacaaa aatatgaccc 2640caaatctgtt tgctctggct tttatttctt caggaagcag acttccactt aaatgccatt 2700ttgtgattgt gtcaatcata cacattttat ttacttcaga gtttgaatag agagtacaca 2760tttcttctgc agatttattt catgatgagt ttgagttgct tagcagggcg tgtgggtccc 2820gttgaagtgc agtttgaagc aactgcttct agatggcact ctttcaggtg gcacaaattg 2880aacctgtatt tgtcatctct gttccacaca ctgcaatgtc aagggatgca gaagtgagta 2940gaattccatc cctgcccttg aggatcttgc tttaacagat gtaaaactga acataaggta 3000tttgcagatt taaacgaact gggggaaata atgaacagtg tgattctagt aataacatta 3060aaatcataga cattgactaa taaggttaaa tgaatcacaa aacctttatg aatttctttt 3120ttctaatagt tcttatatgt tttcctgaaa catgtgagcc tattcttttt tcttctactt 3180tctatatact ttctcccact tgagaaaggg gccttgaggc tgggtccctt catggtatac 3240ctttagactg aacggtttgc aacctagggc ttgggcatta cattccctgg gattcacatg 3300ccctaactaa acctaccttg attttctcag acagcacagg caggcaataa agcgtcacag 3360attgtcccct aaccccatcc agccatgtgt atgagtgtgt tttattcaat gggatagtac 3420tgagcacatg aaagaaatga atgacttctg tcaatctctt ttcattcagt cttctcattc 3480tgtcaattgt tttctcatcc gcagtgcctc tgccagaact gtgctcacat ccattattta 3540agccagatct tttctaagta ttatagaagt gtagaggcac atagaataaa taaaaccaga 3600cttc 360410662PRTHomo sapiens 10Met Val Ser Gln Ser Thr Val Arg Gln Asp Ser Pro Val Glu Pro Trp1 5 10 15Glu Gly Ile Ser Asp His Ser Gly Ile Ile Asp Gly Ser Pro Arg Leu 20 25 30Leu Asn Thr Asp His Pro Pro Cys Gln Leu Asp Ile Arg Leu Met Arg 35 40 45His Lys Ala Val Trp Ile Asn Pro Gln Asp Val Gln Gln Gln Pro Gln 50 55 60Asp Leu Gln Ser Gln Val Pro Ala Ala Gly Asn Ser Gly Thr His Phe65 70 75 80Val Thr Asp Ala Ala Ser Pro Ser Gly Pro Ser Pro Ser Cys Leu Gly 85 90 95Asp Ser Leu Ala Glu Thr Thr Leu Ser Glu Asp Thr Thr Asp Ser Val 100 105 110Gly Ser Ala Ser Pro His Gly Ser Ser Glu Lys Ser Ser Ser Phe Ser 115 120 125Leu Ser Ser Thr Glu Val His Met Val Arg Pro Gly Tyr Ser His Arg 130 135 140Val Ser Leu Pro Thr Ser Pro Gly Ile Leu Ala Thr Ser Pro Tyr Pro145 150 155 160Glu Thr Asp Ser Ala Phe Phe Glu Pro Ser His Leu Thr Ser Ala Ala 165 170 175Asp Glu Gly Ala Val Gln Val Ser Arg Arg Thr Ile Ser Ser Asn Ser 180 185 190Phe Ser Pro Glu Val Phe Val Leu Pro Val Asp Val Glu Lys Glu Asn 195 200 205Ala His Phe Tyr Val Ala Asp Met Ile Ile Ser Ala Met Glu Lys Met 210 215 220Lys Cys Asn Ile Leu Ser Gln Gln Gln Thr Glu Ser Trp Ser Lys Glu225 230 235 240Val Ser Gly Leu Leu Gly Ser Asp Gln Pro Asp Ser Glu Met Thr Phe 245 250 255Asp Thr Asn Ile Lys Gln Glu Ser Gly Ser Ser Thr Ser Ser Tyr Ser 260 265 270Gly Tyr Glu Gly Cys Ala Val Leu Gln Val Ser Pro Val Thr Glu Thr 275 280 285Arg Thr Tyr His Asp Val Lys Glu Ile Cys Lys Cys Asp Val Asp Glu 290 295 300Phe Val Ile Leu Glu Leu Gly Asp Phe Asn Asp Ile Thr Glu Thr Cys305 310 315 320Ser Cys Ser Cys Ser Ser Ser Lys Ser Val Thr Tyr Glu Pro Asp Phe 325 330 335Asn Ser Ala Glu Leu Leu Ala Lys Glu Leu Tyr Arg Val Phe Gln Lys 340 345 350Cys Trp Ile Leu Ser Val Val Asn Ser Gln Leu Ala Gly Ser Leu Ser 355 360 365Ala Ala Gly Ser Ile Val Val Asn Glu Glu Cys Val Arg Lys Asp Phe 370 375 380Glu Ser Ser Met Asn Val Val Gln Glu Ile Lys Phe Lys Ser Arg Ile385 390 395 400Arg Gly Thr Glu Asp Trp Ala Pro Pro Arg Phe Gln Ile Ile Phe Asn 405 410 415Ile His Pro Pro Leu Lys Arg Asp Leu Val Val Ala Ala Gln Asn Phe 420 425 430Phe Cys Ala Gly Cys Gly Thr Pro Val Glu Pro Lys Phe Val Lys Arg 435 440 445Leu Arg Tyr Cys Glu Tyr Leu Gly Lys Tyr Phe Cys Asp Cys Cys His 450 455 460Ser Tyr Ala Glu Ser Cys Ile Pro Ala Arg Ile Leu Met Met Trp Asp465 470 475 480Phe Lys Lys Tyr Tyr Val Ser Asn Phe Ser Lys Gln Leu Leu Asp Ser 485 490 495Ile Trp His Gln Pro Ile Phe Asn Leu Leu Ser Ile Gly Gln Ser Leu 500 505 510Tyr Ala Lys Ala Lys Glu Leu Asp Arg Val Lys Glu Ile Gln Glu Gln 515 520 525Leu Phe His Ile Lys Lys Leu Leu Lys Thr Cys Arg Phe Ala Asn Ser 530 535 540Ala Leu Lys Glu Phe Glu Gln Val Pro Gly His Leu Thr Asp Glu Leu545 550 555 560His Leu Phe Ser Leu Glu Asp Leu Val Arg Ile Lys Lys Gly Leu Leu 565 570 575Ala Pro Leu Leu Lys Asp Ile Leu Lys Ala Ser Leu Ala His Val Ala 580 585 590Gly Cys Glu Leu Cys Gln Gly Lys Gly Phe Ile Cys Glu Phe Cys Gln 595 600 605Asn Thr Thr Val Ile Phe Pro Phe Gln Thr Ala Thr Cys Arg Arg Cys 610 615 620Ser Ala Cys Arg Ala Cys Phe His Lys Gln Cys Phe Gln Ser Ser Glu625 630 635 640Cys Pro Arg Cys Ala Arg Ile Thr Ala Arg Arg Lys Leu Leu Glu Ser 645 650 655Val Ala Ser Ala Ala Thr 660112494DNAHomo sapiens 11ctttctgctg ttaccgggag cgcggtggcc acggaacgct gcccggagcc gcgcgaggga 60ggacccgacg cgcggcgttt acccagcgca gcgttccacc gctcgggttt ggctggaata 120gctctccaga cccccagctg gccatgtggt gagttcaggg cccaaatcaa gtagtaccag 180caatcaggga actcctatct gttttgaatg gattcacacc agccacaagc ctggaaagat 240ggtgtcacaa tctacagtca ggcaggattc tcctgtggag ccctgggaag ggatcagcga 300tcactctggc attattgatg gttcgcccag actcctgaac actgaccatc ctccttgcca 360attagacatc aggctcatga ggcacaaagc tgtctggatt aacccccagg atgtgcagca 420acagccgcag gacttgcaat ctcaggtgcc agcagcaggg aacagtggga cccattttgt 480gacagatgct gcctctccct caggcccttc accttcgtgc ctcggggact ccctggcaga 540gacaacgttg tctgaggata ccacagactc cgttggcagc gcttctcccc atggctcgag 600tgaaaagagt agcagcttct ctctgtcctc aacagaggta cacatggtcc gcccaggata 660ctctcatcgg gtgtctctgc ccacaagccc tgggattttg gccacctccc catatcctga 720gactgacagt gctttttttg agccttccca tctgacatct gctgctgatg aaggtgctgt 780tcaagtcagt agaagaacca tttcttcgaa ttccttctca ccagaggtat ttgtgctgcc 840tgttgatgta gaaaaggaaa atgcccactt ttatgttgca gatatgatta tatcagcaat 900ggagaaaatg aagtgtaaca ttctgagtca acagcagaca gagagctgga gtaaagaagt 960cagtgggtta cttgggagtg atcagcctga ctctgaaatg acttttgata ccaacataaa 1020gcaagagtct gggtcttcta cttcttcata cagtggctat gaaggttgtg ctgtgttaca 1080ggtcagccca gtgactgaaa cacgtactta ccatgatgtg aaagagattt gcaaatgcga 1140tgttgatgaa tttgttattt tagagcttgg agattttaat gatatcacag aaacctgtag 1200ctgttcctgc agctcctcta agagtgtcac ttatgagcca gacttcaatt ctgcagaact 1260attagccaaa gagctgtacc gcgtgttcca gaagtgctgg atactgtcag tagttaattc 1320tcagctggca ggttccctga gtgcagctgg ctcgatagtc gtaaatgaag agtgtgtccg 1380aaaagacttt gaatccagta tgaatgtagt acaggaaatt aaatttaagt ctaggatcag 1440agggactgaa gactgggctc ctcctagatt tcaaatcata tttaatattc atccaccact 1500caagagggac cttgtggtgg cagcccagaa ttttttctgt gccggctgtg gaactccagt 1560agagcctaag tttgtgaagc ggctccggta ctgcgaatac ctagggaagt atttctgtga 1620ctgctgccac tcatatgcag agtcgtgcat ccctgcccga atcctgatga tgtgggactt 1680caagaagtac tacgtcagca atttctccaa acagctgctc gacagcatat ggcaccagcc 1740cattttcaat ttgctgagca tcggccaaag cctgtatgcg aaagccaagg agctggacag 1800agtgaaggaa attcaggagc agctcttcca tatcaagaag ctgttgaaga cctgtaggtt 1860tgctaacagc tgtgtcaagg aaagggcttt atttgtgaat tttgccagaa tacgactgtc 1920atcttcccat ttcagacagc aacatgtaga agatgttcag cgtgcagggc ttgctttcac 1980aaacagtgct tccagtcctc cgagtgcccc cggtgtgcga ggatcacagc gaggagaaaa 2040cttctggaaa gtgtggcctc tgcagcaaca tgatgcccct gagtactgtg aaaaagactg 2100ttcaacatgc cttatgataa caccgatttg tgtctattat tggtgacatt gttttagata 2160ttgggtattg tatattaagg aaaaagatgg tctatattct ctttattgca tatacttaat 2220gtttcaaaag aatgcagatt ctgtgtttaa gcacagggct gatagttgtg gttttgttta 2280caaatgttct gttttggctg ctattggttt tttaaagagg ttttttatac ttttgtattt 2340gaatagttat gtttcactga tgctgagcca gtttgtatgt gtgtgcatat atgtgaactg 2400taactgacaa gatgaattac tcagtttctc tttctctaaa gcttgtttga tgaaactggt 2460tggtcctttc agtgaacaaa aatatgaccc caaa 249412635PRTHomo sapiens 12Met Val Ser Gln Ser Thr Val Arg Gln Asp Ser Pro Val Glu Pro Trp1 5 10 15Glu Gly Ile Ser Asp His Ser Gly Ile Ile Asp Gly Ser Pro Arg Leu 20 25 30Leu Asn Thr Asp His Pro Pro Cys Gln Leu Asp Ile Arg Leu Met Arg 35 40 45His Lys Ala Val Trp Ile Asn Pro Gln Asp Val Gln Gln Gln Pro Gln 50 55 60Asp Leu Gln Ser Gln Val Pro Ala Ala Gly Asn Ser Gly Thr His Phe65 70 75 80Val Thr Asp Ala Ala Ser Pro Ser Gly Pro Ser Pro Ser Cys Leu Gly 85 90 95Asp Ser Leu Ala Glu Thr Thr Leu Ser Glu Asp Thr Thr Asp Ser Val 100 105 110Gly Ser Ala Ser Pro His Gly Ser Ser Glu Lys Ser Ser Ser Phe Ser 115 120 125Leu Ser Ser Thr Glu Val His Met Val Arg Pro Gly Tyr Ser His Arg 130 135 140Val Ser Leu Pro Thr Ser Pro Gly Ile Leu Ala Thr Ser Pro Tyr Pro145 150 155 160Glu Thr Asp Ser Ala Phe Phe Glu Pro Ser His Leu Thr Ser Ala Ala 165 170 175Asp Glu Gly Ala Val Gln Val Ser Arg Arg Thr Ile Ser Ser Asn Ser 180 185 190Phe Ser Pro Glu Val Phe Val Leu Pro Val Asp Val Glu Lys Glu Asn 195 200 205Ala His Phe Tyr Val Ala Asp Met Ile Ile Ser Ala Met Glu Lys Met 210 215 220Lys Cys Asn Ile Leu Ser Gln Gln Gln Thr Glu Ser Trp Ser Lys Glu225 230 235 240Val Ser Gly Leu Leu Gly Ser Asp Gln Pro Asp Ser Glu Met Thr Phe 245 250 255Asp Thr Asn Ile Lys Gln Glu Ser Gly Ser Ser Thr Ser Ser Tyr Ser 260 265 270Gly Tyr Glu Gly Cys Ala Val Leu Gln Val Ser Pro Val Thr Glu Thr 275 280 285Arg Thr Tyr His Asp Val Lys Glu Ile Cys Lys Cys Asp Val Asp Glu 290 295 300Phe Val Ile Leu Glu Leu Gly Asp Phe Asn Asp Ile Thr Glu Thr Cys305 310 315 320Ser Cys Ser Cys Ser Ser Ser Lys Ser Val Thr Tyr Glu Pro Asp Phe 325 330 335Asn Ser Ala Glu Leu Leu Ala Lys Glu Leu Tyr Arg Val Phe Gln Lys 340 345 350Cys Trp Ile Leu Ser Val Val Asn Ser Gln Leu Ala Gly Ser Leu Ser 355 360 365Ala Ala Gly Ser Ile Val Val Asn Glu Glu Cys Val Arg Lys Asp Phe 370 375 380Glu Ser Ser Met Asn Val Val Gln Glu Ile Lys Phe Lys Ser Arg Ile385 390 395 400Arg Gly Thr Glu Asp Trp Ala Pro Pro Arg Phe Gln Ile Ile Phe Asn 405 410 415Ile His Pro Pro Leu Lys Arg Asp Leu Val Val Ala Ala Gln Asn Phe 420 425 430Phe Cys Ala Gly Cys Gly Thr Pro Val Glu Pro Lys Phe Val Lys Arg 435 440 445Leu Arg Tyr Cys Glu Tyr Leu Gly Lys Tyr Phe Cys Asp Cys Cys His 450 455 460Ser Tyr Ala Glu Ser Cys Ile Pro Ala Arg Ile Leu Met Met Trp Asp465 470 475 480Phe Lys Lys Tyr Tyr Val Ser Asn Phe Ser Lys Gln

Leu Leu Asp Ser 485 490 495Ile Trp His Gln Pro Ile Phe Asn Leu Leu Ser Ile Gly Gln Ser Leu 500 505 510Tyr Ala Lys Ala Lys Glu Leu Asp Arg Val Lys Glu Ile Gln Glu Gln 515 520 525Leu Phe His Ile Lys Lys Leu Leu Lys Thr Cys Arg Phe Ala Asn Ser 530 535 540Cys Val Lys Glu Arg Ala Leu Phe Val Asn Phe Ala Arg Ile Arg Leu545 550 555 560Ser Ser Ser His Phe Arg Gln Gln His Val Glu Asp Val Gln Arg Ala 565 570 575Gly Leu Ala Phe Thr Asn Ser Ala Ser Ser Pro Pro Ser Ala Pro Gly 580 585 590Val Arg Gly Ser Gln Arg Gly Glu Asn Phe Trp Lys Val Trp Pro Leu 595 600 605Gln Gln His Asp Ala Pro Glu Tyr Cys Glu Lys Asp Cys Ser Thr Cys 610 615 620Leu Met Ile Thr Pro Ile Cys Val Tyr Tyr Trp625 630 63513148DNAHomo sapiens 13ctggttcacg tgtggagcta gttaatacgt cctgccaaga tgggtaccag ttgactggac 60atgcttatca gatgtgtcaa gatgctgaaa atggaatttg gttcaaaaag attccacttt 120gtaaagttat ccactgcacc ctccacca 148144094DNAHomo sapiens 14ccagagctgc cggacgctcg cgggtctcgg aacgcatccc gccgcggggg cttcggccgt 60ggcatgggcg ccgcgggcct gctcggggtt ttcttggctc tcgtcgcacc gggggtcctc 120gggatttctt gtggctctcc tccgcctatc ctaaatggcc ggattagtta ttattctacc 180cccattgctg ttggtaccgt gataaggtac agttgttcag gtaccttccg cctcattgga 240gaaaaaagtc tattatgcat aactaaagac aaagtggatg gaacctggga taaacctgct 300cctaaatgtg aatatttcaa taaatattct tcttgccctg agcccatagt accaggagga 360tacaaaatta gaggctctac accctacaga catggtgatt ctgtgacatt tgcctgtaaa 420accaacttct ccatgaacgg aaacaagtct gtttggtgtc aagcaaataa tatgtggggg 480ccgacacgac taccaacctg tgtaagtgtt ttccctctcg agtgtccagc acttcctatg 540atccacaatg gacatcacac aagtgagaat gttggctcca ttgctccagg attgtctgtg 600acttacagct gtgaatctgg ttacttgctt gttggagaaa agatcattaa ctgtttgtct 660tcgggaaaat ggagtgctgt cccccccaca tgtgaagagg cacgctgtaa atctctagga 720cgatttccca atgggaaggt aaaggagcct ccaattctcc gggttggtgt aactgcaaac 780tttttctgtg atgaagggta tcgactgcaa ggcccacctt ctagtcggtg tgtaattgct 840ggacagggag ttgcttggac caaaatgcca gtatgtgaag aaattttttg cccatcacct 900ccccctattc tcaatggaag acatataggc aactcactag caaatgtctc atatggaagc 960atagtcactt acacttgtga cccggaccca gaggaaggag tgaacttcat ccttattgga 1020gagagcactc tccgttgtac agttgatagt cagaagactg ggacctggag tggccctgcc 1080ccacgctgtg aactttctac ttctgcggtt cagtgtccac atccccagat cctaagaggc 1140cgaatggtat ctgggcagaa agatcgatat acctataacg acactgtgat atttgcttgc 1200atgtttggct tcaccttgaa gggcagcaag caaatccgat gcaatgccca aggcacatgg 1260gagccatctg caccagtctg tgaaaaggaa tgccaggccc ctcctaacat cctcaatggg 1320caaaaggaag atagacacat ggtccgcttt gaccctggaa catctataaa atatagctgt 1380aaccctggct atgtgctggt gggagaagaa tccatacagt gtacctctga gggggtgtgg 1440acaccccctg taccccaatg caaagtggca gcgtgtgaag ctacaggaag gcaactcttg 1500acaaaacccc agcaccaatt tgttagacca gatgtcaact cttcttgtgg tgaagggtac 1560aagttaagtg ggagtgttta tcaggagtgt caaggcacaa ttccttggtt tatggagatt 1620cgtctttgta aagaaatcac ctgcccacca ccccctgtta tctacaatgg ggcacacacc 1680gggagttcct tagaagattt tccatatgga accacggtca cttacacatg taaccctggg 1740ccagaaagag gagtggaatt cagcctcatt ggagagagca ccatccgttg tacaagcaat 1800gatcaagaaa gaggcacctg gagtggccct gctcccctat gtaaactttc cctccttgct 1860gtccagtgct cacatgtcca tattgcaaat ggatacaaga tatctggcaa ggaagcccca 1920tatttctaca atgacactgt gacattcaag tgttatagtg gatttacttt gaagggcagt 1980agtcagattc gttgcaaacg tgataacacc tgggatcctg aaataccagt ttgtgaaaaa 2040ggctgccagc cacctcctgg gctccaccat ggtcgtcata caggtggaaa tacggtcttc 2100tttgtctctg ggatgactgt agactacact tgtgaccctg gctatttgct tgtgggaaac 2160aaatccattc actgtatgcc ttcaggaaat tggagtcctt ctgccccacg gtgtgaagaa 2220acatgccagc atgtgagaca gagtcttcaa gaacttccag ctggttcacg tgtggagcta 2280gttaatacgt cctgccaaga tgggtaccag ttgactggac atgcttatca gatgtgtcaa 2340gatgctgaaa atggaatttg gttcaaaaag attccacttt gtaaagttat tcactgtcac 2400cctccaccag tgattgtcaa tgggaagcac acaggcatga tggcagaaaa ctttctatat 2460ggaaatgaag tctcttatga atgtgaccaa ggattctatc tcctgggaga gaaaaattgc 2520agtgcagaag tgattctaaa ggcatggatc ttggagcgag ccttcccaca gtgcttacga 2580tctctgtgcc ctaatccaga agtcaaacat gggtacaagc tcaataaaac acattctgca 2640tattcccaca atgacatagt gtatgttgac tgcaatcctg gcttcatcat gaatggtagt 2700cgcgtgatta ggtgtcatac tgataacaca tgggtgccag gtgtgccaac ttgtatcaaa 2760aaagccttca tagggtgtcc acctccgcct aagaccccta acgggaacca tactggtgga 2820aacatagctc gattttctcc tggaatgtca atcctgtaca gctgtgacca aggctacctg 2880gtggtgggag agccactcct tctttgcaca catgagggaa cctggagcca acctgcccct 2940cattgtaaag aggtaaactg tagctcacca gcagatatgg atggaatcca gaaagggctg 3000gaaccaagga aaatgtatca gtatggagct gttgtaactc tggagtgtga agatgggtat 3060atgctggaag gcagtcccca gagccagtgc caatcggatc accaatggaa ccctcccctg 3120gcggtttgca gatcccgttc acttgctcct gtcctttgtg gtattgctgc aggtttgata 3180cttcttacct tcttgattgt cattacctta tacgtgatat caaaacacag agaacgcaat 3240tattatacag atacaagcca gaaagaagct tttcatttag aagcacgaga agtatattct 3300gttgatccat acaacccagc cagctgatca gaagacaaaa ctggtgtgtg cctcattgct 3360tggaattcag cggaatattg attagaaaga aactgctcta atatcagcaa gtctctttat 3420atggcctcaa gatcaatgaa atgatgtcat aagcgatcac ttcctatatg cacttattct 3480caagaagaac atctttatgg taaagatggg agcccagttt cactgccata tactcttcaa 3540ggactttctg aagcctcact tatgagatgc ctgaagccag gccatggcta taaacattac 3600atggctctaa aagttttgcc ctttttaagg aggcactaaa aagagctgtc ctggtatcta 3660gacccatctt ctttttgaaa tcacatactc atgttactat ctgcttttgg ttataatgtg 3720tttttaatta tctaaagtat gaagcatttt ctggggttat gatggcctta cttttattag 3780gaagtatggt tttattttga tagtagcttc cttcctcggt ggtgttaatc atttcgtttt 3840taccctttac cttcggattt gagtttctct cacattactg tatatacttt gccttccata 3900atcactcagt gattgcaatt tgcacaagtt tttttaaatt atgggaatca agatttaatc 3960ctagagattt ggtgtacaat tcaggctttg gatgtttctt tagcagtttt gtgataagtt 4020ctagttgctt gtaaaatttc acttaataat gtgtacatta gtcattcaat aaattgtaat 4080tgtaaagaaa acat 4094151033PRTHomo sapiens 15Met Gly Ala Ala Gly Leu Leu Gly Val Phe Leu Ala Leu Val Ala Pro1 5 10 15Gly Val Leu Gly Ile Ser Cys Gly Ser Pro Pro Pro Ile Leu Asn Gly 20 25 30Arg Ile Ser Tyr Tyr Ser Thr Pro Ile Ala Val Gly Thr Val Ile Arg 35 40 45Tyr Ser Cys Ser Gly Thr Phe Arg Leu Ile Gly Glu Lys Ser Leu Leu 50 55 60Cys Ile Thr Lys Asp Lys Val Asp Gly Thr Trp Asp Lys Pro Ala Pro65 70 75 80Lys Cys Glu Tyr Phe Asn Lys Tyr Ser Ser Cys Pro Glu Pro Ile Val 85 90 95Pro Gly Gly Tyr Lys Ile Arg Gly Ser Thr Pro Tyr Arg His Gly Asp 100 105 110Ser Val Thr Phe Ala Cys Lys Thr Asn Phe Ser Met Asn Gly Asn Lys 115 120 125Ser Val Trp Cys Gln Ala Asn Asn Met Trp Gly Pro Thr Arg Leu Pro 130 135 140Thr Cys Val Ser Val Phe Pro Leu Glu Cys Pro Ala Leu Pro Met Ile145 150 155 160His Asn Gly His His Thr Ser Glu Asn Val Gly Ser Ile Ala Pro Gly 165 170 175Leu Ser Val Thr Tyr Ser Cys Glu Ser Gly Tyr Leu Leu Val Gly Glu 180 185 190Lys Ile Ile Asn Cys Leu Ser Ser Gly Lys Trp Ser Ala Val Pro Pro 195 200 205Thr Cys Glu Glu Ala Arg Cys Lys Ser Leu Gly Arg Phe Pro Asn Gly 210 215 220Lys Val Lys Glu Pro Pro Ile Leu Arg Val Gly Val Thr Ala Asn Phe225 230 235 240Phe Cys Asp Glu Gly Tyr Arg Leu Gln Gly Pro Pro Ser Ser Arg Cys 245 250 255Val Ile Ala Gly Gln Gly Val Ala Trp Thr Lys Met Pro Val Cys Glu 260 265 270Glu Ile Phe Cys Pro Ser Pro Pro Pro Ile Leu Asn Gly Arg His Ile 275 280 285Gly Asn Ser Leu Ala Asn Val Ser Tyr Gly Ser Ile Val Thr Tyr Thr 290 295 300Cys Asp Pro Asp Pro Glu Glu Gly Val Asn Phe Ile Leu Ile Gly Glu305 310 315 320Ser Thr Leu Arg Cys Thr Val Asp Ser Gln Lys Thr Gly Thr Trp Ser 325 330 335Gly Pro Ala Pro Arg Cys Glu Leu Ser Thr Ser Ala Val Gln Cys Pro 340 345 350His Pro Gln Ile Leu Arg Gly Arg Met Val Ser Gly Gln Lys Asp Arg 355 360 365Tyr Thr Tyr Asn Asp Thr Val Ile Phe Ala Cys Met Phe Gly Phe Thr 370 375 380Leu Lys Gly Ser Lys Gln Ile Arg Cys Asn Ala Gln Gly Thr Trp Glu385 390 395 400Pro Ser Ala Pro Val Cys Glu Lys Glu Cys Gln Ala Pro Pro Asn Ile 405 410 415Leu Asn Gly Gln Lys Glu Asp Arg His Met Val Arg Phe Asp Pro Gly 420 425 430Thr Ser Ile Lys Tyr Ser Cys Asn Pro Gly Tyr Val Leu Val Gly Glu 435 440 445Glu Ser Ile Gln Cys Thr Ser Glu Gly Val Trp Thr Pro Pro Val Pro 450 455 460Gln Cys Lys Val Ala Ala Cys Glu Ala Thr Gly Arg Gln Leu Leu Thr465 470 475 480Lys Pro Gln His Gln Phe Val Arg Pro Asp Val Asn Ser Ser Cys Gly 485 490 495Glu Gly Tyr Lys Leu Ser Gly Ser Val Tyr Gln Glu Cys Gln Gly Thr 500 505 510Ile Pro Trp Phe Met Glu Ile Arg Leu Cys Lys Glu Ile Thr Cys Pro 515 520 525Pro Pro Pro Val Ile Tyr Asn Gly Ala His Thr Gly Ser Ser Leu Glu 530 535 540Asp Phe Pro Tyr Gly Thr Thr Val Thr Tyr Thr Cys Asn Pro Gly Pro545 550 555 560Glu Arg Gly Val Glu Phe Ser Leu Ile Gly Glu Ser Thr Ile Arg Cys 565 570 575Thr Ser Asn Asp Gln Glu Arg Gly Thr Trp Ser Gly Pro Ala Pro Leu 580 585 590Cys Lys Leu Ser Leu Leu Ala Val Gln Cys Ser His Val His Ile Ala 595 600 605Asn Gly Tyr Lys Ile Ser Gly Lys Glu Ala Pro Tyr Phe Tyr Asn Asp 610 615 620Thr Val Thr Phe Lys Cys Tyr Ser Gly Phe Thr Leu Lys Gly Ser Ser625 630 635 640Gln Ile Arg Cys Lys Ala Asp Asn Thr Trp Asp Pro Glu Ile Pro Val 645 650 655Cys Glu Lys Glu Thr Cys Gln His Val Arg Gln Ser Leu Gln Glu Leu 660 665 670Pro Ala Gly Ser Arg Val Glu Leu Val Asn Thr Ser Cys Gln Asp Gly 675 680 685Tyr Gln Leu Thr Gly His Ala Tyr Gln Met Cys Gln Asp Ala Glu Asn 690 695 700Gly Ile Trp Phe Lys Lys Ile Pro Leu Cys Lys Val Ile His Cys His705 710 715 720Pro Pro Pro Val Ile Val Asn Gly Lys His Thr Gly Met Met Ala Glu 725 730 735Asn Phe Leu Tyr Gly Asn Glu Val Ser Tyr Glu Cys Asp Gln Gly Phe 740 745 750Tyr Leu Leu Gly Glu Lys Lys Leu Gln Cys Arg Ser Asp Ser Lys Gly 755 760 765His Gly Ser Trp Ser Gly Pro Ser Pro Gln Cys Leu Arg Ser Pro Pro 770 775 780Val Thr Arg Cys Pro Asn Pro Glu Val Lys His Gly Tyr Lys Leu Asn785 790 795 800Lys Thr His Ser Ala Tyr Ser His Asn Asp Ile Val Tyr Val Asp Cys 805 810 815Asn Pro Gly Phe Ile Met Asn Gly Ser Arg Val Ile Arg Cys His Thr 820 825 830Asp Asn Thr Trp Val Pro Gly Val Pro Thr Cys Met Lys Lys Ala Phe 835 840 845Ile Gly Cys Pro Pro Pro Pro Lys Thr Pro Asn Gly Asn His Thr Gly 850 855 860Gly Asn Ile Ala Arg Phe Ser Pro Gly Met Ser Ile Leu Tyr Ser Cys865 870 875 880Asp Gln Gly Tyr Leu Leu Val Gly Glu Ala Leu Leu Leu Cys Thr His 885 890 895Glu Gly Thr Trp Ser Gln Pro Ala Pro His Cys Lys Glu Val Asn Cys 900 905 910Ser Ser Pro Ala Asp Met Asp Gly Ile Gln Lys Gly Leu Glu Pro Arg 915 920 925Lys Met Tyr Gln Tyr Gly Ala Val Val Thr Leu Glu Cys Glu Asp Gly 930 935 940Tyr Met Leu Glu Gly Ser Pro Gln Ser Gln Cys Gln Ser Asp His Gln945 950 955 960Trp Asn Pro Pro Leu Ala Val Cys Arg Ser Arg Ser Leu Ala Pro Val 965 970 975Leu Cys Gly Ile Ala Ala Gly Leu Ile Leu Leu Thr Phe Leu Ile Val 980 985 990Ile Thr Leu Tyr Val Ile Ser Lys His Arg Glu Arg Asn Tyr Tyr Thr 995 1000 1005Asp Thr Ser Gln Lys Glu Ala Phe His Leu Glu Ala Arg Glu Val Tyr 1010 1015 1020Ser Val Asp Pro Tyr Asn Pro Ala Ser1025 103016637DNAHomo sapiensmodified_base(9)n = g, a, c or t 16ctgggcaana ccaagtcaca gtttccagcg tgctgctcag ccctccgagt gtgtgtgctc 60atccttttca tagaagtccc atmkgscatg gagagggttg ggctgcarag ctgwgattgc 120cagaggccct tccttgagaa ctgtggggaa ggaggccctg ggggtttctt ctgtaggcag 180agctcaggcc ccagtcacct ctgccaccct cagcctggca ctgttgtgcc agagcctctg 240ctgcctctct cttcctaccc atctgcagac cagcagaata ttctccccct ctcatcacca 300accaggagtt tggtgtggtt tctggacacg gccagagcag tcactgcggg gctggttttg 360ctgggcttcc ctgtcaaagc aatgctaacg tccagctctc gactcaaggc caggttcttc 420tcccacttgt ggcctcttgg gcttggaggc tgagccaggg gctcctctcc tgctggccgt 480ccaggaacag acatcttcac atcctcagtc ttccaaaccc ggaccatgcc gtcttgactc 540ccggtgatga tgatctggct tgtgtcccat gctgggccct ccatcaggca gcaacaggtt 600atggctcctt ctgggcccca ggctgtggtg atgctgg 637174191DNAHomo sapiens 17agcgagactt ccagtccgag gtcctgcttt ctgctatgga actattccac atgacaagtg 60gaggtgatgc agcgatgttc agagacggca aagagcctca gccaagtgca gaagctgctg 120ctgccccttc tcttgccaac atctcctgct tcacccagaa gctggtggag aagctgtaca 180gtgggatgtt ctcggcagac cccaggcata tcctcctctt catcctggag cacatcatgg 240tggtcattga gactgcctct tctcaaaggg acactgtcct cagcacttta tacagcagtt 300taaataaagt cattctttat tgcctatcca agccccagca gtccctctcc gaatgcctcg 360gccttctcag catcctgggc tttctgcagg agcactggga tgttgtcttt gccacctaca 420attccaacat cagcttcctc ctgtgtctca tgcattgcct tttgctactc aatgagagaa 480gttacccaga aggatttgga ttggagccca agcctagaat gtctacttat catcaagtct 540tcctttcccc aaatgaagac gtgaaagaaa aaagagaaga cttaccaagt ttgagtgatg 600tccaacacaa catccagaag acagtgcaga ctctctggca gcagctggtg gcacaaaggc 660agcagaccct ggaggatgcc ttcaagatcg atctctctgt gaaacctgga gagagggaag 720tgaagattga agaggtcaca ccgctctggg aggagacgat gctcaaggcc tggcagcatt 780acttagcatc tgagaagaag tcactggcaa gtcgttcaaa tgttgcacac cacagcaaag 840tcactttgtg gagtggaagc ctgtcctcag ccatgaagct gatgcccggg cggcaggcca 900aggaccctga gtgcaagaca gaggattttg tgtcatgtat agagaactac agaagaagag 960gacaagagct atatgcatct ttatacaaag accatgtgca aaggcgaaaa tgtggcaaca 1020tcaaggcagc caacgcctgg gccaggatcc aggagcagct ttttggggag ctgggcttgt 1080ggagccaggg ggaagaaacc aagccctgtt ccccatggga actcgactgg agagaaggac 1140cagctcgaat gaggaaacgc atcaaacgct tgtctccttt ggaggccctg agctcaggaa 1200ggcacaagga aagccaagac aaaaatgatc atatttctca aacaaatgct gaaaaccaag 1260atgaactgac actgagggag gctgagggcg agccggacga ggtgggggtg gactgcaccc 1320agctgacctt cttcccagcc ttacacgaaa gtctgcactc agaagacttc ttggaactgt 1380gtcgggaaag acaagttatt ttacaagagc ttcttgataa agaaaaggtg acgcagaagt 1440tctccctggt gattgtgcag ggccacctgg tgtcagaagg ggtcctgctt tttggccacc 1500aacacttcta catctgcgag aacttcacac tgtctcccac gggtgatgtc tactgtaccc 1560gtcactgctt atccaacatc agcgatccgt tcattttcaa cctgtgcagc aaagacaggt 1620ccactgacca ttactcgtgc cagtgccaca gctacgctga catgcgggag ctacggcagg 1680ctcgcttcct cctgcaggac atcgccctgg agatcttctt ccacaatgga tattccaagt 1740ttcttgtctt ctacaacaat gatcggagta aggcctttaa aagcttctgc tctttccaac 1800ccagcctgaa ggggaaagcc acctcggagg acaccctcaa tctaaggaga taccccggct 1860ctgacaggat catgctgcag aagtggcaga aaagggacat cagcaatttt gagtatctca 1920tgtacctcaa caccgcggct gggagaacct gcaatgacta catgcagtac ccagtgttcc 1980cctgggtcct cgcagactac acctcagaga cattgaactt ggcaaatccg aagattttcc 2040gggatctttc aaagcccatg ggggctcaga ccaaggaaag gaagctgaaa tttatccaga 2100ggtttaaaga agttgagaaa actgaaggag acatgactgt ccagtgccac tactacaccc 2160actactcctc ggccatcatc gtggcctcct acctggtccg gatgccaccc ttcacccagg 2220ccttctgcgc tctgcagggc ggaagcttcg acgtggcaga cagaatgttc cacagtgtga 2280agagcacgtg ggagtcggcc tccagagaga acatgagtga cgtcagggag ctgaccccag 2340agttcttcta cctgcctgag ttcttaacca actgcaacgg ggtagagttc ggctgcatgc 2400aggacgggac tgtgctagga gacgtgcagc tccctccctg ggctgatggg gaccctcgga 2460aattcatcag cctgcacaga aaggccctgg aaagtgactt tgtcagtgcc aacctccacc 2520attggataga ccttattttt gggtacaagc agcaggggcc agccgcagtg gatgctgtta 2580atatcttcca cccctacttc tacggtgaca gaatggacct cagcagcatc actgaccccc 2640tcatcaaaag caccatcctg gggtttgtca gcaactttgg acaggtgccc aaacagctct 2700ttaccaaacc tcacccagcc aggactgcag cagggaagcc tctgcctgga aaggatgtct

2760ccacccccgt gagcctgcct ggccacccac agcccttttt ctacagcctg cagtcgctga 2820ggccctccca ggtcacggtc aaagatatgt acctcttttc tctaggctca gagtccccca 2880aaggggccat tggccacatt gtctctactg agaagaccat tctggctgta gagaggaaca 2940aagtgctgct gcctcctctc tggaacagga ccttcagctg gggctttgat gacttcagct 3000gctgcttggg gagctacggc tccgacaagg tcctgatgac attcgagaac ctggctgcct 3060ggggccgctg tctgtgcgcc gtgtgcccat ccccaacaac gattgtcacc tctgggacca 3120gcactgtggt gtgtgtgtgg gagctcagca tgaccaaagg ccgcccgagg ggcttgcgcc 3180tccggcaggc cttgtatgga cacacacagg ctgtcacgtg cctggcagcg tcagtcacct 3240tcagcctcct ggtgagcggc tcccaggact gcacctgtat cctgtgggat ctggaccacc 3300tcacccacgt gacccgcctg cccgcccatc gggaaggcat ctcagccatc accatcagtg 3360acgtctcagg caccattgtc tcctgtgcgg gagcacactt gtccctgtgg aatgtcaatg 3420gacagcccct ggccagcatc accacagcct ggggcccaga aggagccata acctgttgct 3480gcctgatgga gggcccagca tgggacacaa gccagatcat catcaccggg agtcaagacg 3540gcatggtccg ggtttggaag actgaggatg tgaagatgtc tgttcctgga cggccagcag 3600gagaggagcc cctggctcag cctccaagcc caagaggcca caagtgggag aagaacctgg 3660ccttgagtcg agagctggac gttagcattg ctttgacagg gaagcccagc aaaaccagcc 3720ccgcagtgac tgctctggcc gtgtccagaa accacaccaa actcctggtt ggtgatgaga 3780gggggagaat attctgctgg tctgcagatg ggtaggaaga gagaggcagc agaggctctg 3840gcacaacagt gccaggctga gggtggcaga ggtgactggg gcctgagctc tgcctacaga 3900agaaaccccc agggcctcct tccccacagt tctcaaggaa gggcctctgg caatcacagc 3960tctgcagccc aaccctctcc atggccgatg ggacttctat gaaaaggatg agcacacaca 4020ctcggagggc tgagcagcac gctggaaact gtgacttggt gatgcccagc tgcacacgaa 4080attacacatg actcacctta ttaagggcta ttgcactgaa aaaaaaaaaa agatgggtcg 4140cttactggaa attattgtat tgtctttatt ttattaaagc aactatgttt t 4191181606PRTHomo sapiens 18Met Asn Ile Ser Ser Arg Asp Asn Ala Met Pro Val Phe Leu Leu Arg1 5 10 15Asn Cys Ala Gly His Leu Ser Gly Ser Leu Arg Thr Ile Gly Ala Val 20 25 30Ala Val Gly Gln Leu Gly Val Arg Val Phe His Ser Ser Pro Ala Ala 35 40 45Ser Ser Leu Asp Phe Ile Gly Gly Pro Ala Ile Leu Leu Gly Leu Ile 50 55 60Ser Leu Ala Thr Asp Asp His Thr Met Tyr Ala Ala Val Lys Val Leu65 70 75 80His Ser Val Leu Thr Ser Asn Ala Met Cys Asp Phe Leu Met Gln His 85 90 95Ile Cys Gly Tyr Gln Ile Met Ala Phe Leu Leu Arg Lys Lys Ala Ser 100 105 110Leu Leu Asn His Arg Ile Phe Gln Leu Ile Leu Ser Val Ala Gly Thr 115 120 125Val Glu Leu Gly Phe Arg Ser Ser Ala Ile Thr Asn Thr Gly Val Phe 130 135 140Gln His Ile Leu Cys Asn Phe Glu Leu Trp Met Asn Thr Ala Asp Asn145 150 155 160Leu Glu Leu Ser Leu Phe Ser His Leu Leu Glu Ile Leu Gln Ser Pro 165 170 175Arg Glu Gly Pro Arg Asn Ala Glu Ala Ala His Gln Ala Gln Leu Ile 180 185 190Pro Lys Leu Ile Phe Leu Phe Asn Glu Pro Ser Leu Ile Pro Ser Lys 195 200 205Ile Pro Thr Ile Ile Gly Ile Leu Ala Cys Gln Leu Arg Gly His Phe 210 215 220Ser Thr Gln Asp Leu Leu Arg Ile Gly Leu Phe Val Val Tyr Thr Leu225 230 235 240Lys Pro Ser Ser Val Asn Glu Arg Gln Ile Cys Met Asp Gly Ala Leu 245 250 255Asp Pro Ser Leu Pro Ala Gly Ser Gln Thr Ser Gly Lys Thr Ile Trp 260 265 270Leu Arg Asn Gln Leu Leu Glu Met Leu Leu Ser Val Ile Ser Ser Pro 275 280 285Gln Leu His Leu Ser Ser Glu Ser Lys Glu Glu Met Phe Leu Lys Leu 290 295 300Gly Pro Asp Trp Phe Leu Leu Leu Leu Gln Gly His Leu His Ala Ser305 310 315 320Thr Thr Val Leu Ala Leu Lys Leu Leu Leu Tyr Phe Leu Ala Ser Pro 325 330 335Ser Leu Arg Thr Arg Phe Arg Asp Gly Leu Cys Ala Gly Ser Trp Val 340 345 350Glu Arg Ser Thr Glu Gly Val Asp Ile Val Met Asp Asn Leu Lys Ser 355 360 365Gln Ser Pro Leu Pro Glu Gln Ser Pro Cys Leu Leu Pro Gly Phe Arg 370 375 380Val Leu Asn Asp Phe Leu Ala His His Val His Ile Pro Glu Val Tyr385 390 395 400Leu Ile Val Ser Thr Phe Phe Leu Gln Thr Pro Leu Thr Glu Leu Met 405 410 415Asp Gly Pro Lys Asp Ser Leu Asp Ala Met Leu Gln Trp Leu Leu Gln 420 425 430Arg His His Gln Glu Glu Val Leu Gln Ala Gly Leu Cys Thr Glu Gly 435 440 445Ala Leu Leu Leu Leu Glu Met Leu Lys Ala Thr Met Ser Gln Pro Leu 450 455 460Ala Gly Ser Glu Asp Gly Ala Trp Ala Gln Thr Phe Pro Ala Ser Val465 470 475 480Leu Gln Phe Leu Ser Leu Val His Arg Thr Tyr Pro Gln Asp Pro Ala 485 490 495Trp Arg Ala Pro Glu Phe Leu Gln Thr Leu Ala Ile Ala Ala Phe Pro 500 505 510Leu Gly Ala Gln Lys Gly Val Gly Ala Glu Ser Thr Arg Asn Thr Ser 515 520 525Ser Pro Glu Ala Ala Ala Glu Gly Asp Ser Thr Val Glu Gly Leu Gln 530 535 540Ala Pro Thr Lys Ala His Pro Ala Arg Arg Lys Leu Arg Glu Phe Thr545 550 555 560Gln Leu Leu Leu Arg Glu Leu Leu Leu Gly Ala Ser Ser Pro Lys Gln 565 570 575Trp Leu Pro Leu Glu Val Leu Leu Glu Ala Ser Pro Asp His Ala Thr 580 585 590Ser Gln Gln Lys Arg Asp Phe Gln Ser Glu Val Leu Leu Ser Ala Met 595 600 605Glu Leu Phe His Met Thr Ser Gly Gly Asp Ala Ala Met Phe Arg Asp 610 615 620Gly Lys Glu Pro Gln Pro Ser Ala Glu Ala Ala Ala Ala Pro Ser Leu625 630 635 640Ala Asn Ile Ser Cys Phe Thr Gln Lys Leu Val Glu Lys Leu Tyr Ser 645 650 655Gly Met Phe Ser Ala Asp Pro Arg His Ile Leu Leu Phe Ile Leu Glu 660 665 670His Ile Met Val Val Ile Glu Thr Ala Ser Ser Gln Arg Asp Thr Val 675 680 685Leu Ser Thr Leu Tyr Ser Ser Leu Asn Lys Val Ile Leu Tyr Cys Leu 690 695 700Ser Lys Pro Gln Gln Ser Leu Ser Glu Cys Leu Gly Leu Leu Ser Ile705 710 715 720Leu Gly Phe Leu Gln Glu His Trp Asp Val Val Phe Ala Thr Tyr Asn 725 730 735Ser Asn Ile Ser Phe Leu Leu Cys Leu Met His Cys Leu Leu Leu Leu 740 745 750Asn Glu Arg Ser Tyr Pro Glu Gly Phe Gly Leu Glu Pro Lys Pro Arg 755 760 765Met Ser Thr Tyr His Gln Val Phe Leu Ser Pro Asn Glu Asp Val Lys 770 775 780Glu Lys Arg Glu Asp Leu Pro Ser Leu Ser Asp Val Gln His Asn Ile785 790 795 800Gln Lys Thr Val Gln Thr Leu Trp Gln Gln Leu Val Ala Gln Arg Gln 805 810 815Gln Thr Leu Glu Asp Ala Phe Lys Ile Asp Leu Ser Val Lys Pro Gly 820 825 830Glu Arg Glu Val Lys Ile Glu Glu Val Thr Pro Leu Trp Glu Glu Thr 835 840 845Met Leu Lys Ala Trp Gln His Tyr Leu Ala Ser Glu Lys Lys Ser Leu 850 855 860Ala Ser Arg Ser Asn Val Ala His His Ser Lys Val Thr Leu Trp Ser865 870 875 880Gly Ser Leu Ser Ser Ala Met Lys Leu Met Pro Gly Arg Gln Ala Lys 885 890 895Asp Pro Glu Cys Lys Thr Glu Asp Phe Val Ser Cys Ile Glu Asn Tyr 900 905 910Arg Arg Arg Gly Gln Glu Leu Tyr Ala Ser Leu Tyr Lys Asp His Val 915 920 925Gln Arg Arg Lys Cys Gly Asn Ile Lys Ala Ala Asn Ala Trp Ala Arg 930 935 940Ile Gln Glu Gln Leu Phe Gly Glu Leu Gly Leu Trp Ser Gln Gly Glu945 950 955 960Glu Thr Lys Pro Cys Ser Pro Trp Glu Leu Asp Trp Arg Glu Gly Pro 965 970 975Ala Arg Met Arg Lys Arg Ile Lys Arg Leu Ser Pro Leu Glu Ala Leu 980 985 990Ser Ser Gly Arg His Lys Glu Ser Gln Asp Lys Asn Asp His Ile Ser 995 1000 1005Gln Thr Asn Ala Glu Asn Gln Asp Glu Leu Thr Leu Arg Glu Ala Glu 1010 1015 1020Gly Glu Pro Asp Glu Val Gly Val Asp Cys Thr Gln Leu Thr Phe Phe1025 1030 1035 1040Pro Ala Leu His Glu Ser Leu His Ser Glu Asp Phe Leu Glu Leu Cys 1045 1050 1055Arg Glu Arg Gln Val Ile Leu Gln Glu Leu Leu Asp Lys Glu Lys Val 1060 1065 1070Thr Gln Lys Phe Ser Leu Val Ile Val Gln Gly His Leu Val Ser Glu 1075 1080 1085Gly Val Leu Leu Phe Gly His Gln His Phe Tyr Ile Cys Glu Asn Phe 1090 1095 1100Thr Leu Ser Pro Thr Gly Asp Val Tyr Cys Thr Arg His Cys Leu Ser1105 1110 1115 1120Asn Ile Ser Asp Pro Phe Ile Phe Asn Leu Cys Ser Lys Asp Arg Ser 1125 1130 1135Thr Asp His Tyr Ser Cys Gln Cys His Ser Tyr Ala Asp Met Arg Glu 1140 1145 1150Leu Arg Gln Ala Arg Phe Leu Leu Gln Asp Ile Ala Leu Glu Ile Phe 1155 1160 1165Phe His Asn Gly Tyr Ser Lys Phe Leu Val Phe Tyr Asn Asn Asp Arg 1170 1175 1180Ser Lys Ala Phe Lys Ser Phe Cys Ser Phe Gln Pro Ser Leu Lys Gly1185 1190 1195 1200Lys Ala Thr Ser Glu Asp Thr Leu Asn Leu Arg Arg Tyr Pro Gly Ser 1205 1210 1215Asp Arg Ile Met Leu Gln Lys Trp Gln Lys Arg Asp Ile Ser Asn Phe 1220 1225 1230Glu Tyr Leu Met Tyr Leu Asn Thr Ala Ala Gly Arg Thr Cys Asn Asp 1235 1240 1245Tyr Met Gln Tyr Pro Val Phe Pro Trp Val Leu Ala Asp Tyr Thr Ser 1250 1255 1260Glu Thr Leu Asn Leu Ala Asn Pro Lys Ile Phe Arg Asp Leu Ser Lys1265 1270 1275 1280Pro Met Gly Ala Gln Thr Lys Glu Arg Lys Leu Lys Phe Ile Gln Arg 1285 1290 1295Phe Lys Glu Val Glu Lys Thr Glu Gly Asp Met Thr Val Gln Cys His 1300 1305 1310Tyr Tyr Thr His Tyr Ser Ser Ala Ile Ile Val Ala Ser Tyr Leu Val 1315 1320 1325Arg Met Pro Pro Phe Thr Gln Ala Phe Cys Ala Leu Gln Gly Gly Ser 1330 1335 1340Phe Asp Val Ala Asp Arg Met Phe His Ser Val Lys Ser Thr Trp Glu1345 1350 1355 1360Ser Ala Ser Arg Glu Asn Met Ser Asp Val Arg Glu Leu Thr Pro Glu 1365 1370 1375Phe Phe Tyr Leu Pro Glu Phe Leu Thr Asn Cys Asn Gly Val Glu Phe 1380 1385 1390Gly Cys Met Gln Asp Gly Thr Val Leu Gly Asp Val Gln Leu Pro Pro 1395 1400 1405Trp Ala Asp Gly Asp Pro Arg Lys Phe Ile Ser Leu His Arg Lys Ala 1410 1415 1420Leu Glu Ser Asp Phe Val Ser Ala Asn Leu His His Trp Ile Asp Leu1425 1430 1435 1440Ile Phe Gly Tyr Lys Gln Gln Gly Pro Ala Ala Val Asp Ala Val Asn 1445 1450 1455Ile Phe His Pro Tyr Phe Tyr Gly Asp Arg Met Asp Leu Ser Ser Ile 1460 1465 1470Thr Asp Pro Leu Ile Lys Ser Thr Ile Leu Gly Phe Val Ser Asn Phe 1475 1480 1485Gly Gln Val Pro Lys Gln Leu Phe Thr Lys Pro His Pro Ala Arg Thr 1490 1495 1500Ala Ala Gly Lys Pro Leu Pro Gly Lys Asp Val Ser Thr Pro Val Ser1505 1510 1515 1520Leu Pro Gly His Pro Gln Pro Phe Phe Tyr Ser Leu Gln Ser Leu Arg 1525 1530 1535Pro Ser Gln Val Thr Val Lys Asp Met Tyr Leu Phe Ser Leu Gly Ser 1540 1545 1550Glu Ser Pro Lys Gly Ala Ile Gly His Ile Val Ser Thr Glu Lys Thr 1555 1560 1565Ile Leu Ala Val Glu Arg Asn Lys Val Leu Leu Pro Pro Leu Trp Asn 1570 1575 1580Arg Thr Phe Ser Trp Gly Phe Asp Asp Phe Ser Cys Cys Leu Gly Ser1585 1590 1595 1600Tyr Gly Ser Asp Lys Ser 160519426DNAHomo sapiens 19ctgcccttcc atgtcgtcac aggcataccg ggcgtggacc cactgcttgg tgcccttgcc 60gaagtagtag cacttccgtt ggaaattgat ccacttttca gggcacgtgt tgcacacaaa 120gccgctggac acctgcaact ccatccttag ctttgtcacc tcctcccgga gtctttccag 180caaatctgaa gcttcgttcc tctcgttcaa ttcctgggac ttgaagctgc tcagatctgc 240ttgaagcccg ttcaggttcc aggacagctc caagtcctga gatttcaatc tctgctgttc 300agctcgaagt tcctccagtt cctgtgaaat ctgcgtggac tgggatttct gcgccatctg 360gtcaccgtgg tggctttcca agttcttgga aacttgagag acgttccggg cagccctctc 420ttccag 426201569DNAHomo sapiens 20ggcacgaggc tgcttaaacc tctgtctctg acggtccctg ccaatcgctc tggtcgaccc 60caacacacta ggaggacaga cacaggctcc aaactccact aaccagagct gtgattgtgc 120ccgctgagtg gactgcgttg tcagggagtg agtgctccat catcgggaga atccaagcag 180gaccgccatg gaggaaggtc aatattcaga gatcgaggag cttcccagga ggcggtgttg 240caggcgtggg actcagatcg tgctgctggg gctggtgacc gccgctctgt gggctgggct 300gctgactctg cttctcctgt ggcactggga caccacacag agtctaaaac agctggaaga 360gagggctgcc cggaacgtct ctcaagtttc caagaacttg gaaagccacc acggtgacca 420gatggcgcag aaatcccagt ccacgcagat ttcacaggaa ctggaggaac ttcgagctga 480acagcagaga ttgaaatctc aggacttgga gctgtcctgg aacctgaacg ggcttcaagc 540agatctgagc agcttcaagt cccaggaatt gaacgagagg aacgaagctt cagatttgct 600ggaaagactc cgggaggagg tgacaaagct aaggatggag ttgcaggtgt ccagcggctt 660tgtgtgcaac acgtgccctg aaaagtggat caatttccaa cggaagtgct actacttcgg 720caagggcacc aagcagtggg tccacgcccg gtatgcctgt gacgacatgg aagggcagct 780ggtcagcatc cacagcccgg aggagcagga cttcctgacc aagcatgcca gccacaccgg 840ctcctggatt ggccttcgga acttggacct gaagggggag tttatctggg tggatgggag 900ccacgtggac tacagcaact gggctccagg ggagcccacc agccggagcc agggcgagga 960ctgcgtgatg atgcggggct ccggtcgctg gaacgacgcc ttctgcgacc gtaagctggg 1020cgcctgggtg tgcgaccggc tggccacatg cacgccgcca gccagcgaag gttccgcgga 1080gtccatggga cctgattcaa gaccagaccc tgacggccgc ctgcccaccc cctctgcccc 1140tctccactct tgagcatgga tacagccagg cccagagcaa gaccctgaag acccccaacc 1200acggcctaaa agcctctttg tggctgaaag gtccctgtga cattttctgc cacccaaacg 1260gaggcagctg acacatctcc cgctcctcta tggcccctgc cttcccagga gtacacccca 1320acagcaccct ctccagatgg gagtgccccc aacagcaccc tctccagatg agagtacacc 1380ccaacagcac cctctccaga tgagagtaca ccccaacagc accctctcca gatgagagta 1440caccccaaca gcaccctctc cagatgcagc cccatctcct cagcacccca ggacctgagt 1500atccccagct caggtggtga gtcctcctgt ccagcctgca tcaataaaat ggggcagtga 1560tggcctccc 156921321PRTHomo sapiens 21Met Glu Glu Gly Gln Tyr Ser Glu Ile Glu Glu Leu Pro Arg Arg Arg1 5 10 15Cys Cys Arg Arg Gly Thr Gln Ile Val Leu Leu Gly Leu Val Thr Ala 20 25 30Ala Leu Trp Ala Gly Leu Leu Thr Leu Leu Leu Leu Trp His Trp Asp 35 40 45Thr Thr Gln Ser Leu Lys Gln Leu Glu Glu Arg Ala Ala Arg Asn Val 50 55 60Ser Gln Val Ser Lys Asn Leu Glu Ser His His Gly Asp Gln Met Ala65 70 75 80Gln Lys Ser Gln Ser Thr Gln Ile Ser Gln Glu Leu Glu Glu Leu Arg 85 90 95Ala Glu Gln Gln Arg Leu Lys Ser Gln Asp Leu Glu Leu Ser Trp Asn 100 105 110Leu Asn Gly Leu Gln Ala Asp Leu Ser Ser Phe Lys Ser Gln Glu Leu 115 120 125Asn Glu Arg Asn Glu Ala Ser Asp Leu Leu Glu Arg Leu Arg Glu Glu 130 135 140Val Thr Lys Leu Arg Met Glu Leu Gln Val Ser Ser Gly Phe Val Cys145 150 155 160Asn Thr Cys Pro Glu Lys Trp Ile Asn Phe Gln Arg Lys Cys Tyr Tyr 165 170 175Phe Gly Lys Gly Thr Lys Gln Trp Val His Ala Arg Tyr Ala Cys Asp 180 185 190Asp Met Glu Gly Gln Leu Val Ser Ile His Ser Pro Glu Glu Gln Asp 195 200 205Phe Leu Thr Lys His Ala Ser His Thr Gly Ser Trp Ile Gly Leu Arg 210 215 220Asn Leu Asp Leu Lys Gly Glu Phe Ile Trp Val Asp Gly Ser His Val225 230 235 240Asp Tyr Ser Asn Trp Ala Pro Gly Glu Pro Thr Ser Arg Ser Gln Gly 245 250 255Glu Asp Cys Val Met Met Arg Gly Ser Gly Arg Trp Asn Asp Ala Phe 260

265 270Cys Asp Arg Lys Leu Gly Ala Trp Val Cys Asp Arg Leu Ala Thr Cys 275 280 285Thr Pro Pro Ala Ser Glu Gly Ser Ala Glu Ser Met Gly Pro Asp Ser 290 295 300Arg Pro Asp Pro Asp Gly Arg Leu Pro Thr Pro Ser Ala Pro Leu His305 310 315 320Ser221076DNAHomo sapiensmodified_base(1)..(1076)n = g, a, c or t 22atcagcacga atacattcac gtccaacaac acatcaacta ccaacaccat caccacgagc 60acattcatgc ccaacaacac atcaaccacc aacacctttg ccacaaacac attcatgtct 120aacaacacat caattaccaa caccagcgcc acgaacacat tcacgtccat caacacatca 180accaccaaca ccatcagcac gagcacattc acatccaata acacatcaac taccaacacc 240agcaccatga acacattcat gcccaacaac acgtaaaccc ctaacactgt caccacaaac 300accttacagc cagcagaacg ccagtcacta acaccatcgc catcagcact tcgtggttag 360caacacctca gctgacgcca atgtcaccac aaacacctca tggccagaag cagctcaacc 420accaacaccg ttattataaa tacattcttg accatcaatg cttcaactgc tgacaccatt 480accataaata catccatggc cagcaatact tcaatcacca acaccatgac cagcagcacg 540tcagcggcca tcactgtcac cacaaacacc ttcatatcca ataacacttc aaccaccatc 600atcaccacaa acacctcata gccaacagtg cctcagccac caaccccatc atgacaaaca 660cctcatggcc agcagcactt caaccaccaa caaacccctc caaggtcagc aacaccttca 720tcaccgacat cattaacaga ggtacccacc accagcagca gcttatctcc accacccaca 780ccacagccaa caccatcttt accaaccaca ccagccgtgt cttcatcact ggcaccgaca 840gcaaaaccag tgctgtggcc aggtccacca gcgattactt tccccaagca ccatccctac 900caacagccct ggtcatcatc actggcagaa cccgccaaac cagcactcct agccaatgtc 960tgggaaattg ngatnatttt cttccagtgg gaggctntgg tcaggagagc caatgggatt 1020gcaagactag gtcccacaat ccctcaatat ggtctctttn tccccttccc cccacc 107623476DNAHomo sapiens 23aggtacgcgg ggacgttcaa cgacttactg gggagagaaa gaaaaggaac gggagctgag 60agctgggagt ggagtatgaa gaccaaggaa ttctcttaaa gacctgagca gttatctgga 120actcctcaca aaatcacagt aatggatatt atttcccagt tcctactcta cactgggcat 180agatgttatg gacatcttct gagtcccaca acacccctgc aaggcagata tgatacactc 240ctttcaccta tggagaacgg aggctcaaag aggctaggtg accctcagga aacacagatg 300agaggtcccc gcccagtctg cccagctctg aaatcttcca tgccaactcc cttagggcga 360tcctgagtct agcctgtaca ggcagttcat gtggttgtat ttgaataaaa tccctttcct 420ccagaataaa aaaaaaaaaa taaaaaaaaa aatgaaaaat tgaaagggaa aaaaaa 47624421DNAHomo sapiens 24ccaccaacag tcagaggcca aggaagctgt tggctgaaaa ggtggtctat gttggcgtct 60ggatccctgc cctcctgctg actattcccg acttcatctt tgccaacgtc agtgaggcag 120atgacagata tatctgtgac cgcttctacc ccaatgactt gtgggtggtt gtgttccagt 180ttcagcacat catggttggc cttatcctgc ctggtattgt catcctgtcc tgctattgca 240ttatcatctc caagctgtca cactccaagg gccaccagaa gcgcaaggcc ctcaagacca 300cagtcatcct catcctggct ttcttcgcct gttggctgcc ttactacatt gggatcagca 360tcgactcctt catcctcctg gaaatcatca agcaagggtg tgagtttgag aacactgtgc 420a 421258747DNAHomo sapiens 25caattctgaa tcctgccttt tgcacttaat gtttcataag tatttcccca tgtcactaaa 60aattcttcca aataacattc acgatgtcca tatggaattt cagatgtgga tgaaccaaaa 120tcttgtcaac tattccacta acagtggtta tttagggatg ttcagacatt tcactattta 180aaaaaaaatg tttccacaaa tacctttgtg gcataagttt ttatgagtgg agttactgtt 240ctgaagttcc tgctgaatag aaaatgcttt ccagtgaggc tgtcccaagc cacattccca 300tcagtgacaa gcgagagaca gctggtcttt tcaaatccgg agaccaaata ttatctttga 360aaaaaaatgg atttttgcct aatttggtag tcaccaaata gcatctcatt gttcttttaa 420ttatctgctt ccttttagta gagatcccta aaaagatctg aaaggagtct tcagataaag 480gaaggagctt tcttttgtct gtctacaatc aacaaatatt tattatgcaa accattttgc 540tccgagtttt ctcctctttc cctttttgga cagatttggg agatctcacc tttcaggttt 600tagacatcgt gcagggagga gttttgaggt agggtgcagc ttacggtcca ggataaaaca 660tactgattct gccactacca ggctttgtga aaagcaagtc atgaaaacgc tctgaaattc 720tagaccttca gtagatagga tctaccgtgt ctataaaaat atgaagatcc ttaagtttta 780ttaaagattc gaaaaaagta aaagtgtttt tacggtttta ttttcatttt tatttcttac 840cgttatcgtt tattataaag gatattataa aggatacaga tgaagagata cgtaatgcaa 900ggcctgtgag aaggggcgtg gagcttccga aacctcttcc agccaccacc ctccaagaac 960ctggagtttc tttttttttt tttaattcta caaatgtaat attagaattg attttatctg 1020gccattagtg tgtgtcctaa ctcgttcgtt tctgagagtc ccatctcccg gcccgggata 1080tcatctttcc tgtgtcagtg aaagtgcaga gtagatgaga acctttaacc accaacatta 1140gggaggggtc ccagacaaag ggggtaagtc atgctctgta gagaaaaggt tccctgcctc 1200cgaactacct ctggaacact ccagtaaatg tttcctcttt tgatatagaa aagagggatc 1260gtgtgtagag tgcagtctgg gcaatccctc tcctcgggac catttcgggg taggggcctc 1320tggggtccgt gtcgcgacgc acgcgcctcg gtcccagcta tctccgcagc gggccacccc 1380gcctgcggac gcagtttctc ggccccgccc cacactcgct cccccgcccc acccagtctc 1440cgcgccggag ggaagtggcg cgagggggaa agcactgtct gcgcgcccac tgcaaacctc 1500agccagtctg agatcgcttt aaacgtctga cccccacccc cactccgccc cgcccagttc 1560ttcaacctaa tttctgattc gtgccaaagc ttgtcctctg ctcaaaatcg tggaagacgc 1620cgagtatggg gaccgaagac ctgggttcaa gcccggcttg gaatccctgc ccatccctgg 1680catttcatct ctccgggctt atttgctggt ttctccgaat gcgggccttg tctggttcac 1740gctggatccc caacgcctag aacagtgcgt ggcacgcagt tcgtccttct ataaatatcg 1800gactaaatgc atctctgtga tggtaatacc cacacggtgt tgtgagaatg aatgagtgat 1860tctgtgcaag ttcctagtga tctgttacaa aaagtactgg tcgctaaatt actcttataa 1920taaagcatac ttttaggata ataaagcact attcgcgaat tggttaccgc tattatgaaa 1980ttactgagca atacatatct acatctgatc agtctccaga attatgccaa atcctacctt 2040cttctgaaag tatctcctaa ttatctgcac ctgaccctag tgatgctgtg aatgtgcaag 2100tatagctaca tcctccgaag gaaaggatct ttactccttt tacctcctga atgggctgcg 2160tctgctgaaa gcgcggggga atgggcggtt ggaagcttgg ccctacttcc agcattgccg 2220cctactggtt gggttactcc agcaagtcac tccccttccc tgggcctcag tgtctctact 2280gtagcattcc caggtctgga attccatcca ctttagcaag gatggacgcg ccacagagag 2340acgcgttcct agcccgcgct tcccacctgt cttcaggcgc atcccgcttc cctcaaactt 2400aggaaatgcc tctgggaggt cctgtccggc tccggactca ctaccgacca cccgcaaaca 2460gcagggtccc ctgggcttcc caagccgcgc acctctccgc cccgcccctg cgccctcctt 2520cctcgcgtct gcccctctcc cccaccccgc cttctccctc cccgccccag cggcgcatgc 2580gccgcgctcg gagcgtgttt ttataaaagt ccggccgcgg ccagaaactt cagtttgttg 2640gctgcggcag caggtagcaa agtgacgccg agggcctgag tgctccagta gccaccgcat 2700ctggagaacc agcggttacc atggagggga tcagtgtaag tccagtttca acctgctttg 2760tcataaatgt acaaacgttt gaacttagag cgcagcccct ctccgagcgg gcagaagcgg 2820ccaggacatt ggaggtaccc gtactccaaa aaagggtcac cgaaaggagt tttcttgacc 2880atgcctatat agtgcgggtg ggtggggggg gagcaggatt ggaatctttt tctctgtgag 2940tcgaggagaa acgactggaa agagcgttcc agtggctgca tgtgtctccc ccttgagtcc 3000cgccgcgcgc ggcggcttgc acgctgtttg caaacgtaag aacattctgt gcacaagtgc 3060agagaaggcg tgcgcgctgc ctcgggactc agaccaccgg tctcttcctt ggggaagcgg 3120ggatgtcttg gagcgagtta cattgtctga atttagaggc ggagggcggc gtgcctgggc 3180tgagttccca ggaggagatt gcgcccgctt taacttcggg gttaagcgcc tggtgactgt 3240tcttgacact gggtgcgtgt ttgttaaact ctgtgcggcc gacggagctg tgccagtctc 3300ccagcacagt aggcagaggg cgggagaggc gggtggaccc accgcgccga tcctctgagg 3360ggatcgagtg gtggcagcag ctaggagttg atccgcccgc gcgctttggg tttgaggggg 3420aaaccttccc gccgtccgaa gcgcgcctct tccccacggc cgcgagtggg tcctgcagtt 3480cgagagtttg gggtcgtgca gaggtcagcg gagtggtttg acctcccctt tgacaccgcg 3540cagctgccag ccctgagatt tgcgctccgg ggataggagc gggtacgggg tgaggggcgg 3600gggcggttaa gaccgcacct gggctgccag gtcgccgccg cgaagactgg caggtgcaag 3660tggggaaacc gtttggctct ctccgagtcc agttgtgatg tttaaccgtc ggtggtttcc 3720agaaaccttt tgaaaccctc ttgctaggga gtttttggtt tcctgcagcg gcgcgcaatt 3780caaagacgct cgcggcggag ccgcccagtc gctccccagc accctgtggg acagagcctg 3840gcgtgtcgcc cagcggagcc cctgcagcgc tgcttgcggg cggttggcgt gggtgtagtg 3900ggcagccgcg gcggcccggg gctggacgac ccggcccccc gcgtgcccac cgcctggagg 3960cttccagctg cccacctccg gccgggttaa ctggatcagt ggcggggtaa tgggaaacca 4020cccgggagag tgaggaaatg aaacttgggg cgaggaccac gggtgcagac cccgttacct 4080tctccaccca ggaaaatgcc ccgctcccta acgtcccaaa cgcgccaagt gataaacacg 4140aggatggcaa gagacccaca caccggagga gcgcccgctt gggggaggag gtgccgtttg 4200ttcattttct gacactcccg cccaatatac cccaagcacc gaagggcctt cgttttaaga 4260ccgcattctc tttacccact acaagttgct tgaagcccag aatggtttgt atttaggcag 4320gcgtgggaaa attaagtttt tgcgccttag gagaatgagt ctttgcaacg cccccgccct 4380ccccccgtga tcctcccttc tcccctcttc cctccctggg cgaaaaactt cttacaaaaa 4440gttaatcact gcccctccta gcagcaccca ccccaccccc cacgccgcct gggagtggcc 4500tctttgtgtg tatttttttt ttcctcctaa ggaaggtttt ttttcttccc tctagtgggc 4560ggggcagagg agttagccaa gatgtgactt tgaaaccctc agcgtctcag tgcccttttg 4620ttctaaacaa agaattttgt aattggttct accaaagaag gatataatga agtcactatg 4680ggaaaagatg gggaggagag ttgtaggatt ctacattaat tctcttgtgc ccttagccca 4740ctacttcaga atttcctgaa gaaagcaagc ctgaattggt tttttaaatt gctttaaaaa 4800atttttttaa ctgggttaat gcttgctgaa ttggaagtga atgtccattc ctttgcctct 4860tttgcagata tacacttcag ataactacac cgaggaaatg ggctcagggg actatgactc 4920catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc aataaaatct tcctgcccac 4980catctactcc atcatcttct taactggcat tgtgggcaat ggattggtca tcctggtcat 5040gggttaccag aagaaactga gaagcatgac ggacaagtac aggctgcacc tgtcagtggc 5100cgacctcctc tttgtcatca cgcttccctt ctgggcagtt gatgccgtgg caaactggta 5160ctttgggaac ttcctatgca aggcagtcca tgtcatttac acagtcaacc tctacagcag 5220tgtcctcatc ctggccttca tcagtctgga ccgctacctg gccatcgtcc acgccaccaa 5280cagtcagagg ccaaggaagc tgttggctga aaaggtggtc tatgttggcg tctggatccc 5340tgccctcctg ctgactattc ccgacttcat ctttgccaac gtcagtgagg cagatgacag 5400atatatctgt gaccgcttct accccaatga cttgtgggtg gttgtgttcc agtttcagca 5460catcatggtt ggccttatcc tgcctggtat tgtcatcctg tcctgctatt gcattatcat 5520ctccaagctg tcacactcca agggccacca gaagcgcaag gccctcaaga ccacagtcat 5580cctcatcctg gctttcttcg cctgttggct gccttactac attgggatca gcatcgactc 5640cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt gagaacactg tgcacaagtg 5700gatttccatc accgaggccc tagctttctt ccactgttgt ctgaacccca tcctctatgc 5760tttccttgga gccaaattta aaacctctgc ccagcacgca ctcacctctg tgagcagagg 5820gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga cattcatctg tttccactga 5880gtctgagtct tcaagttttc actccagcta acacagatgt aaaagacttt tttttatacg 5940ataaataact tttttttaag ttacacattt ttcagatata aaagactgac caatattgta 6000cagtttttat tgcttgttgg atttttgtct tgtgtttctt tagtttttgt gaagtttaat 6060tgacttattt atataaattt tttttgtttc atattgatgt gtgtctaggc aggacctgtg 6120gccaagttct tagttgctgt atgtctcgtg gtaggactgt agaaaaggga actgaacatt 6180ccagagcgtg tagtgaatca cgtaaagcta gaaatgatcc ccagctgttt atgcatagat 6240aatctctcca ttcccgtgga acgtttttcc tgttcttaag acgtgatttt gctgtagaag 6300atggcactta taaccaaagc ccaaagtggt atagaaatgc tggtttttca gttttcagga 6360gtgggttgat ttcagcacct acagtgtaca gtcttgtatt aagttgttaa taaaagtaca 6420tgttaaactt acttagtgtt atgttctgat ttctgttgac attcttttgg ctagtagaag 6480acaaaagtaa tacatttatg gtatgcaaag cactatccta ggtatttcat tgtaatattt 6540tacttacccc ttatcacaac tctgatagat tctgcttctg ttactaatta cattttatag 6600aagaggaaac ggaggcacag aaagcctaag taacttggtt aaaggcatgt agtaagtatc 6660aaatcctgta ttttaaacca ggtaacatga cttaacgaat ctgaagcctt caccacttta 6720aattcaaatg gaagtttaga aatggccagc cagcacctat ttgtatgaaa ggtcatcttt 6780cagaggataa gcatgtataa agaagaaaag gtatgcagtc gtgtttggat tttactccac 6840catccacttg tgaaacccag gtctgtgcaa tgccagacgg tgtgtgcttt cctcatccag 6900tatcctcagt gtagataacc atcactccct tttcacagac aagagaactg agattcagag 6960actttccata cattgcactt tcaagggggc aaagccaaga actaattctg tttattgttc 7020cagctcttgc tcttaactct tacctactat tgcccttcag aacacctggg cataagtcaa 7080ctgaactgct aataaagaaa gccaaaagtg aatgttttct tcataaaatt aaccatgacc 7140aaaatactcc tcttgtaata tcttctatgc aaatctcaac acttttattc ttaaactatc 7200gcaacaccta gcacctcctc aaggactcag ccaagcagct acaagttaat actgatattt 7260gttagagtca gaaggaaggt ccactgaagc aagctccctg ttgctcacat tttgcacaag 7320attttggaga cttatgtaac cacccgttgc tattaacacg accattgtgc aagccccagg 7380ctcttgagta aatttcagct ttggtttcta tttaaagata atttctaaac tctagccata 7440cctacctcac attggaacac aaacagggta cactccaggc atgcactcag ataataagta 7500ggatataatt acgacaatat ttggtctact tttagtaatt gtttctggca cagaaaatcc 7560attttggagg aaaaattgca atgccttatc tttctgaggc aaatcacatt tgttcaaggc 7620aaattataga tcctgtgaag ggaaataact taattactta aaatagaatc caatttggct 7680gtacattttt gctgccgtct atggatctgg ggtaattcaa agtggtattc atattctact 7740tgaggacaca attagatttc agataggaaa ttatcttgag gtttcttggt tttccctgag 7800aagcctaatt ggatcaccct tcatttaagc atagttttac atgcactctc tcaaaggctt 7860agtcttaaag ccacaaccat tgagacagac ttcacttgaa ccctctctat aaatatttat 7920tctccgggag acaatagaag aaatccttgg aaggcatgct ttttctttct catcttggct 7980tgaaacctcc ttaccccaga ttcctctcct ttaccgtgga gtcacaacaa aaggaactga 8040gccaaaacaa aattcccagt gtcaccagtc ttaatggata tttcattctc ccttggaaca 8100aagatggaat agcttttttt ccaaaagaaa aacaagcctt ggctctctcc ctgccccaaa 8160agggtgcccc ccacccccat cattctctgt cccaaccctg ccatgttaga gcgtctccaa 8220agccttccct gtgtcgtggt ttgtctgaca atgtggggaa acccagtctg ctggccagcc 8280cttgcatgaa gtagctgatt gttccctctc ctcatccctt atgaatgggg cccttgaagt 8340tcagtcatgt agattcagtt gtataatgaa agctaaaata tttaaattgt atgcatgctg 8400ccaataacag catacatctg acatctaact tattaataac attaagcctg caactagggg 8460ggaaagtgga tgttttttct tgcaaagcct ttgttttcct aaaatgacac ttgaaaattt 8520atctccccct actgcaggct tcccagcccc cttttataat tatgcttaaa ttaaaataat 8580gattctggga tactcttttg gggagatacc ctacaggctt tattttaata attgaactaa 8640gtgtttgtga ctttctccta gatattgtca aatattaaat aaaggctcca taaacaattg 8700agctgtctta ttcccagata atacccattt aggaggggca aggatcc 874726568PRTHomo sapiens 26Met Asp Val Asp Glu Gly Gln Asp Met Ser Gln Val Ser Gly Lys Glu1 5 10 15Ser Pro Pro Val Ser Asp Thr Pro Asp Glu Gly Asp Glu Pro Met Pro 20 25 30Val Pro Glu Asp Leu Ser Thr Thr Ser Gly Ala Gln Gln Asn Ser Lys 35 40 45Ser Asp Arg Gly Met Ala Ser Asn Val Lys Val Glu Thr Gln Ser Asp 50 55 60Glu Glu Asn Gly Arg Ala Cys Glu Met Asn Gly Glu Glu Cys Ala Glu65 70 75 80Asp Leu Arg Met Leu Asp Ala Ser Gly Glu Lys Met Asn Gly Ser His 85 90 95Arg Asp Gln Gly Ser Ser Ala Leu Ser Gly Val Gly Gly Ile Arg Leu 100 105 110Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys Gly Ile Val Cys Ile Gly 115 120 125Pro Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu Arg Pro 130 135 140Phe Gln Cys Asn Gln Cys Ser Ser Ala Leu Ser Gly Val Gly Gly Ile145 150 155 160Arg Leu Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys Gly Ile Val Cys 165 170 175Ile Gly Pro Asn Val Leu Met Val His Lys Arg Ser His Thr Gly Glu 180 185 190Arg Pro Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly 195 200 205Asn Leu Leu Arg His Ile Lys Leu His Ser Gly Glu Lys Pro Phe Lys 210 215 220Cys His Leu Cys Asn Tyr Ala Cys Arg Arg Arg Asp Ala Leu Thr Gly225 230 235 240His Leu Arg Thr His Ser Val Gly Lys Pro His Lys Cys Gly Tyr Cys 245 250 255Gly Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg 260 265 270Cys His Asn Tyr Leu Glu Ser Met Gly Leu Pro Gly Met Tyr Pro Val 275 280 285Ile Lys Glu Glu Thr Asn His Asn Glu Met Ala Glu Asp Leu Cys Lys 290 295 300Ile Gly Ala Glu Arg Ser Leu Val Leu Asp Arg Leu Ala Ser Asn Val305 310 315 320Ala Lys Arg Lys Ser Ser Met Pro Gln Lys Phe Leu Gly Asp Lys Cys 325 330 335Leu Ser Asp Met Pro Tyr Asp Ser Ala Asn Tyr Glu Lys Glu Asp Met 340 345 350Met Thr Ser His Val Met Asp Gln Ala Ile Asn Asn Ala Ile Asn Tyr 355 360 365Leu Gly Ala Glu Ser Leu Arg Pro Leu Val Gln Thr Pro Pro Gly Ser 370 375 380Ser Glu Val Val Pro Val Ile Ser Ser Met Tyr Gln Leu His Lys Pro385 390 395 400Pro Ser Asp Gly Pro Pro Arg Ser Asn His Ser Ala Gln Asp Ala Val 405 410 415Asp Asn Leu Leu Leu Leu Ser Lys Ala Lys Ser Val Ser Ser Glu Arg 420 425 430Glu Ala Ser Pro Ser Asn Ser Cys Gln Asp Ser Thr Asp Thr Glu Ser 435 440 445Asn Ala Glu Glu Gln Arg Ser Gly Leu Ile Tyr Leu Thr Asn His Ile 450 455 460Asn Pro His Ala Arg Asn Gly Leu Ala Leu Lys Glu Glu Gln Arg Ala465 470 475 480Tyr Glu Val Leu Arg Ala Ala Ser Glu Asn Ser Gln Asp Ala Phe Arg 485 490 495Val Val Ser Thr Ser Gly Glu Gln Leu Lys Val Tyr Lys Cys Glu His 500 505 510Cys Arg Val Leu Phe Leu Asp His Val Met Tyr Thr Ile His Met Gly 515 520 525Cys His Gly Cys His Gly Phe Arg Asp Pro Phe Glu Cys Asn Met Cys 530 535 540Gly Tyr His Ser Gln Asp Arg Tyr Glu Phe Ser Ser His Ile Thr Arg545 550 555 560Gly Glu His Arg Tyr His Leu Ser 56527350DNAHomo sapiens 27ccagagagta agaataggag gagaaaacat gctgcagatg taggcggggc ccagattgta 60gacagcatag aaataatttt gggcttttcc tgttaaattc ctctagcttc taggatacat 120tttttttaac ttttgtcttt gagataattt tagatttaca gaagagttgc aaaaagagta 180gagagagttc ctgtacaccc ttcacccagc ttcctctact gctaacatct tacataatca 240tagtttcaac ctgagaaatt agcatggggt acagtcctat taatgaaacc ccaggcttta 300ttcagatttc accaggtttt cagtaacatc ctttatctgt ttcagaattt

35028850DNAHomo sapiens 28gaattccggc aaaatgcatg acagtaacaa tgtggagaaa gacattacac catctgaatt 60gcctgcaaac ccaggttgtc tgcattcaaa agagcattct attaaagcta ccttaatttg 120gcgcttattt ttcttaatca tgtttctgac aatcatagtg tgtggaatgg ttgctgcttt 180aagcgcaata agagctaact gccatcaaga gccatcagta tgtcttcaag ctgcatgccc 240agaaagctgg attggttttc aaagaaagtg tttctatttt tctgatgaca ccaagaactg 300gacatcaagt cagaggtttt gtgactcaca agatgctgat cttgctcagg ttgaaagctt 360ccaggaactg aatttcctgt tgagatataa aggcccatct gatcactgga ttgggctgag 420cagagaacaa ggccaaccat ggaaatggat aaatggtact gaatggacaa gacagtttcc 480tatcctggga gcaggagagt gtgcctattt gaatgacaaa ggtgccagta gtgccaggca 540ctacacagag aggaagtgga tttgttccaa atcagatata catgtctaga tgttacagca 600aagccccaac taatctttag aagcatattg gaactgataa ctccatttta aaatgagcaa 660agaatttatt tcttatacca acaggtatat gaaaatatgc tcaatatcac taataactgg 720gaaaatacaa atcaaaatca tagtaaaata ttacctgttt tcatggtgct aatattacct 780gttctcccac tgctaatgac atacccgaga atgagtaatt tataaataaa agagatttaa 840ttgaaaaaaa 85029191PRTHomo sapiens 29Met His Asp Ser Asn Asn Val Glu Lys Asp Ile Thr Pro Ser Glu Leu1 5 10 15Pro Ala Asn Pro Gly Cys Leu His Ser Lys Glu His Ser Ile Lys Ala 20 25 30Thr Leu Ile Trp Arg Leu Phe Phe Leu Ile Met Phe Leu Thr Ile Ile 35 40 45Val Cys Gly Met Val Ala Ala Leu Ser Ala Ile Arg Ala Asn Cys His 50 55 60Gln Glu Pro Ser Val Cys Leu Gln Ala Ala Cys Pro Glu Ser Trp Ile65 70 75 80Gly Phe Gln Arg Lys Cys Phe Tyr Phe Ser Asp Asp Thr Lys Asn Trp 85 90 95Thr Ser Ser Gln Arg Phe Cys Asp Ser Gln Asp Ala Asp Leu Ala Gln 100 105 110Val Glu Ser Phe Gln Glu Leu Asn Phe Leu Leu Arg Tyr Lys Gly Pro 115 120 125Ser Asp His Trp Ile Gly Leu Ser Arg Glu Gln Gly Gln Pro Trp Lys 130 135 140Trp Ile Asn Gly Thr Glu Trp Thr Arg Gln Phe Pro Ile Leu Gly Ala145 150 155 160Gly Glu Cys Ala Tyr Leu Asn Asp Lys Gly Ala Ser Ser Ala Arg His 165 170 175Tyr Thr Glu Arg Lys Trp Ile Cys Ser Lys Ser Asp Ile His Val 180 185 19030558DNAHomo sapiensmodified_base(1)..(558)n = g, a, c or t 30ccatgggatg gctcttctga ccattggggg ccaggccagg ccaggccagg cttagggcag 60caaggaccag gccaaagggg cagggcctcc tttggagggg ttgaggggta catcctcggc 120tggtgtttgc atccaggggt ccagcaggat ctcttccagt gagggtcggg aagaaggttt 180gggggccagg caccggcgga ttagggcaca gcaatcttgg ggaaaacatg ggcttgggaa 240gtggagctca gcttccagaa tctcctggtc cctctcaaag ggaatgtccc cacacaccat 300gtcatagagg aggatgccca gtgaccagac agtggccggg agtgcatggt actggtgtcg 360agagatccac tctggggggc tgtacaccct tgtcccatca aagtcagtgt agggttcatc 420atgaagcagg gcaccagaac caaaatcaat gagtttggca cagccacggc gtaggtctat 480caggatgntc tcatccttga tgtcacgatg gacaactnca cgggaaatgg cagtgctgga 540tggctgccac tactttgg 558312088DNAHomo sapiens 31gaattcggca cgagcgcgcg gcgaatctca acgctgcgcc gtctgcgggc gcttccgggc 60caccagtttc tctgctttcc accctggcgc cccccagccc tggctcccca gctgcgctgc 120cccgggcgtc cacgccctgc gggcttagcg ggttcagtgg gctcaatctg cgcagcgcca 180cctccatgtt gaccaagcct ctacaggggc ctcccgcgcc ccccgggacc cccacgccgc 240cgccaggagg caaggatcgg gaagcgttcg aggccgagta tcgactcggc cccctcctgg 300gtaagggggg ctttggcacc gtcttcgcag gacaccgcct cacagatcga ctccaggtgg 360ccatcaaagt gattccccgg aatcgtgtgc tgggctggtc ccccttgtca gactcagtca 420catgcccact cgaagtcgca ctgctatgga aagtgggtgc aggtggtggg caccctggcg 480tgatccgcct gcttgactgg tttgagacac aggaaggctt catgctggtc ctcgagcggc 540ctttgcccgc ccaggatctc tttgactata tcacagagaa gggcccactg ggtgaaggcc 600caagccgctg cttctttggc caagtagtgg cagccatcca gcactgccat tcccgtggag 660ttgtccatcg tgacatcaag gatgagaaca tcctgataga cctacgccgt ggctgtgcca 720aactcattga ttttggttct ggtgccctgc ttcatgatga accctacact gactttgatg 780ggacaagggt gtacagcccc ccagagtgga tctctcgaca ccagtaccat gcactcccgg 840ccactgtctg gtcactgggc atcctcctct atgacatggt gtgtggggac attccctttg 900agagggacca ggagattctg gaagctgagc tccacttccc agcccatgtc tccccagact 960gctgtgccct aatccgccgg tgcctggccc ccaaaccttc ttcccgaccc tcactggaag 1020agatcctgct ggacccctgg atgcaaacac cagccgagga tgttacccct caacccctcc 1080aaaggaggcc ctgccccttt ggcctggtcc ttgctaccct aagcctggcc tggcctggcc 1140tggcccccaa tggtcagaag agccatccca tggccatgtc acagggatag atggacattt 1200gttgacttgg ttttacaggt cattaccagt cattaaagtc cagtattact aaggtaaggg 1260attgaggatc aggggttaga agacataaac caagtttgcc cagttccctt cccaatccta 1320caaaggagcc ttcctcccag aacctgtggt ccctgatttt ggagggggaa cttcttgctt 1380ctcattttgc taaggaagtt tattttggtg aagttgttcc cattttgagc cccgggactc 1440ttattttgat gatgtgtcac cccacattgg cacctcctac taccaccaca caaacttagt 1500tcatatgctt ttacttgggc aagggtgctt tccttccaat accccagtag cttttatttt 1560agtaaaggga ccctttcccc tagcctaggg tcccatattg ggtcaagctg cttacctgcc 1620tcagcccagg attttttatt ttgggggagg taatgccctg ttgttacccc aaggcttctt 1680tttttttttt tttttttttg ggtgagggga ccctactttg ttatcccaag tgctcttatt 1740ctggtgagaa gaaccttaat tccataattt gggaaggaat ggaagatgga caccaccgga 1800caccaccaga caataggatg ggatggatgg ttttttgggg gatgggctag gggaaataag 1860gcttgctgtt tgttttcctg gggcgctccc tccaattttg cagatttttg caacctcctc 1920ctgagccggg attgtccaat tactaaaatg taaataatca cgtattgtgg ggaggggagt 1980tccaagtgtg ccctcctttt ttttcctgcc tggattattt aaaaagccat gtgtggaaac 2040ccactattta ataaaagtaa tagaatcaga aaaaaaaaaa aaaaaaaa 208832334PRTHomo sapiens 32Met Leu Thr Lys Pro Leu Gln Gly Pro Pro Ala Pro Pro Gly Thr Pro1 5 10 15Thr Pro Pro Pro Gly Gly Lys Asp Arg Glu Ala Phe Glu Ala Glu Tyr 20 25 30Arg Leu Gly Pro Leu Leu Gly Lys Gly Gly Phe Gly Thr Val Phe Ala 35 40 45Gly His Arg Leu Thr Asp Arg Leu Gln Val Ala Ile Lys Val Ile Pro 50 55 60Arg Asn Arg Val Leu Gly Trp Ser Pro Leu Ser Asp Ser Val Thr Cys65 70 75 80Pro Leu Glu Val Ala Leu Leu Trp Lys Val Gly Ala Gly Gly Gly His 85 90 95Pro Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Thr Gln Glu Gly Phe 100 105 110Met Leu Val Leu Glu Arg Pro Leu Pro Ala Gln Asp Leu Phe Asp Tyr 115 120 125Ile Thr Glu Lys Gly Pro Leu Gly Glu Gly Pro Ser Arg Cys Phe Phe 130 135 140Gly Gln Val Val Ala Ala Ile Gln His Cys His Ser Arg Gly Val Val145 150 155 160His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Arg Arg Gly 165 170 175Cys Ala Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu His Asp Glu 180 185 190Pro Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu Trp 195 200 205Ile Ser Arg His Gln Tyr His Ala Leu Pro Ala Thr Val Trp Ser Leu 210 215 220Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro Phe Glu Arg225 230 235 240Asp Gln Glu Ile Leu Glu Ala Glu Leu His Phe Pro Ala His Val Ser 245 250 255Pro Asp Cys Cys Ala Leu Ile Arg Arg Cys Leu Ala Pro Lys Pro Ser 260 265 270Ser Arg Pro Ser Leu Glu Glu Ile Leu Leu Asp Pro Trp Met Gln Thr 275 280 285Pro Ala Glu Asp Val Thr Pro Gln Pro Leu Gln Arg Arg Pro Cys Pro 290 295 300Phe Gly Leu Val Leu Ala Thr Leu Ser Leu Ala Trp Pro Gly Leu Ala305 310 315 320Pro Asn Gly Gln Lys Ser His Pro Met Ala Met Ser Gln Gly 325 330331215DNAHomo sapiens 33ggggggactt gagtatcctt tgttaccctc aggagatcct gaaaccagtc ccccatggat 60actgagggct gactgtatag tcctatcctc acggaacttt cattctaatg ggggaagact 120gactataaac aaaatatatg taataggtgg tggtaagtac cgtggagaag taacaaatgg 180ggcaaagtga gttatacagc tccattctta gaaaccttgg agtacttttc ttagtttata 240ctcgtggtgg tttccttttg tctcctttat tacatgggac tctgacatgt gcccatagct 300agggtgacag taggatctac ccgatagtag ggtggcagta ggatctaccc aaaaagcgtc 360ctgctgatac aggaccaaag catcctgttg ttctcgagcc tataaaaaga gctaatggtg 420ttgcttctct taactgtggc ctcctacact gtgttttgga tgattggtga tgtcttggat 480attctgtttc tttggaactt tgaatataca acactttact agggaattag caatggaagc 540agagcaaaga tgtacagagg aaacaatgcg taactctgat ggaattgaag tcatgaggca 600gcagagagct taaattacag ctttaaaaat ttttattttt tagagggaat ttacttggga 660gtaacagcag taatagttaa cggagccaga atgcttgagt catataattg caaagcagag 720ttgggagcaa cagatgctaa agagtagttg ctgtagttcc tctttgggtc gtaggagcag 780ttgtcatatt actatatagc tactgcatga agaagagttc ttagtgaggc ctgggtgatc 840agctcttctt agtattctgt gtgaccccat ttgacctttt aacaaatccc taagtaaata 900aatagcccct caggaaaact aagtttttct ctgctgtttt tttgcttgag agagctataa 960ctgtaataga cttatatttc tgaacatttt agtgcttgcc aatatttggt aatatttatg 1020tttcctatat ttgtaatgaa cattcttctt ccggtacatt ttttgttaaa ttattgtttg 1080atggataaaa gttcaccttt tattgtataa aattgactga gattaattta tacacattga 1140caatgggtaa atagaatttt tcagattatt aaaagctgaa ggatgcccac gtaagcaaaa 1200aaaaaaaaaa aaaaa 1215343144DNAHomo sapiens 34tcctctttcc gtgcgcgagt gcacagctcc ggaggcccga gccgaccctg gggcgtccgg 60tccggtggtc ttgcagcctc caaaccccga gtgctatacc gaactgcgcg ccaagggtgg 120gagagctgac ggcctgggcc acccttcttc cttcactggg caggctttga ggtgcttgtc 180ggtctggact gatgaaaatc catatgacct gaaagatgtc tgaaaattcc agtgacagtg 240attcatcttg tggttggact gtcatcagtc atgaggggtc agatatagaa atgttgaatt 300ctgtgacccc cactgacagc tgtgagcccg ccccagaatg ttcatcttta gagcaagagg 360agcttcaagc attgcagata gagcaaggag aaagcagcca aaatggcaca gtgcttatgg 420aagaaactgc ttatccagct ttggaggaaa ccagctcaac aattgaggca gaggaacaaa 480agatacccga agacagtatc tatattggaa ctgccagtga tgattctgat attgttaccc 540ttgagccacc taagttagaa gaaattggaa atcaagaagt tgtcattgtt gaagaagcac 600agagttcaga agactttaac atgggctctt cctctagcag ccagtatact ttctgtcagc 660cagaaactgt attttcatct cagcctagtg acgatgaatc aagtagtgat gaaaccagta 720atcagcccag tcctgccttt agacgacgcc gtgctaggaa gaagaccgtt tctgcttcag 780aatctgaaga ccggctagtt gctgaacaag aaactgaacc ttctaaggag ttgagtaaac 840gtcagttcag tagtggtctc aataagtgtg ttatacttgc tttggtgatt gcaatcagca 900tgggatttgg ccatttctat ggcacaattc agattcagaa gcgtcaacag ttagtcagaa 960agatacatga agatgaattg aatgatatga aggattatct ttcccagtgt caacaggaac 1020aagaatcttt tatagattat aagtcattga aagaaaatct tgcaaggtgt tggacactta 1080ctgaagcaga gaagatgtcc tttgaaactc agaaaacgaa ccttgctaca gaaaatcagt 1140atttaagagt atccctggag aaggaagaaa aagccttatc ctcattacag gaagagttaa 1200acaaactaag agaacagatt agaatattgg aagataaagg gacaagtact gaattagtta 1260aagaaaatca gaaacttaag cagcatttgg aagaggaaaa gcagaaaaaa cacagctttc 1320ttagtcaaag ggagactctg ttgacagaag caaagatgct aaagagagaa ctggagagag 1380aacgactagt aactacggct ttaagggggg aactccagca gttaagtggt agtcagttac 1440atggcaagtc agattctccc aatgtatata ctgaaaaaaa ggaaatagca atcttacggg 1500aaagactcac tgagctggaa cggaagctaa ccttcgaaca gcagcgttct gatttgtggg 1560aaagattgta tgttgaggca aaagatcaaa atggaaaaca aggaacagat ggaaaaaaga 1620aagggggcag aggaagccac agggctaaaa ataagtcaaa ggaaacattt ttgggttcag 1680ttaaggaaac atttgatgcc atgaagaatt ctaccaagga gtttgtaagg catcataaag 1740agaaaattaa gcaggctaaa gaagctgtga aggaaaatct gaaaaaattc tcagattcag 1800ttaaatccac tttcagacac tttaaagata ccaccaagaa tatctttgat gaaaagggta 1860ataaaagatt tggtgctaca aaagaagcag ctgaaaaacc aagaacagtt tttagtgact 1920atttacatcc acagtataag gcacctacag aaaaccattc aaggccctac tatgcaaaaa 1980gatggaagga agaaaagcca gttcacttta aagaattcag aaaaaataca aattcaaaga 2040aatgcagtcc tgggcatgat tgtagagaaa attctcattc tttcagaaag gcttgttctg 2100gtgtatttga ttgtgctcaa caagagtcca tgagcctttt taacacagtg gtgatcccta 2160taaggatgga tgaatttaga cagataattc aaaggtacat gttaaaagaa ctggatactt 2220tttgtcgctg gaacgaactt gatcagttca tcaataagtt tttcctaaac ggtgtcttta 2280tacatgatca gaagctcttc actgactttg ttaatgatgt taagattatc ttaggaaaca 2340tgaaggaata tgaagtagat aatgatggag tatttgagaa gttggatgaa tatatatata 2400gacacttctt tggtcacact ttttcccctc catatggacc caggtcggtt tacataaaac 2460cgtgtcatta cagtagtttg taacatttgt agattggata cgatttttat gatttgatga 2520gtttcttgta aggttaccgt ttctaagagt tgtgctttat ggccactgag agaattcaga 2580ataaattgaa agatggagtc taaaaattat tagctgttac aaatggaaca atttcattat 2640aacgtgatca ctttgacttg agcaaatggt ttaattttta tcttaaaatc agttaagaat 2700atataaaatc ctacctttgg ccaagtttgt ttcttttcat tatagtttat atgaaaagat 2760caccttaagt gaaattattt tccttatttt cctttaatct tttatgtatt tattcacttc 2820tggaagctag gaatgagcaa cacaaatttt actctgaagt cagaagagct catatatata 2880attctaatgt cccacctatg tccattccat gtaccagctt agttatatac tagtcacata 2940attatctttg ataaaggtag aggcacaaag aggcaaacta acaagtcaaa ttctaatgtg 3000tgtacttcat aataattttt tatccatttt catcttcttt atctttatat tctgtaacat 3060gaaacttacc taatcttcaa atgttagctt cattttttac ctttgaaata cttaatcttt 3120ctgaataaat ataatggtct ataa 314435755PRTHomo sapiens 35Met Ser Glu Asn Ser Ser Asp Ser Asp Ser Ser Cys Gly Trp Thr Val1 5 10 15Ile Ser His Glu Gly Ser Asp Ile Glu Met Leu Asn Ser Val Thr Pro 20 25 30Thr Asp Ser Cys Glu Pro Ala Pro Glu Cys Ser Ser Leu Glu Gln Glu 35 40 45Glu Leu Gln Ala Leu Gln Ile Glu Gln Gly Glu Ser Ser Gln Asn Gly 50 55 60Thr Val Leu Met Glu Glu Thr Ala Tyr Pro Ala Leu Glu Glu Thr Ser65 70 75 80Ser Thr Ile Glu Ala Glu Glu Gln Lys Ile Pro Glu Asp Ser Ile Tyr 85 90 95Ile Gly Thr Ala Ser Asp Asp Ser Asp Ile Val Thr Leu Glu Pro Pro 100 105 110Lys Leu Glu Glu Ile Gly Asn Gln Glu Val Val Ile Val Glu Glu Ala 115 120 125Gln Ser Ser Glu Asp Phe Asn Met Gly Ser Ser Ser Ser Ser Gln Tyr 130 135 140Thr Phe Cys Gln Pro Glu Thr Val Phe Ser Ser Gln Pro Ser Asp Asp145 150 155 160Glu Ser Ser Ser Asp Glu Thr Ser Asn Gln Pro Ser Pro Ala Phe Arg 165 170 175Arg Arg Arg Ala Arg Lys Lys Thr Val Ser Ala Ser Glu Ser Glu Asp 180 185 190Arg Leu Val Ala Glu Gln Glu Thr Glu Pro Ser Lys Glu Leu Ser Lys 195 200 205Arg Gln Phe Ser Ser Gly Leu Asn Lys Cys Val Ile Leu Ala Leu Val 210 215 220Ile Ala Ile Ser Met Gly Phe Gly His Phe Tyr Gly Thr Ile Gln Ile225 230 235 240Gln Lys Arg Gln Gln Leu Val Arg Lys Ile His Glu Asp Glu Leu Asn 245 250 255Asp Met Lys Asp Tyr Leu Ser Gln Cys Gln Gln Glu Gln Glu Ser Phe 260 265 270Ile Asp Tyr Lys Ser Leu Lys Glu Asn Leu Ala Arg Cys Trp Thr Leu 275 280 285Thr Glu Ala Glu Lys Met Ser Phe Glu Thr Gln Lys Thr Asn Leu Ala 290 295 300Thr Glu Asn Gln Tyr Leu Arg Val Ser Leu Glu Lys Glu Glu Lys Ala305 310 315 320Leu Ser Ser Leu Gln Glu Glu Leu Asn Lys Leu Arg Glu Gln Ile Arg 325 330 335Ile Leu Glu Asp Lys Gly Thr Ser Thr Glu Leu Val Lys Glu Asn Gln 340 345 350Lys Leu Lys Gln His Leu Glu Glu Glu Lys Gln Lys Lys His Ser Phe 355 360 365Leu Ser Gln Arg Glu Thr Leu Leu Thr Glu Ala Lys Met Leu Lys Arg 370 375 380Glu Leu Glu Arg Glu Arg Leu Val Thr Thr Ala Leu Arg Gly Glu Leu385 390 395 400Gln Gln Leu Ser Gly Ser Gln Leu His Gly Lys Ser Asp Ser Pro Asn 405 410 415Val Tyr Thr Glu Lys Lys Glu Ile Ala Ile Leu Arg Glu Arg Leu Thr 420 425 430Glu Leu Glu Arg Lys Leu Thr Phe Glu Gln Gln Arg Ser Asp Leu Trp 435 440 445Glu Arg Leu Tyr Val Glu Ala Lys Asp Gln Asn Gly Lys Gln Gly Thr 450 455 460Asp Gly Lys Lys Lys Gly Gly Arg Gly Ser His Arg Ala Lys Asn Lys465 470 475 480Ser Lys Glu Thr Phe Leu Gly Ser Val Lys Glu Thr Phe Asp Ala Met 485 490 495Lys Asn Ser Thr Lys Glu Phe Val Arg His His Lys Glu Lys Ile Lys 500 505 510Gln Ala Lys Glu Ala Val Lys Glu Asn Leu Lys Lys Phe Ser Asp Ser 515 520 525Val Lys Ser Thr Phe Arg His Phe Lys Asp Thr Thr Lys Asn Ile Phe 530 535 540Asp Glu Lys Gly Asn Lys Arg Phe Gly Ala Thr Lys Glu Ala Ala Glu545 550 555 560Lys Pro Arg Thr Val Phe Ser Asp Tyr Leu His Pro Gln Tyr Lys Ala 565 570 575Pro Thr Glu Asn His Ser Arg Pro Tyr Tyr Ala Lys Arg Trp Lys Glu 580 585 590Glu Lys Pro Val His Phe Lys Glu Phe Arg Lys

Asn Thr Asn Ser Lys 595 600 605Lys Cys Ser Pro Gly His Asp Cys Arg Glu Asn Ser His Ser Phe Arg 610 615 620Lys Ala Cys Ser Gly Val Phe Asp Cys Ala Gln Gln Glu Ser Met Ser625 630 635 640Leu Phe Asn Thr Val Val Ile Pro Ile Arg Met Asp Glu Phe Arg Gln 645 650 655Ile Ile Gln Arg Tyr Met Leu Lys Glu Leu Asp Thr Phe Cys Arg Trp 660 665 670Asn Glu Leu Asp Gln Phe Ile Asn Lys Phe Phe Leu Asn Gly Val Phe 675 680 685Ile His Asp Gln Lys Leu Phe Thr Asp Phe Val Asn Asp Val Lys Ile 690 695 700Ile Leu Gly Asn Met Lys Glu Tyr Glu Val Asp Asn Asp Gly Val Phe705 710 715 720Glu Lys Leu Asp Glu Tyr Ile Tyr Arg His Phe Phe Gly His Thr Phe 725 730 735Ser Pro Pro Tyr Gly Pro Arg Ser Val Tyr Ile Lys Pro Cys His Tyr 740 745 750Ser Ser Leu 75536558DNAHomo sapiensmodified_base(1)..(558)n = g, a, c or t 36ccatgggatg gctcttctga ccattggggg ccaggccagg ccaggccagg cttagggcag 60caaggaccag gccaaagggg cagggcctcc tttggagggg ttgaggggta catcctcggc 120tggtgtttgc atccaggggt ccagcaggat ctcttccagt gagggtcggg aagaaggttt 180gggggccagg caccggcgga ttagggcaca gcaatcttgg ggaaaacatg ggcttgggaa 240gtggagctca gcttccagaa tctcctggtc cctctcaaag ggaatgtccc cacacaccat 300gtcatagagg aggatgccca gtgaccagac agtggccggg agtgcatggt actggtgtcg 360agagatccac tctggggggc tgtacaccct tgtcccatca aagtcagtgt agggttcatc 420atgaagcagg gcaccagaac caaaatcaat gagtttggca cagccacggc gtaggtctat 480caggatgntc tcatccttga tgtcacgatg gacaactnca cgggaaatgg cagtgctgga 540tggctgccac tactttgg 5583786PRTHomo sapiensMOD_RES(1)..(86)Xaa = any amino acid 37Gln Val Val Ala Xaa Ile Gln His Cys His Ser Arg Gly Val Val His1 5 10 15Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Arg Arg Gly Cys 20 25 30Ala Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu His Asp Glu Pro 35 40 45Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu Trp Ile 50 55 60Ser Arg His Gln Tyr His Ala Leu Pro Ala Thr Val Trp Ser Leu Gly65 70 75 80Ile Xaa Leu Tyr Asp Met 8538584DNAHomo sapiensmodified_base(1)..(584)n = g, a, c or t 38aaataatcca ggcaggagaa gagaggaggg cacacttgga actcccctcc ccacaatacg 60tgattattta cattttagta attggacaat cccggctcag gaggaggttg caagaatctg 120caaaagttgg agggagcgcc ccaggagaac aaacagcaag ccttatttcc cctagcccat 180cccccaaaaa accatccatc ccatcctagt gtctggtggt gtccggtggt gtccatcttc 240cattccttcc caaattatgg aagtaaggtt cttctcacca gaataagagc acttgggata 300acagagtagg gtcccctcac ccaaaaaaaa aaaaaaaaan gaagccttgg ggtaacaaca 360gggcattacc tcccccagaa taaagaatcc tgggctgagg caggtaagca gcttgaccca 420atatgggacc ctaggctagg ggaaagggtc cctttactaa aataaaagct actggggtat 480tggaaggaaa gcacccttgc ccaagtaaga gcatatgaac taagtttgng tggnggtagt 540aggaggngcc aatgtggggt gacacatcat cagaataaga gtcc 584392052DNAHomo sapiens 39cgcgcgcggc gaatctcaac gctgcgccgt ctgcgggcgc ttccgggcca ccagtttctc 60tgctttccac cctggcgccc cccagccctg gctccccagc tgcgctgccc cgggcgtcca 120cgccctgcgg gcttagcggg ttcagtgggc tcaatctgcg cagcgccacc tccatgttga 180ccaagcctct acaggggcct cccgcgcccc ccgggacccc cacgccgccg ccaggaggca 240aggatcggga agcgttcgag gccgagtatc gactcggccc cctcctgggt aaggggggct 300ttggcaccgt cttcgcagga caccgcctca cagatcgact ccaggtggcc atcaaagtga 360ttccccggaa tcgtgtgctg ggctggtccc ccttgtcaga ctcagtcaca tgcccactcg 420aagtcgcact gctatggaaa gtgggtgcag gtggtgggca ccctggcgtg atccgcctgc 480ttgactggtt tgagacacag gagggcttca tgctggtcct cgagcggcct ttgcccgccc 540aggatctctt tgactatatc acagagaagg gcccactggg tgaaggccca agccgctgct 600tctttggcca agtagtggca gccatccagc actgccattc ccgtggagtt gtccatcgtg 660acatcaagga tgagaacatc ctgatagacc tacgccgtgg ctgtgccaaa ctcattgatt 720ttggttctgg tgccctgctt catgatgaac cctacactga ctttgatggg acaagggtgt 780acagcccccc agagtggatc tctcgacacc agtaccatgc actcccggcc actgtctggt 840cactgggcat cctcctctat gacatggtgt gtggggacat tccctttgag agggaccagg 900agattctgga agctgagctc cacttcccag cccatgtctc cccagactgc tgtgccctaa 960tccgccggtg cctggccccc aaaccttctt cccgaccctc actggaagag atcctgctgg 1020acccctggat gcaaacacca gccgaggatg tacccctcaa cccctccaaa ggaggccctg 1080cccctttggc ctggtccttg ctaccctaag cctggcctgg cctggcctgg cccccaatgg 1140tcagaagagc catcccatgg ccatgtcaca gggatagatg gacatttgtt gacttggttt 1200tacaggtcat taccagtcat taaagtccag tattactaag gtaagggatt gaggatcagg 1260ggttagaaga cataaaccaa gtctgcccag ttcccttccc aatcctacaa aggagccttc 1320ctcccagaac ctgtggtccc tgattctgga gggggaactt cttgcttctc attttgctaa 1380ggaagtttat tttggtgaag ttgttcccat tctgagcccc gggactctta ttctgatgat 1440gtgtcacccc acattggcac ctcctactac caccacacaa acttagttca tatgctctta 1500cttgggcaag ggtgctttcc ttccaatacc ccagtagctt ttattttagt aaagggaccc 1560tttcccctag cctagggtcc catattgggt caagctgctt acctgcctca gcccaggatt 1620ctttattctg ggggaggtaa tgccctgttg ttaccccaag gcttcttttt tttttttttt 1680tgggtgaggg gaccctactc tgttatccca agtgctctta ttctggtgag aagaacctta 1740cttccataat ttgggaagga atggaagatg gacaccaccg gacaccacca gacactagga 1800tgggatggat ggttttttgg gggatgggct aggggaaata aggcttgctg tttgttctcc 1860tggggcgctc cctccaactt ttgcagattc ttgcaacctc ctcctgagcc gggattgtcc 1920aattactaaa atgtaaataa tcacgtattg tggggagggg agttccaagt gtgccctcct 1980ctcttctcct gcctggatta tttaaaaagc catgtgtgga aacccactat ttaataaaag 2040taatagaatc ag 205240311PRTHomo sapiens 40Met Leu Thr Lys Pro Leu Gln Gly Pro Pro Ala Pro Pro Gly Thr Pro1 5 10 15Thr Pro Pro Pro Gly Gly Lys Asp Arg Glu Ala Phe Glu Ala Glu Tyr 20 25 30Arg Leu Gly Pro Leu Leu Gly Lys Gly Gly Phe Gly Thr Val Phe Ala 35 40 45Gly His Arg Leu Thr Asp Arg Leu Gln Val Ala Ile Lys Val Ile Pro 50 55 60Arg Asn Arg Val Leu Gly Trp Ser Pro Leu Ser Asp Ser Val Thr Cys65 70 75 80Pro Leu Glu Val Ala Leu Leu Trp Lys Val Gly Ala Gly Gly Gly His 85 90 95Pro Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Thr Gln Glu Gly Phe 100 105 110Met Leu Val Leu Glu Arg Pro Leu Pro Ala Gln Asp Leu Phe Asp Tyr 115 120 125Ile Thr Glu Lys Gly Pro Leu Gly Glu Gly Pro Ser Arg Cys Phe Phe 130 135 140Gly Gln Val Val Ala Ala Ile Gln His Cys His Ser Arg Gly Val Val145 150 155 160His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Arg Arg Gly 165 170 175Cys Ala Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu His Asp Glu 180 185 190Pro Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro Pro Glu Trp 195 200 205Ile Ser Arg His Gln Tyr His Ala Leu Pro Ala Thr Val Trp Ser Leu 210 215 220Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro Phe Glu Arg225 230 235 240Asp Gln Glu Ile Leu Glu Ala Glu Leu His Phe Pro Ala His Val Ser 245 250 255Pro Asp Cys Cys Ala Leu Ile Arg Arg Cys Leu Ala Pro Lys Pro Ser 260 265 270Ser Arg Pro Ser Leu Glu Glu Ile Leu Leu Asp Pro Trp Met Gln Thr 275 280 285Pro Ala Glu Asp Val Pro Leu Asn Pro Ser Lys Gly Gly Pro Ala Pro 290 295 300Leu Ala Trp Ser Leu Leu Pro305 31041105DNAHomo sapiensmodified_base(1)..(105)n = g, a, c or t 41ctggaactgc acntagtccc agctctcctc ggccgcggtc tcctcggggn tggtgccgta 60cttttggatg gttttctcta cnacntcccg caagcttccn tccag 105421125DNAHomo sapiens 42gtctccccca ctgtcagcac ctcttctgtg tggtgagtgg accgcttacc ccactaggtg 60aagatgtcag cccaggagag ctgcctcagc ctcatcaagt acttcctctt cgttttcaac 120ctcttcttct tcgtcctcgg cagcctgatc ttctgcttcg gcatctggat cctcatcgac 180aagaccagct tcgtgtcctt tgtgggcttg gccttcgtgc ctctgcagat ctggtccaaa 240gtcctggcca tctcaggaat cttcaccatg ggcatcgccc tcctgggttg tgtgggggcc 300ctcaaggagc tccgctgcct cctgggcctg tattttggga tgctgctgct cctgtttgcc 360acacagatca ccctgggaat cctcatctcc actcagcggg cccagctgga gcgaagcttg 420cgggacgtcg tagagaaaac catccaaaag tacggcacca accccgagga gaccgcggcc 480gaggagagct gggactatgt gcagttccag ctgcgctgct gcggctggca ctacccgcag 540gactggttcc aagtcctcat cctgagaggt aacgggtcgg aggcgcaccg cgtgccctgc 600tcctgctaca acttgtcggc gaccaacgac tccacaatcc tagataaggt gatcttgccc 660cagctcagca ggcttggaca cctggcgcgg tccagacaca gtgcagacat ctgcgctgtc 720cctgcagaga gccacatcta ccgcgagggc tgcgcgcagg gcctccagaa gtggctgcac 780aacaacctta tttccatagt gggcatttgc ctgggcgtcg gcctactcga gctcgggttc 840atgacgctct cgatattcct gtgcagaaac ctggaccacg tctacaaccg gctcgctcga 900taccgttagg ccccgccctc cccaaagtcc cgccccgccc ccgtcacgtg cgctgggcac 960ttccctgctg cctgtaaata tttgtttaat ccccagttcg cctggagccc tccgccttca 1020cattcccctg gggacccacg tggctgcgtg cccctgctgc tgtcacctct cccacgggac 1080ctggggcttt cgtccacagc ttcctgtccc catctgtcgg cctac 112543281PRTHomo sapiens 43Met Ser Ala Gln Glu Ser Cys Leu Ser Leu Ile Lys Tyr Phe Leu Phe1 5 10 15Val Phe Asn Leu Phe Phe Phe Val Leu Gly Ser Leu Ile Phe Cys Phe 20 25 30Gly Ile Trp Ile Leu Ile Asp Lys Thr Ser Phe Val Ser Phe Val Gly 35 40 45Leu Ala Phe Val Pro Leu Gln Ile Trp Ser Lys Val Leu Ala Ile Ser 50 55 60Gly Ile Phe Thr Met Gly Ile Ala Leu Leu Gly Cys Val Gly Ala Leu65 70 75 80Lys Glu Leu Arg Cys Leu Leu Gly Leu Tyr Phe Gly Met Leu Leu Leu 85 90 95Leu Phe Ala Thr Gln Ile Thr Leu Gly Ile Leu Ile Ser Thr Gln Arg 100 105 110Ala Gln Leu Glu Arg Ser Leu Arg Asp Val Val Glu Lys Thr Ile Gln 115 120 125Lys Tyr Gly Thr Asn Pro Glu Glu Thr Ala Ala Glu Glu Ser Trp Asp 130 135 140Tyr Val Gln Phe Gln Leu Arg Cys Cys Gly Trp His Tyr Pro Gln Asp145 150 155 160Trp Phe Gln Val Leu Ile Leu Arg Gly Asn Gly Ser Glu Ala His Arg 165 170 175Val Pro Cys Ser Cys Tyr Asn Leu Ser Ala Thr Asn Asp Ser Thr Ile 180 185 190Leu Asp Lys Val Ile Leu Pro Gln Leu Ser Arg Leu Gly His Leu Ala 195 200 205Arg Ser Arg His Ser Ala Asp Ile Cys Ala Val Pro Ala Glu Ser His 210 215 220Ile Tyr Arg Glu Gly Cys Ala Gln Gly Leu Gln Lys Trp Leu His Asn225 230 235 240Asn Leu Ile Ser Ile Val Gly Ile Cys Leu Gly Val Gly Leu Leu Glu 245 250 255Leu Gly Phe Met Thr Leu Ser Ile Phe Leu Cys Arg Asn Leu Asp His 260 265 270Val Tyr Asn Arg Leu Ala Arg Tyr Arg 275 280442915DNAHomo sapiens 44agccccgccg cgatgcccgc gcgcccagga cgcctcctcc cgctgctggc ccggccggcg 60gccctgactg cgctgctgct gctgctgctg ggccatggcg gcggcgggcg ctggggcgcc 120cgggcccagg aggcggcggc ggcggcggcg gacgggcccc ccgcggcaga cggcgaggac 180ggacaggacc cgcacagcaa gcacctgtac acggccgaca tgttcacgca cgggatccag 240agcgccgcgc acttcgtcat gttcttcgcg ccctggtgtg gacactgcca gcggctgcag 300ccgacttgga atgacctggg agacaaatac aacagcatgg aagatgccaa agtctatgtg 360gctaaagtgg actgcacggc ccactccgac gtgtgctccg cccagggggt gcgaggatac 420cccaccttaa agcttttcaa gccaggccaa gaagctgtga agtaccaggg tcctcgggac 480ttccagacac tggaaaactg gatgctgcag acactgaacg aggagccagt gacaccagag 540ccggaagtgg aaccgcccag tgcccccgag ctcaagcaag ggctgtatga gctctcagca 600agcaactttg agctgcacgt tgcacaaggc gaccacttta tcaagttctt cgctccgtgg 660tgtggtcact gcaaagccct ggctccaacc tgggagcagc tggctctggg ccttgaacat 720tccgaaactg tcaagattgg caaggttgat tgtacacagc actatgaact ctgctccgga 780aaccaggttc gtggctatcc cactcttctc tggttccgag atgggaaaaa ggtggatcag 840tacaagggaa agcgggattt ggagtcactg agggagtacg tggagtcgca gctgcagcgc 900acagagactg gagcgacgga gaccgtcacg ccctcagagg ccccggtgct ggcagctgag 960cccgaggctg acaagggcac tgtgttggca ctcactgaaa ataacttcga tgacaccatt 1020gcagaaggaa taaccttcat caagttttat gctccatggt gtggtcattg taggactctg 1080gctcctactt gggaggaact ctctaaaaag gaattccctg gtctggcggg ggtcaagatc 1140gccgaagtag actgcactgc tgaacggaat atctgcagca agtattcggt acgaggctac 1200cccacgttat tgcttttccg aggagggaag aaagtcagtg agcacagtgg aggcagagac 1260cttgactcgt tacaccgctt tgtcctgagc caagcgaaag acgaacttta ggaacacagt 1320tggaggtcac ctctcctgcc cagctcccgc accctgcgtt taggagttca gtcccacaga 1380ggccactggg ttcccagtgg tggctgttca gaaagcagaa catactaagc gtgaggtatc 1440ttctttgtgt gtgtgttttc caagccaaca cactctacag attctttatt aaatgtgtaa 1500ctcatggtca ctgtgtaaac attttcagtg gcgatatatc ccctttgacc ttctcttgat 1560gaaatttaca tggtttcctt tgagactaaa atagcgttga gggaaatgaa attgctggac 1620tatttgtggc tcctgagttg agtgattttg gtgaaagaaa gcacatccaa agcatagttt 1680acctgcccac gagttctgga aaggttgcct tgtggcagta ttgacgttcc tctgatctta 1740aggtcacagt tgactcaata ctgtgttggt ccgtagcatg gagcagattg aaatgcaaaa 1800acccacacct ctggaggata ccttcacggc cgctgctgga gcttctgttg ctgtgaatac 1860ttctctcagt gtgagaggtt agccgtgatg aaagcagcgt tacttctgac cgtgcctgag 1920taagagaatg ctgatgccat aactttatgt gtcgatactt gtcaaatcag ttactgttca 1980ggggatcctt ctgtttctca cggggtgaaa catgtcttta gttcctcatg ttaacacgaa 2040gccagagccc acatgaactg ttggatgtct tccttagaaa gggtaggcat ggaaaattcc 2100acgaggctca ttctcagtat ctcattaact cattgaaaga ttccagttgt atttgtcacc 2160tggggtgaca agaccagaca ggctttccca ggcctgggta tccagggagg ctctgcagcc 2220ctgctgaagg gccctaacta gagttctaga gtttctgatt ctgtttctca gtagtccttt 2280tagaggcttg ctatacttgg tctgcttcaa ggaggtcgac cttctaatgt atgaagaatg 2340ggatgcattt gatctcaaga ccaaagacag atgtcagtgg gctgctctgg ccctggtgtg 2400cacggctgtg gcagctgttg atgccagtgt cctctaactc atgctgtcct tgtgattaaa 2460cacctctatc tcccttggga ataagcacat acaggcttaa gctctaagat agataggtgt 2520ttgtcctttt accatcgagc tacttcccat aataaccact ttgcatccaa cactcttcac 2580ccacctccca tacgcaaggg gatgtggata cttggcccaa agtaactggt ggtaggaatc 2640ttagaaacaa gaccacttat actgtctgtc tgaggcagaa gataacagca gcatctcgac 2700cagcctctgc cttaaaggaa atctttatta atcacgtatg gttcacagat aattcttttt 2760ttaaaaaaac ccaacctcct agagaagcac aactgtcaag agtcttgtac acacaacttc 2820agctttgcat cacgagtctt gtattccaag aaaatcaaag tggtacaatt tgtttgttta 2880cactatgata ctttctaaat aaactccttt ttttt 291545432PRTHomo sapiens 45Met Pro Ala Arg Pro Gly Arg Leu Leu Pro Leu Leu Ala Arg Pro Ala1 5 10 15Ala Leu Thr Ala Leu Leu Leu Leu Leu Leu Gly His Gly Gly Gly Gly 20 25 30Arg Trp Gly Ala Arg Ala Gln Glu Ala Ala Ala Ala Ala Ala Asp Gly 35 40 45Pro Pro Ala Ala Asp Gly Glu Asp Gly Gln Asp Pro His Ser Lys His 50 55 60Leu Tyr Thr Ala Asp Met Phe Thr His Gly Ile Gln Ser Ala Ala His65 70 75 80Phe Val Met Phe Phe Ala Pro Trp Cys Gly His Cys Gln Arg Leu Gln 85 90 95Pro Thr Trp Asn Asp Leu Gly Asp Lys Tyr Asn Ser Met Glu Asp Ala 100 105 110Lys Val Tyr Val Ala Lys Val Asp Cys Thr Ala His Ser Asp Val Cys 115 120 125Ser Ala Gln Gly Val Arg Gly Tyr Pro Thr Leu Lys Leu Phe Lys Pro 130 135 140Gly Gln Glu Ala Val Lys Tyr Gln Gly Pro Arg Asp Phe Gln Thr Leu145 150 155 160Glu Asn Trp Met Leu Gln Thr Leu Asn Glu Glu Pro Val Thr Pro Glu 165 170 175Pro Glu Val Glu Pro Pro Ser Ala Pro Glu Leu Lys Gln Gly Leu Tyr 180 185 190Glu Leu Ser Ala Ser Asn Phe Glu Leu His Val Ala Gln Gly Asp His 195 200 205Phe Ile Lys Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala 210 215 220Pro Thr Trp Glu Gln Leu Ala Leu Gly Leu Glu His Ser Glu Thr Val225 230 235 240Lys Ile Gly Lys Val Asp Cys Thr Gln His Tyr Glu Leu Cys Ser Gly 245 250 255Asn Gln Val Arg Gly Tyr Pro Thr Leu Leu Trp Phe Arg Asp Gly Lys 260 265 270Lys Val Asp Gln Tyr Lys Gly Lys Arg Asp Leu Glu Ser Leu Arg Glu 275 280 285Tyr Val Glu Ser Gln Leu Gln Arg Thr Glu Thr Gly Ala Thr Glu Thr 290 295 300Val Thr Pro Ser Glu Ala Pro Val Leu Ala Ala Glu Pro Glu Ala Asp305 310 315 320Lys Gly Thr Val Leu Ala Leu Thr Glu Asn Asn Phe Asp Asp Thr Ile 325 330 335Ala Glu Gly Ile Thr Phe Ile Lys Phe Tyr Ala Pro Trp Cys Gly His 340 345 350Cys Arg Thr Leu Ala Pro Thr Trp Glu Glu Leu

Ser Lys Lys Glu Phe 355 360 365Pro Gly Leu Ala Gly Val Lys Ile Ala Glu Val Asp Cys Thr Ala Glu 370 375 380Arg Asn Ile Cys Ser Lys Tyr Ser Val Arg Gly Tyr Pro Thr Leu Leu385 390 395 400Leu Phe Arg Gly Gly Lys Lys Val Ser Glu His Ser Gly Gly Arg Asp 405 410 415Leu Asp Ser Leu His Arg Phe Val Leu Ser Gln Ala Lys Asp Glu Leu 420 425 43046551DNAHomo sapiens 46ccagccagtg acagaaaaaa gagtgaatgt gcctttaaga agaagagcaa tgagacacag 60tgtttcaact tcatccgtgt cctggtttct tacaatgtca cccatctcta cacctgcggc 120accttcgcct tcagccctgc ttgtaccttc attgaacttc aagattccta cctgttgccc 180atctcggagg acaaggtcat ggagggaaaa ggccaaagcc cctttgaccc cgctcacaag 240catacggctg tcttggtgga tgggatgctc tattctggta ctatgaacaa cttcctgggc 300agtgagccca tcctgatgcg cacactggga tcccagcctg tcctcaagac cgacaacttc 360ctccgctggc tgcatcatga cgcctccttt gtggcagcca tcccttcgac ccaggtcgtc 420tacttcttct tcgaggagac agccagcgag tttgacttct ttgagaggct ccacacatcg 480cgggtggcta gagtctgcaa gaatgacgtg ggcggcgaaa agctgctgca gaagaagtgg 540accaccttcc t 551473252DNAHomo sapiens 47aggatgatga aagtgagacc gtcttagggc ccttccagat agtgaacctt ctctgcccca 60atgccccacc cctgccacca atacacacgc ttctgctgcc tggggctctc ctattggtcc 120tcggggggat gtggtaagaa ctgctcaccc agaaagtgcc cgggtgcctg tttccccaga 180cctccctggt gacagtctgt ggctgagcat ggccctccca gccctgggcc tggacccctg 240gagcctcctg ggccttttcc tcttccaact gcttcagctg ctgctgccga cgacgaccgc 300ggggggaggc gggcaggggc ccatgcccag ggtcagatac tatgcagggg atgaacgtag 360ggcacttagc ttcttccacc agaagggcct ccaggatttt gacactctgc tcctgagtgg 420tgatggaaat actctctacg tgggggctcg agaagccatt ctggccttgg atatccagga 480tccaggggtc cccaggctaa agaacatgat accgtggcca gccagtgaca gaaaaaagag 540tgaatgtgcc tttaagaaga agagcaatga gacacagtgt ttcaacttca tccgtgtcct 600ggtttcttac aatgtcaccc atctctacac ctgcggcacc ttcgccttca gccctgcttg 660taccttcatt gaacttcaag attcctacct gttgcccatc tcggaggaca aggtcatgga 720gggaaaaggc caaagcccct ttgaccccgc tcacaagcat acggctgtct tggtggatgg 780gatgctctat tctggtacta tgaacaactt cctgggcagt gagcccatcc tgatgcgcac 840actgggatcc cagcctgtcc tcaagaccga caacttcctc cgctggctgc atcatgacgc 900ctcctttgtg gcagccatcc cttcgaccca ggtcgtctac ttcttcttcg aggagacagc 960cagcgagttt gacttctttg agaggctcca cacatcgcgg gtggctagag tctgcaagaa 1020tgacgtgggc ggcgaaaagc tgctgcagaa gaagtggacc accttcctga aggcccagct 1080gctctctgca cccagccggg gcagctgccc ttcaacgtca tccgccacgc ggtcctgctc 1140cccgccgatt ctcccacagc tccccacatc tacgcagtct tcacctccca gtgggcaggt 1200tggcgggacc aggagctctg cggtttgtgc cttctctctc ttggacattg aacgtgtctt 1260taaggggaaa ttcaaagagt tgaacaaaga aacttcacgc tggactactt ataggggccc 1320tgagaccaac ccccggccag gcagttgctc agtgggcccc tcctctgata aggccctgac 1380cttcatgaag gaccatttcc tgatggatga gcaagtggtg gggacgcccc tgctggtgaa 1440atctggcgtg gagtatacac ggcttgcagt ggagacagcc cagggccttg atgggcacag 1500ccatcttgtc atgtacctgg gaaccaccac agggtcgctc cacaaggctg tggtaagtgg 1560ggacagcagt gctcatctgg tggaagagat tcagctgttc cctgaccctg aacctgttcg 1620caacctgcag ctggccccca cccagggtgc agtgtttgta ggcttctcag gaggtgtctg 1680gagggtgccc cgagccaact gtagtgtcta tgagagctgt gtggactgtg tccttgcccg 1740ggacccccac tgtgcctggg accctgagtc ccgaacctgt tgcctcctgt ctgcccccaa 1800cctgaactcc tggaagcagg acatggagcg ggggaaccca gagtgggcat gtgccagtgg 1860ccccatgagc aggagccttc ggcctcagag ccgcccgcaa atcattaaag aagtcctggc 1920tgtccccaac tccatcctgg agctcccctg cccccacctg tcagccttgg cctcttatta 1980ttggagtcat ggcccagcag cagtcccaga agcctcttcc actgtctaca atggctccct 2040cttgctgata gtgcaggatg gagttggggg tctctaccag tgctgggcaa ctgagaatgg 2100cttttcatac cctgtgatct cctactgggt ggacagccag gaccagaccc tggccctgga 2160tcctgaactg gcaggcatcc cccgggagca tgtgaaggtc ccgttgacca gggtcagtgg 2220tggggccgcc ctggctgccc agcagtccta ctggccccac tttgtcactg tcactgtcct 2280ctttgcctta gtgctttcag gagccctcat catcctcgtg gcctccccat tgagagcact 2340ccgggctcgg ggcaaggttc agggctgtga gaccctgcgc cctggggaga aggccccgtt 2400aagcagagag caacacctcc agtctcccaa ggaatgcagg acctctgcca gtgatgtgga 2460cgctgacaac aactgcctag gcactgaggt agcttaaact ctaggcacag gccggggctg 2520cggtgcaggc acctggccat gctggctggg cggcccaagc acagccctga ctaggatgac 2580agcagcacaa aagaccacct ttctcccctg agaggagctt ctgctactct gcatcactga 2640tgacactcag cagggtgatg cacagcagtc tgcctcccct atgggactcc cttctaccaa 2700gcacatgagc tctctaacag ggtgggggct acccccagac ctgctcctac actgatattg 2760aagaacctgg agaggatcct tcagttctgg ccattccagg gaccctccag aaacacagtg 2820tttcaagaga ccctaaaaaa cctgcctgtc ccaggaccct atggtaatga acaccaaaca 2880tctaaacaat catatgctaa catgccactc ctggaaactc cactctgaag ctgccgcttt 2940ggacaccaac actcccttct cccagggtca tgcagggatc tgctccctcc tgcttccctt 3000accagtcgtg caccgctgac tcccaggaag tctttcctga agtctgacca cctttcttct 3060tgcttcagtt ggggcagact ctgatccctt ctgccctggc agaatggcag gggtaatctg 3120agccttcttc actcctttac cctagctgac cccttcacct ctccccctcc cttttccttt 3180gttttgggat tcagaaaact gcttgtcaga gactgtttat tttttattaa aaatataagg 3240cttatgtatg at 325248762PRTHomo sapiens 48Met Ala Leu Pro Ala Leu Gly Leu Asp Pro Trp Ser Leu Leu Gly Leu1 5 10 15Phe Leu Phe Gln Leu Leu Gln Leu Leu Leu Pro Thr Thr Thr Ala Gly 20 25 30Gly Gly Gly Gln Gly Pro Met Pro Arg Val Arg Tyr Tyr Ala Gly Asp 35 40 45Glu Arg Arg Ala Leu Ser Phe Phe His Gln Lys Gly Leu Gln Asp Phe 50 55 60Asp Thr Leu Leu Leu Ser Gly Asp Gly Asn Thr Leu Tyr Val Gly Ala65 70 75 80Arg Glu Ala Ile Leu Ala Leu Asp Ile Gln Asp Pro Gly Val Pro Arg 85 90 95Leu Lys Asn Met Ile Pro Trp Pro Ala Ser Asp Arg Lys Lys Ser Glu 100 105 110Cys Ala Phe Lys Lys Lys Ser Asn Glu Thr Gln Cys Phe Asn Phe Ile 115 120 125Arg Val Leu Val Ser Tyr Asn Val Thr His Leu Tyr Thr Cys Gly Thr 130 135 140Phe Ala Phe Ser Pro Ala Cys Thr Phe Ile Glu Leu Gln Asp Ser Tyr145 150 155 160Leu Leu Pro Ile Ser Glu Asp Lys Val Met Glu Gly Lys Gly Gln Ser 165 170 175Pro Phe Asp Pro Ala His Lys His Thr Ala Val Leu Val Asp Gly Met 180 185 190Leu Tyr Ser Gly Thr Met Asn Asn Phe Leu Gly Ser Glu Pro Ile Leu 195 200 205Met Arg Thr Leu Gly Ser Gln Pro Val Leu Lys Thr Asp Asn Phe Leu 210 215 220Arg Trp Leu His His Asp Ala Ser Phe Val Ala Ala Ile Pro Ser Thr225 230 235 240Gln Val Val Tyr Phe Phe Phe Glu Glu Thr Ala Ser Glu Phe Asp Phe 245 250 255Phe Glu Arg Leu His Thr Ser Arg Val Ala Arg Val Cys Lys Asn Asp 260 265 270Val Gly Gly Glu Lys Leu Leu Gln Lys Lys Trp Thr Thr Phe Leu Lys 275 280 285Ala Gln Leu Leu Ser Ala Pro Ser Arg Gly Ser Cys Pro Ser Thr Ser 290 295 300Ser Ala Thr Arg Ser Cys Ser Pro Pro Ile Leu Pro Gln Leu Pro Thr305 310 315 320Ser Thr Gln Ser Ser Pro Pro Ser Gly Gln Val Gly Gly Thr Arg Ser 325 330 335Ser Ala Val Cys Ala Phe Ser Leu Leu Asp Ile Glu Arg Val Phe Lys 340 345 350Gly Lys Phe Lys Glu Leu Asn Lys Glu Thr Ser Arg Trp Thr Thr Tyr 355 360 365Arg Gly Pro Glu Thr Asn Pro Arg Pro Gly Ser Cys Ser Val Gly Pro 370 375 380Ser Ser Asp Lys Ala Leu Thr Phe Met Lys Asp His Phe Leu Met Asp385 390 395 400Glu Gln Val Val Gly Thr Pro Leu Leu Val Lys Ser Gly Val Glu Tyr 405 410 415Thr Arg Leu Ala Val Glu Thr Ala Gln Gly Leu Asp Gly His Ser His 420 425 430Leu Val Met Tyr Leu Gly Thr Thr Thr Gly Ser Leu His Lys Ala Val 435 440 445Val Ser Gly Asp Ser Ser Ala His Leu Val Glu Glu Ile Gln Leu Phe 450 455 460Pro Asp Pro Glu Pro Val Arg Asn Leu Gln Leu Ala Pro Thr Gln Gly465 470 475 480Ala Val Phe Val Gly Phe Ser Gly Gly Val Trp Arg Val Pro Arg Ala 485 490 495Asn Cys Ser Val Tyr Glu Ser Cys Val Asp Cys Val Leu Ala Arg Asp 500 505 510Pro His Cys Ala Trp Asp Pro Glu Ser Arg Thr Cys Cys Leu Leu Ser 515 520 525Ala Pro Asn Leu Asn Ser Trp Lys Gln Asp Met Glu Arg Gly Asn Pro 530 535 540Glu Trp Ala Cys Ala Ser Gly Pro Met Ser Arg Ser Leu Arg Pro Gln545 550 555 560Ser Arg Pro Gln Ile Ile Lys Glu Val Leu Ala Val Pro Asn Ser Ile 565 570 575Leu Glu Leu Pro Cys Pro His Leu Ser Ala Leu Ala Ser Tyr Tyr Trp 580 585 590Ser His Gly Pro Ala Ala Val Pro Glu Ala Ser Ser Thr Val Tyr Asn 595 600 605Gly Ser Leu Leu Leu Ile Val Gln Asp Gly Val Gly Gly Leu Tyr Gln 610 615 620Cys Trp Ala Thr Glu Asn Gly Phe Ser Tyr Pro Val Ile Ser Tyr Trp625 630 635 640Val Asp Ser Gln Asp Gln Thr Leu Ala Leu Asp Pro Glu Leu Ala Gly 645 650 655Ile Pro Arg Glu His Val Lys Val Pro Leu Thr Arg Val Ser Gly Gly 660 665 670Ala Ala Leu Ala Ala Gln Gln Ser Tyr Trp Pro His Phe Val Thr Val 675 680 685Thr Val Leu Phe Ala Leu Val Leu Ser Gly Ala Leu Ile Ile Leu Val 690 695 700Ala Ser Pro Leu Arg Ala Leu Arg Ala Arg Gly Lys Val Gln Gly Cys705 710 715 720Glu Thr Leu Arg Pro Gly Glu Lys Ala Pro Leu Ser Arg Glu Gln His 725 730 735Leu Gln Ser Pro Lys Glu Cys Arg Thr Ser Ala Ser Asp Val Asp Ala 740 745 750Asp Asn Asn Cys Leu Gly Thr Glu Val Ala 755 76049182DNAHomo sapiensmodified_base(1)..(182)n = g, a, c or t 49accagcagtc ctgcggcacc tacctccgcg tgcgccagcc gccccccagg cccttcctgg 60acatggggga gggcaccaag aaccgaatca tcacagccga ggggatcatc ctcctgttct 120gcgcggtggt gcctgggacg ctgctgctgt tnaggaaacg atggcaagaa cganaactcn 180gg 1825060PRTHomo sapiensMOD_RES(1)..(60)Xaa = any amino acid 50Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro Pro Pro Arg1 5 10 15Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala 20 25 30Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu 35 40 45Leu Xaa Arg Lys Arg Trp Gln Glu Arg Xaa Leu Xaa 50 55 6051182DNAHomo sapiensmodified_base(1)..(182)n = g, a, c or t 51accagcagtc ctgcggcacc tacctccgcg tgcgccagcc gccccccagg cccttcctgg 60acatggggga gggcaccaag aaccgaatca tcacagccga ggggatcatc ctcctgttct 120gcgcggtggt gcctgggacg ctgctgctgt tnaggaaacg atggcaagaa cganaactcn 180gg 1825260PRTHomo sapiensMOD_RES(1)..(60)Xaa = any amino acid 52Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro Pro Pro Arg1 5 10 15Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala 20 25 30Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu 35 40 45Leu Xaa Arg Lys Arg Trp Gln Glu Arg Xaa Leu Xaa 50 55 60531107DNAHomo sapiens 53tgctgcaact caaactaacc aacccactgg gagaagatgc ctgggggtcc aggagtcctc 60caagctctgc ctgccaccat cttcctcctc ttcctgctgt ctgctgtcta cctgggccct 120gggtgccagg ccctgtggat gcacaaggtc ccagcatcat tgatggtgag cctgggggaa 180gacgcccact tccaatgccc gcacaatagc agcaacaacg ccaacgtcac ctggtggcgc 240gtcctccatg gcaactacac gtggccccct gagttcttgg gcccgggcga ggaccccaat 300ggtacgctga tcatccagaa tgtgaacaag agccatgggg gcatatacgt gtgccgggtc 360caggagggca acgagtcata ccagcagtcc tgcggcacct acctccgcgt gcgccagccg 420ccccccaggc ccttcctgga catgggggag ggcaccaaga accgaatcat cacagccgag 480gggatcatcc tcctgttctg cgcggtggtg cctgggacgc tgctgctgtt caggaaacga 540tggcagaacg agaagctcgg gttggatgcc ggggatgaat atgaagatga aaacctttat 600gaaggcctga acctggacga ctgctccatg tatgaggaca tctcccgggg cctccagggc 660acctaccagg atgtgggcag cctcaacata ggagatgtcc agctggagaa gccgtgacac 720ccctactcct gccaggctgc ccccgcctgc tgtgcaccca gctccagtgt ctcagctcac 780ttccctggga cattctcctt tcagcccttc tgggggcttc cttagtcata ttcccccagt 840ggggggtggg agggtaacct cactcttctc caggccaggc ctccttggac tcccctgggg 900gtgtcccact cttcttccct ctaaactgcc ccacctccta acctaatccc cacgccccgc 960tgcctttccc aggctcccct cacccagcgg gtaatgagcc cttaatcgct gcctctaggg 1020gagctgattg tagcagcctc gttagtgtca ccccctcctc cctgatctgt cagggccact 1080tagtgataat aaattcttcc caactgc 110754226PRTHomo sapiens 54Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe1 5 10 15Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe65 70 75 80Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90 95Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro 115 120 125Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly145 150 155 160Thr Leu Leu Leu Phe Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220Lys Pro225551038DNAHomo sapiens 55atgtacaagg actgcatcga gtccactgga gactattttc ttctctgtga cgccgagggg 60ccatggggca tcattctgga gtccctggcc atacttggca tcgtggtcac aattctgcta 120ctcttagcat ttctcttcct catgcgaaag atccaagact gcagccagtg gaatgtcctc 180cccacccagc tcctcttcct cctgagtgtc ctggggctct tcggactcgc ttttgccttc 240atcatcgagc tcaatcaaca aactgccccc gtacgctact ttctctttgg ggttctcttt 300gctctctgtt tctcatgcct cttagctcat gcctccaatc tagtgaagct ggttcggggt 360tgtgtctcct tctcctggac gacaattctg tgcattgcta ttggttgcag tctgttgcaa 420atcattattg ccactgagta tgtgactctc atcatgacca gaggtatgat gtttgtgaat 480atgacaccct gccagctcaa tgtggacttt gttgtactcc tggtctatgt cctcttcctg 540atggccctca cattcttcgt ctccaaagcc accttctgtg gcccgtgtga gaactggaag 600cagcatggaa ggctcatctt tatcactgtg ctcttctcca tcatcatctg ggtggtgtgg 660atctccatgc tcctgagagg caacccgcag ttccagcgac agccccagtg ggacgacccg 720gtcgtctgca ttgctctggt caccaacgca tgggttttcc tgctgctgta catcgtccct 780gagctctgca ttctctacag atcgtgtaga caggagtgcc ctttacaagg caatgcctgc 840cccgtcacag cctaccaaca cagcttccaa gtggagaacc aggagctctc cagagcccga 900gacagtgatg gagctgagga ggatgtagca ttaacttcat atggtactcc cattcagccg 960cagactgttg atcccacaca agagtgtttc atcccacagg ctaaactaag cccccagcaa 1020gatgcaggag gagtataa 103856345PRTHomo sapiens 56Met Tyr Lys Asp Cys Ile Glu Ser Thr Gly Asp Tyr Phe Leu Leu Cys1 5 10 15Asp Ala Glu Gly Pro Trp Gly Ile Ile Leu Glu Ser Leu Ala Ile Leu 20 25 30Gly Ile Val Val Thr Ile Leu Leu Leu Leu Ala Phe Leu Phe Leu Met 35 40 45Arg Lys Ile Gln Asp Cys Ser Gln Trp Asn Val Leu Pro Thr Gln Leu 50 55 60Leu Phe Leu Leu Ser Val Leu Gly Leu Phe Gly Leu Ala Phe Ala Phe65 70 75 80Ile Ile Glu Leu Asn Gln Gln Thr Ala Pro Val Arg Tyr Phe Leu Phe 85 90 95Gly Val Leu Phe Ala Leu Cys Phe Ser Cys Leu Leu Ala His Ala Ser 100 105 110Asn Leu Val Lys Leu Val Arg Gly Cys Val Ser Phe Ser Trp Thr Thr 115 120 125Ile Leu Cys Ile Ala Ile Gly Cys Ser Leu Leu Gln Ile Ile Ile Ala 130 135 140Thr Glu Tyr Val Thr Leu Ile Met Thr Arg Gly Met Met Phe Val

Asn145 150 155 160Met Thr Pro Cys Gln Leu Asn Val Asp Phe Val Val Leu Leu Val Tyr 165 170 175Val Leu Phe Leu Met Ala Leu Thr Phe Phe Val Ser Lys Ala Thr Phe 180 185 190Cys Gly Pro Cys Glu Asn Trp Lys Gln His Gly Arg Leu Ile Phe Ile 195 200 205Thr Val Leu Phe Ser Ile Ile Ile Trp Val Val Trp Ile Ser Met Leu 210 215 220Leu Arg Gly Asn Pro Gln Phe Gln Arg Gln Pro Gln Trp Asp Asp Pro225 230 235 240Val Val Cys Ile Ala Leu Val Thr Asn Ala Trp Val Phe Leu Leu Leu 245 250 255Tyr Ile Val Pro Glu Leu Cys Ile Leu Tyr Arg Ser Cys Arg Gln Glu 260 265 270Cys Pro Leu Gln Gly Asn Ala Cys Pro Val Thr Ala Tyr Gln His Ser 275 280 285Phe Gln Val Glu Asn Gln Glu Leu Ser Arg Ala Arg Asp Ser Asp Gly 290 295 300Ala Glu Glu Asp Val Ala Leu Thr Ser Tyr Gly Thr Pro Ile Gln Pro305 310 315 320Gln Thr Val Asp Pro Thr Gln Glu Cys Phe Ile Pro Gln Ala Lys Leu 325 330 335Ser Pro Gln Gln Asp Ala Gly Gly Val 340 345572457DNAHomo sapiens 57ggcacgagga agggcctgtg ggtttattat aaggcggagc tcggcgggag aggtgcgggc 60cgaatccgag ccgagcggag aggaatccgg cagtagagag cggactccag ccggcggacc 120ctgcagccct cgcctgggac agcggcgcgc tgggcaggcg cccaagagag catcgagcag 180cggaacccgc gaagccggcc cgcagccgcg acccgcgcag cctgccgctc tcccgccgcc 240ggtccgggca gcatgaggcg cgcggcgctc tggctctggc tgtgcgcgct ggcgctgagc 300ctgcagccgg ccctgccgca aattgtggct actaatttgc cccctgaaga tcaagatggc 360tctggggatg actctgacaa cttctccggc tcaggtgcag gtgctttgca agatatcacc 420ttgtcacagc agaccccctc cacttggaag gacacgcagc tcctgacggc tattcccacg 480tctccagaac ccaccggcct ggaggctaca gctgcctcca cctccaccct gccggctgga 540gaggggccca aggagggaga ggctgtagtc ctgccagaag tggagcctgg cctcaccgcc 600cgggagcagg aggccacccc ccgacccagg gagaccacac agctcccgac cactcatcag 660gcctcaacga ccacagccac cacggcccag gagcccgcca cctcccaccc ccacagggac 720atgcagcctg gccaccatga gacctcaacc cctgcaggac ccagccaagc tgaccttcac 780actccccaca cagaggatgg aggtccttct gccaccgaga gggctgctga ggatggagcc 840tccagtcagc tcccagcagc agagggctct ggggagcagg acttcacctt tgaaacctcg 900ggggagaata cggctgtagt ggccgtggag cctgaccgcc ggaaccagtc cccagtggat 960cagggggcca cgggggcctc acagggcctc ctggacagga aagaggtgct gggaggggtc 1020attgccgtag gcctcgtggg gctcatcttt gctgtgtgcc tggtgggttt catgctgtac 1080cgcatgaaga agaaggacga aggcagctac tccttggagg agccgaaaca agccaacggc 1140ggggcctacc agaagcccac caaacaggag gaattctatg cctgacgcgg gagccatgcg 1200ccccctccgc cctgccactc actaggcccc cacttgcctc ttccttgaag aactgcaggc 1260cctggcctcc cctgccacca ggccacctcc ccagcattcc agcccctctg gtcgctcctg 1320cccacggagt cgtggggtgt gctgggagct ccactctgct tctctgactt ctgcctggag 1380acttagggca ccaggggttt ctcgcatagg acctttccac cacagccagc acctggcatc 1440gcaccattct gactcggttt ctccaaactg aagcagcctc tccccaggtc cagctctgga 1500ggggaggggg atccgactgc tttggaccta aatggcctca tgtggctgga agatcctgcg 1560ggtggggctt ggggctcaca cacctgtagc acttactggt aggaccaagc atcttggggg 1620ggtggccgct gagtggcagg ggacaggagt ccactttgtt tcgtggggag gtctaatcta 1680gatatcgact tgtttttgca catgtttcct ctagttcttt gttcatagcc cagtagacct 1740tgttacttct gaggtaagtt aagtaagttg attcggtatc cccccatctt gcttccctaa 1800tctatggtcg ggagacagca tcagggttaa gaagactttt tttttttttt tttttaaact 1860aggagaacca aatctggaag ccaaaatgta ggcttagttt gtgtgttgtc tcttgagttt 1920gtcgctcatg tgtgcaacag ggtatggact atctgtctgg tggccccgtt tctggtggtc 1980tgttggcagg ctggccagtc caggctgccg tggggccgcc gcctctttca agcagtcgtg 2040cctgtgtcca tgcgctcagg gccatgctga ggcctgggcc gctgccacgt tggagaagcc 2100cgtgtgagaa gtgaatgctg ggactcagcc ttcagacaga gaggactgta gggagggcgg 2160caggggcctg gagatcctcc tgcagaccac gcccgtcctg cctgtggcgc cgtctccagg 2220ggctgcttcc tcctggaaat tgacgagggg tgtcttgggc agagctggct ctgagcgcct 2280ccatccaagg ccaggttctc cgttagctcc tgtggcccca ccctgggccc tgggctggaa 2340tcaggaatat tttccaaaga gtgatagtct tttgcttttg gcaaaactct acttaatcca 2400atgggttttt ccctgtacag tagattttcc aaatgtaata aactttaata taaagta 245758310PRTHomo sapiens 58Met Arg Arg Ala Ala Leu Trp Leu Trp Leu Cys Ala Leu Ala Leu Ser1 5 10 15Leu Gln Pro Ala Leu Pro Gln Ile Val Ala Thr Asn Leu Pro Pro Glu 20 25 30Asp Gln Asp Gly Ser Gly Asp Asp Ser Asp Asn Phe Ser Gly Ser Gly 35 40 45Ala Gly Ala Leu Gln Asp Ile Thr Leu Ser Gln Gln Thr Pro Ser Thr 50 55 60Trp Lys Asp Thr Gln Leu Leu Thr Ala Ile Pro Thr Ser Pro Glu Pro65 70 75 80Thr Gly Leu Glu Ala Thr Ala Ala Ser Thr Ser Thr Leu Pro Ala Gly 85 90 95Glu Gly Pro Lys Glu Gly Glu Ala Val Val Leu Pro Glu Val Glu Pro 100 105 110Gly Leu Thr Ala Arg Glu Gln Glu Ala Thr Pro Arg Pro Arg Glu Thr 115 120 125Thr Gln Leu Pro Thr Thr His Gln Ala Ser Thr Thr Thr Ala Thr Thr 130 135 140Ala Gln Glu Pro Ala Thr Ser His Pro His Arg Asp Met Gln Pro Gly145 150 155 160His His Glu Thr Ser Thr Pro Ala Gly Pro Ser Gln Ala Asp Leu His 165 170 175Thr Pro His Thr Glu Asp Gly Gly Pro Ser Ala Thr Glu Arg Ala Ala 180 185 190Glu Asp Gly Ala Ser Ser Gln Leu Pro Ala Ala Glu Gly Ser Gly Glu 195 200 205Gln Asp Phe Thr Phe Glu Thr Ser Gly Glu Asn Thr Ala Val Val Ala 210 215 220Val Glu Pro Asp Arg Arg Asn Gln Ser Pro Val Asp Gln Gly Ala Thr225 230 235 240Gly Ala Ser Gln Gly Leu Leu Asp Arg Lys Glu Val Leu Gly Gly Val 245 250 255Ile Ala Val Gly Leu Val Gly Leu Ile Phe Ala Val Cys Leu Val Gly 260 265 270Phe Met Leu Tyr Arg Met Lys Lys Lys Asp Glu Gly Ser Tyr Ser Leu 275 280 285Glu Glu Pro Lys Gln Ala Asn Gly Gly Ala Tyr Gln Lys Pro Thr Lys 290 295 300Gln Glu Glu Phe Tyr Ala305 31059357DNAHomo sapiens 59ctggggctga ggatggagtc caagactgag aaatggatgg aacgaataca cctcaatgtc 60tctgaagggc cttttccacc tcatatccag ctccctccag aaattcaaga gtcccaggaa 120gtcactctga cctgcttgct gaatttctcc tgctatgggt atccgatcca attgcagtgg 180ctcctagagg gggttccaat gaggcaggct gctgtcacct cgacctcctt gaccatcaag 240tctgtcttca cccggagcga gctcaagttc tccccacagt ggagtcacca tgggaagatt 300gtgacctgcc agcttcagga tgcagatggg aagttcctct ccaatgacac ggtgcag 357603260DNAHomo sapiens 60ccatcccata gtgagggaag acacgcggaa acaggcttgc acccagacac gacaccatgc 60atctcctcgg cccctggctc ctgctcctgg ttctagaata cttggctttc tctgactcaa 120gtaaatgggt ttttgagcac cctgaaaccc tctacgcctg ggagggggcc tgcgtctgga 180tcccctgcac ctacagagcc ctagatggtg acctggaaag cttcatcctg ttccacaatc 240ctgagtataa caagaacacc tcgaagtttg atgggacaag actctatgaa agcacaaagg 300atgggaaggt tccttctgag cagaaaaggg tgcaattcct gggagacaag aataagaact 360gcacactgag tatccacccg gtgcacctca atgacagtgg tcagctgggg ctgaggatgg 420agtccaagac tgagaaatgg atggaacgaa tacacctcaa tgtctctgaa aggccttttc 480cacctcatat ccagctccct ccagaaattc aagagtccca ggaagtcact ctgacctgct 540tgctgaattt ctcctgctat gggtatccga tccaattgca gtggctccta gagggggttc 600caatgaggca ggctgctgtc acctcgacct ccttgaccat caagtctgtc ttcacccgga 660gcgagctcaa gttctcccca cagtggagtc accatgggaa gattgtgacc tgccagcttc 720aggatgcaga tgggaagttc ctctccaatg acacggtgca gctgaacgtg aagcacaccc 780cgaagttgga gatcaaggtc actcccagtg atgccatagt gagggagggg gactctgtga 840ccatgacctg cgaggtcagc agcagcaacc cggagtacac gacggtatcc tggctcaagg 900atgggacctc gctgaagaag cagaatacat tcacgctaaa cctgcgcgaa gtgaccaagg 960accagagtgg gaagtactgc tgtcaggtct ccaatgacgt gggcccggga aggtcggaag 1020aagtgttcct gcaagtgcag tatgccccgg aaccttccac ggttcagatc ctccactcac 1080cggctgtgga gggaagtcaa gtcgagtttc tttgcatgtc actggccaat cctcttccaa 1140caaattacac gtggtaccac aatgggaaag aaatgcaggg aaggacagag gagaaagtcc 1200acatcccaaa gatcctcccc tggcacgctg ggacttattc ctgtgtggca gaaaacattc 1260ttggtactgg acagaggggc ccgggagctg agctggatgt ccagtatcct cccaagaagg 1320tgaccacagt gattcaaaac cccatgccga ttcgagaagg agacacagtg accctttcct 1380gtaactacaa ttccagtaac cccagtgtta cccggtatga atggaaaccc catggcgcct 1440gggaggagcc atcgcttggg gtgctgaaga tccaaaacgt tggctgggac aacacaacca 1500tcgcctgcgc acgttgtaat agttggtgct cgtgggcctc ccctgtcgcc ctgaatgtcc 1560agtatgcccc ccgagacgtg agggtccgga aaatcaagcc cctttccgag attcactctg 1620gaaactcggt cagcctccaa tgtgacttct caagcagcca ccccaaagaa gtccagttct 1680tctgggagaa aaatggcagg cttctgggga aagaaagcca gctgaatttt gactccatct 1740ccccagaaga tgctgggagt tacagctgct gggtgaacaa ctccatagga cagacagcgt 1800ccaaggcctg gacacttgaa gtgctgtatg cacccaggag gctgcgtgtg tccatgagcc 1860cgggggacca agtgatggag gggaagagtg caaccctgac ctgtgagagt gacgccaacc 1920ctcccgtctc ccactacacc tggtttgact ggaataacca aagcctcccc caccacagcc 1980agaagctgag attggagccg gtgaaggtcc agcactcggg tgcctactgg tgccagggga 2040ccaacagtgt gggcaagggc cgttcgcctc tcagcaccct tactgtctac tatagcccgg 2100agaccatcgg caggcgagtg gctgtgggac tcgggtcctg cctcgccatc ctcatcctgg 2160caatctgtgg gctcaagctc cagcgacgtt ggaagaggac acagagccag caggggcttc 2220aggagaattc cagcggccag agcttctttg tgaggaataa aaaggttaga agggcccccc 2280tctctgaagg cccccactcc ctgggatgct acaatccaat gatggaagat ggcattagct 2340acaccaccct gcgctttccc gagatgaaca taccacgaac tggagatgca gagtcctcag 2400agatgcagag acctccccgg acctgcgatg acacggtcac ttattcagca ttgcacaagc 2460gccaagtggg cgactatgag aacgtcattc cagattttcc agaagatgag gggattcatt 2520actcagagct gatccagttt ggggtcgggg agcggcctca ggcacaagaa aatgtggact 2580atgtgatcct caaacattga cactggatgg gctgcagcag aggcactggg ggcagcgggg 2640gccagggaag tccccgagtt tccccagaca ccgccacatg gcttcctcct gcgtgcatgt 2700gcgcacacac acacacacac gcacacacac acacacacac tcactgcgga gaaccttgtg 2760cctggctcag agccagtctt tttggtgagg gtaaccccaa acctccaaaa ctcctgcccc 2820tgttctcttc cactctcctt gctacccaga aatcatctaa atacctgccc tgacatgcac 2880acctcccctg ccccaccagc ccactggcca tctccacccg gagctgctgt gtcctctgga 2940tctgctcgtc attttccttc ccttctccat ctctctggcc ctctacccct gatctgacat 3000ccccactcac gaatattatg cccagtttct gcctctgagg gaaagcccag aaaaggacag 3060aaacgaagta gaaaggggcc cagtcctggc ctggcttctc ctttggaagt gaggcattgc 3120acggggagac gtacgtatca gcggcccctt gactctgggg actccgggtt tgagatggac 3180acactggtgt ggattaacct gccagggaga cagagctcac aataaaaatg gctcagatgc 3240cacttcaaag aaaaaaaaaa 326061847PRTHomo sapiens 61Met His Leu Leu Gly Pro Trp Leu Leu Leu Leu Val Leu Glu Tyr Leu1 5 10 15Ala Phe Ser Asp Ser Ser Lys Trp Val Phe Glu His Pro Glu Thr Leu 20 25 30Tyr Ala Trp Glu Gly Ala Cys Val Trp Ile Pro Cys Thr Tyr Arg Ala 35 40 45Leu Asp Gly Asp Leu Glu Ser Phe Ile Leu Phe His Asn Pro Glu Tyr 50 55 60Asn Lys Asn Thr Ser Lys Phe Asp Gly Thr Arg Leu Tyr Glu Ser Thr65 70 75 80Lys Asp Gly Lys Val Pro Ser Glu Gln Lys Arg Val Gln Phe Leu Gly 85 90 95Asp Lys Asn Lys Asn Cys Thr Leu Ser Ile His Pro Val His Leu Asn 100 105 110Asp Ser Gly Gln Leu Gly Leu Arg Met Glu Ser Lys Thr Glu Lys Trp 115 120 125Met Glu Arg Ile His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro His 130 135 140Ile Gln Leu Pro Pro Glu Ile Gln Glu Ser Gln Glu Val Thr Leu Thr145 150 155 160Cys Leu Leu Asn Phe Ser Cys Tyr Gly Tyr Pro Ile Gln Leu Gln Trp 165 170 175Leu Leu Glu Gly Val Pro Met Arg Gln Ala Ala Val Thr Ser Thr Ser 180 185 190Leu Thr Ile Lys Ser Val Phe Thr Arg Ser Glu Leu Lys Phe Ser Pro 195 200 205Gln Trp Ser His His Gly Lys Ile Val Thr Cys Gln Leu Gln Asp Ala 210 215 220Asp Gly Lys Phe Leu Ser Asn Asp Thr Val Gln Leu Asn Val Lys His225 230 235 240Thr Pro Lys Leu Glu Ile Lys Val Thr Pro Ser Asp Ala Ile Val Arg 245 250 255Glu Gly Asp Ser Val Thr Met Thr Cys Glu Val Ser Ser Ser Asn Pro 260 265 270Glu Tyr Thr Thr Val Ser Trp Leu Lys Asp Gly Thr Ser Leu Lys Lys 275 280 285Gln Asn Thr Phe Thr Leu Asn Leu Arg Glu Val Thr Lys Asp Gln Ser 290 295 300Gly Lys Tyr Cys Cys Gln Val Ser Asn Asp Val Gly Pro Gly Arg Ser305 310 315 320Glu Glu Val Phe Leu Gln Val Gln Tyr Ala Pro Glu Pro Ser Thr Val 325 330 335Gln Ile Leu His Ser Pro Ala Val Glu Gly Ser Gln Val Glu Phe Leu 340 345 350Cys Met Ser Leu Ala Asn Pro Leu Pro Thr Asn Tyr Thr Trp Tyr His 355 360 365Asn Gly Lys Glu Met Gln Gly Arg Thr Glu Glu Lys Val His Ile Pro 370 375 380Lys Ile Leu Pro Trp His Ala Gly Thr Tyr Ser Cys Val Ala Glu Asn385 390 395 400Ile Leu Gly Thr Gly Gln Arg Gly Pro Gly Ala Glu Leu Asp Val Gln 405 410 415Tyr Pro Pro Lys Lys Val Thr Thr Val Ile Gln Asn Pro Met Pro Ile 420 425 430Arg Glu Gly Asp Thr Val Thr Leu Ser Cys Asn Tyr Asn Ser Ser Asn 435 440 445Pro Ser Val Thr Arg Tyr Glu Trp Lys Pro His Gly Ala Trp Glu Glu 450 455 460Pro Ser Leu Gly Val Leu Lys Ile Gln Asn Val Gly Trp Asp Asn Thr465 470 475 480Thr Ile Ala Cys Ala Arg Cys Asn Ser Trp Cys Ser Trp Ala Ser Pro 485 490 495Val Ala Leu Asn Val Gln Tyr Ala Pro Arg Asp Val Arg Val Arg Lys 500 505 510Ile Lys Pro Leu Ser Glu Ile His Ser Gly Asn Ser Val Ser Leu Gln 515 520 525Cys Asp Phe Ser Ser Ser His Pro Lys Glu Val Gln Phe Phe Trp Glu 530 535 540Lys Asn Gly Arg Leu Leu Gly Lys Glu Ser Gln Leu Asn Phe Asp Ser545 550 555 560Ile Ser Pro Glu Asp Ala Gly Ser Tyr Ser Cys Trp Val Asn Asn Ser 565 570 575Ile Gly Gln Thr Ala Ser Lys Ala Trp Thr Leu Glu Val Leu Tyr Ala 580 585 590Pro Arg Arg Leu Arg Val Ser Met Ser Pro Gly Asp Gln Val Met Glu 595 600 605Gly Lys Ser Ala Thr Leu Thr Cys Glu Ser Asp Ala Asn Pro Pro Val 610 615 620Ser His Tyr Thr Trp Phe Asp Trp Asn Asn Gln Ser Leu Pro His His625 630 635 640Ser Gln Lys Leu Arg Leu Glu Pro Val Lys Val Gln His Ser Gly Ala 645 650 655Tyr Trp Cys Gln Gly Thr Asn Ser Val Gly Lys Gly Arg Ser Pro Leu 660 665 670Ser Thr Leu Thr Val Tyr Tyr Ser Pro Glu Thr Ile Gly Arg Arg Val 675 680 685Ala Val Gly Leu Gly Ser Cys Leu Ala Ile Leu Ile Leu Ala Ile Cys 690 695 700Gly Leu Lys Leu Gln Arg Arg Trp Lys Arg Thr Gln Ser Gln Gln Gly705 710 715 720Leu Gln Glu Asn Ser Ser Gly Gln Ser Phe Phe Val Arg Asn Lys Lys 725 730 735Val Arg Arg Ala Pro Leu Ser Glu Gly Pro His Ser Leu Gly Cys Tyr 740 745 750Asn Pro Met Met Glu Asp Gly Ile Ser Tyr Thr Thr Leu Arg Phe Pro 755 760 765Glu Met Asn Ile Pro Arg Thr Gly Asp Ala Glu Ser Ser Glu Met Gln 770 775 780Arg Pro Pro Arg Thr Cys Asp Asp Thr Val Thr Tyr Ser Ala Leu His785 790 795 800Lys Arg Gln Val Gly Asp Tyr Glu Asn Val Ile Pro Asp Phe Pro Glu 805 810 815Asp Glu Gly Ile His Tyr Ser Glu Leu Ile Gln Phe Gly Val Gly Glu 820 825 830Arg Pro Gln Ala Gln Glu Asn Val Asp Tyr Val Ile Leu Lys His 835 840 84562340DNAHomo sapiens 62ctggggggtc cgggaaaggg gttgggccat gagccaggca gctccgaagc agtcactgag 60gccagggagc ctgcacccag gtcatggggc gacctggctc tcactcctgg cctgggtgct 120cacctacaga ccacttcact tcccctgtcc gcagcgtcac tatgtcctca taggtggctg 180tctggtcaat gtccaggccc tcgtaggtgt gatcttcctc catgccagcc ttgctgtcat 240ccttgtccag cagcaggaag ataggcacga tgatgaagag gatgatcagc agcgtctgga 300tcatgatgat accatccttc agcgtgttcc tctgcttcag 3406379PRTHomo sapiens 63Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly Ile Ile Met Ile Gln Thr1 5 10 15Leu Leu Ile Ile Leu Phe Ile Ile Val Pro Ile Phe Leu Leu Leu Asp 20 25 30Lys Asp Asp Ser

Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu Gly 35 40 45Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu Asp Ile Val Thr Leu Arg 50 55 60Thr Gly Glu Val Lys Trp Ser Val Gly Glu His Pro Gly Gln Glu65 70 7564340DNAHomo sapiens 64ctggggggtc cgggaaaggg gttgggccat gagccaggca gctccgaagc agtcactgag 60gccagggagc ctgcacccag gtcatggggc gacctggctc tcactcctgg cctgggtgct 120cacctacaga ccacttcact tcccctgtcc gcagcgtcac tatgtcctca taggtggctg 180tctggtcaat gtccaggccc tcgtaggtgt gatcttcctc catgccagcc ttgctgtcat 240ccttgtccag cagcaggaag ataggcacga tgatgaagag gatgatcagc agcgtctgga 300tcatgatgat accatccttc agcgtgttcc tctgcttcag 340651226DNAHomo sapiens 65ccacgcgtcc gcccacgcgt ccgcagagcg gtgaccatgg ccaggctggc gttgtctcct 60gtgcccagcc actggatggt ggcgttgctg ctgctgctct cagctgagcc agtaccagca 120gccagatcgg aggaccggta ccggaatccc aaaggtagtg cttgttcgcg gatctggcag 180agcccacgtt tcatagccag gaaacggggc ttcacggtga aaatgcactg ctacatgaac 240agcgcctccg gcaatgtgag ctggctctgg aagcaggaga tggacgagaa tccccagcag 300ctgaagctgg aaaagggccg catggaagag tcccagaacg aatctctcgc caccctcacc 360atccaaggca tccggtttga ggacaatggc atctacttct gccagcagaa gtgcaacaac 420acctcggagg tctaccaggg ctgcggcaca gagctgcgag tcatgggatt cagcaccttg 480gcacagctga agcagaggaa cacgctgaag gatggtatca tcatgatcca gacgctgctg 540atcatcctct tcatcatcgt gcctatcttc ctgctgctgg acaaggatga cagcaaggct 600ggcatggagg aagatcacac ctacgagggc ctggacattg accagacagc cacctatgag 660gacatagtga cgctgcggac aggggaagtg aagtggtctg taggtgagca cccaggccag 720gagtgagagc caggtcgccc catgacctgg gtgcaggctc cctggcctca gtgactgctt 780cggagctgcc tggctcatgg cccaacccct ttcccggacc ccccagctgg cctctgaagc 840tggcccacca gagctgccat ttgtctccag cccctggtcc ccagctcttg ccaaagggcc 900tggagtagaa ggacaacagg gcagcaactt ggagggagtt ctctggggat ggacgggacc 960cagccttctg ggggtgctat gaggtgatcc gtccccacac atgggatggg ggaggcagag 1020actggtccag agcccgcaaa tggactcgga gccgagggcc tcccagcaga gcttgggaag 1080ggccatggac ccaactgggc cccagaagag ccacaggaac atcattcctc tcccgcaacc 1140actcccaccc cagggaggcc ctggcctcca gtgccttccc ccgtggaata aacggtgtgt 1200cctgagaaac caaaaaaaaa aaaaaa 122666229PRTHomo sapiens 66Met Ala Arg Leu Ala Leu Ser Pro Val Pro Ser His Trp Met Val Ala1 5 10 15Leu Leu Leu Leu Leu Ser Ala Glu Pro Val Pro Ala Ala Arg Ser Glu 20 25 30Asp Arg Tyr Arg Asn Pro Lys Gly Ser Ala Cys Ser Arg Ile Trp Gln 35 40 45Ser Pro Arg Phe Ile Ala Arg Lys Arg Gly Phe Thr Val Lys Met His 50 55 60Cys Tyr Met Asn Ser Ala Ser Gly Asn Val Ser Trp Leu Trp Lys Gln65 70 75 80Glu Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu Lys Gly Arg Met 85 90 95Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr Ile Gln Gly Ile 100 105 110Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn 115 120 125Thr Ser Glu Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly 130 135 140Phe Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly145 150 155 160Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val Pro 165 170 175Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly Met Glu Glu 180 185 190Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr Ala Thr Tyr Glu 195 200 205Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu 210 215 220His Pro Gly Gln Glu22567449DNAHomo sapiensmodified_base(16)n = g, a, c or t 67aaaattgatc acaacnaggg aaaacaaaat aaaattaggg ggcaaagggt aggagtatgg 60ggggagggga gagcaaacct atcgaatata tcttagaatt ttgctcagaa atcactgctg 120cctctcaagt gttgcattgt ccctgcctaa accaagaagg ctaaacaaag cccctcctgt 180ttgaattctt aaggtaagaa atttctaagc taagaaaaca ctattgccta aaaccaatga 240tagtggagct catttacaaa taggcatgcc tcacacacac agtccaaagg caagacactg 300gctttgaaat taggctcatg atgtgattcc tattatatgt acctgatttt tttaggcccc 360aggtatgtgg accagagtta atgtcatgac tcttcaaaga tatgatgaaa agttgcccta 420gaaatctaga gatgcatgtt tatttaatt 449682359DNAHomo sapiens 68ctttcaagaa aatacatctg tgctgtattt tccccttccc tcaggccatg atctctgctg 60ttttccttac taactggcat gtcagtacaa gagtgattgt gaagctgctc cggaagggct 120ttatgctaac ctctgttgct tgatgacatg tcctcaggac tctgatatta aaactcaatc 180cttagataac aggtagcttt atcatggaag taggtagcaa tttggaatta gaccattctt 240agttattttt ttcttaatga attgatacat gcactttaaa aaatattttt gttattttgg 300gaagaaaaac tcagactttt aaaaaagtgt atattgtccc attataatat gtatatggaa 360gagtgaaatc tgaacgctgt cttatattaa gcagtagaat taggtattat cataaaaagt 420cttaatctgt agggaatatg agtttatgtt tatgagtcct gctcagtccc tctttgagag 480aattagttga aacccagact ctaaagtctg cttttatatt tgtttgttaa gaccacttat 540ctgcagaagg ttgcctttta accccagtgg ttctaaggtg tggaattgag tgaccctaat 600atttacataa gagacttgtt ttagtggagc ataagggagg ggcataagtt acaccgtttt 660gtgctgcttg agaactgtct tttaaaattg atcacaacga gggaaaacaa aataaaatta 720gggggcaaag ggtaggagta tggggggagg ggagagcaaa cctatcgaat atatcttaga 780attttgctca gaaatcactg ctgcctctca agtgttgcat tgtccctgcc taaaccaaga 840aggctaaaca aagcccctcc tgtttgaatt cttaaggtaa gaaatttcta agctaagaaa 900acactattgc ctaaaaccaa tgatagtgga gctcatttac aaataggcat gcctcacaca 960cacagtccaa aggcaagaca ctggctttga aattaggctc atgatgtgat tcctattata 1020tgtacctgat ttttttaggc cccaggtatg tggaccagag ttaatgtcat gactcttcaa 1080agatatgatg aaaagttgcc ctagaaatct agagatgcat gtttatttaa ttccatagtt 1140taaaaaaaaa tttaagcagg tagttgtggc ttatctgggg gcaaaataat atatgtgaaa 1200ttgcttccag aggacaaagt atattttcta aagtcctgaa ataggatcat gaacccttct 1260gaagttttgg tttgaaatat tatagtatat gatattacca aagagccctt aattcagagt 1320ttaaggggct ctcttcctga actctcttca tcactcaggg ttgaatgtgt aatgttcctt 1380gctattgatt gttattgttg attcttagga tcaggccaag aatcatctgg aaaacattat 1440cttaattccg tctctcatat cctaaacagt acattttact aagaaattcc atatgaaaaa 1500ctccactcat gtctcctgag attatcctgt aagtgaagta gctttcattt aaccaagcta 1560aattatttcc atttagccat gttaaagaga agccaagtct agagaaagca atcctgtaac 1620ccatgaatct ggtgtaccca ttttccctta acgtaacggg aagtgttttg aaattcccag 1680aagagagctg ttttgtaatc aaagtgatgg attataagaa agccagactt tggaaaagga 1740taattggaat aaagggaggt gcttgaagat tttccaaact actttatgtc atttagcttc 1800tattttctga agggctttct ttggtgccat gtactcagat cagtcagttg actgaaagat 1860gatcatgttt tcttcgtaaa gatttaagca attggcaact acaaagacat tattttctta 1920ctgttctata tcatgtactg ttgctgacat tacaaaaagg gtctggaagg gaaaccgtgt 1980cactgtttta tcttttttct ttaaaataca aaagtatccc aactaatcat ttattatggt 2040cagcttgttt tacatgtccc ctatgatgag aaatgctatc aacatctgtg atttctaaga 2100gtcttaccaa attgttactt taattcttgt gtcctgctga gtggtttttc ttttaaaata 2160ccatttttat caccctgtgg cactgggtgt gttactgcga ttacactgat gattctgagc 2220tgtgcttctt caagtagctc agttcttgcg ttttatatta ggtaacagtt ttgtgatgct 2280tttgtgcatt ctttgtcatc tcttctgagt tttcgaatct gtcataaata aactttttca 2340ctatgcacct ggtaaaaaa 235969240DNAHomo sapiens 69cctaagccgc ctaaggggct gcctcggctg tccatcagtt acctcgtttc ctgagcagag 60taattgggtg agattgttca tggaggcatt gctggctctc tagtcctgga acctacagtt 120ggtccaattc attatgccaa agggtccgtc taggaggttc ttgttccaag tattgagatt 180cccgagagaa gtaggtcccc ttagatagaa gcagagtttc tcagaggtat ttagcagcag 24070980DNAHomo sapiens 70gccgctgccg ctccaggaga caggttccca tgcaggaatg aaagacatgg aagggaagag 60gggggccagc tccctgagtc ctgtgtccac cagctgctgc taaatacctc tgagaaactc 120tgcttctatc taaggggacc tacttctctc gggaatctca atacttggaa caagaacctc 180ctagacggac cctttggcat aatgaattgg accaactgta ggttccagga ctagagagcc 240agcaatgcct ccatgaacaa tctcacccaa ttactctgct caggaaacga ggtaactgat 300ggacagccga ggcagcccct taggcggctt aggcctcccc tgtggagcat ccctgaggcg 360gactccggcc agcccgagtg atgcgatcca aagagcactc ccgggtagga aattgccccg 420gtggaatgcc tcaccagagc agcgtgtagc agttccctgt ggaggattaa cacagtggct 480gaacaccggg aaggaactgg cacttggagt ccggacatct gaaacttgta gactgggagc 540tgtacatgga tgggagcagc ttcaccaacc cctgcaaagt gactctgaag aagacgacaa 600gccctgctcc agtcacaccc ggaagctgac tggtccacgc acagctgaag catgaggaaa 660ctcatcgcgg gactaatttt ccttaaaatt tagacttgca cagtaaggac ttcaactgac 720cttcctcaga ctgagaactg tttccagtat atacatcaag tcactgaggt aggacaaaag 780attgctacat tcctattatt ttaaggttac atttttgggg acccctcttt cttctgttct 840agctattacc tttcttgtgt cacctagaaa aggaccagtc cttaattgta ttttaaaaac 900tgtgatcatg ggaagcttta aattggttca ataacacgca tcaagttggt tatttcctgg 960gctacatacc ttggatagat 98071118PRTHomo sapiens 71Met Asp Ser Arg Gly Ser Pro Leu Gly Gly Leu Gly Leu Pro Cys Gly1 5 10 15Ala Ser Leu Arg Arg Thr Pro Ala Ser Pro Ser Asp Ala Ile Gln Arg 20 25 30Ala Leu Pro Gly Arg Lys Leu Pro Arg Trp Asn Ala Ser Pro Glu Gln 35 40 45Arg Val Ala Val Pro Cys Gly Gly Leu Thr Gln Trp Leu Asn Thr Gly 50 55 60Lys Glu Leu Ala Leu Gly Val Arg Thr Ser Glu Thr Cys Arg Leu Gly65 70 75 80Ala Val His Gly Trp Glu Gln Leu His Gln Pro Leu Gln Ser Asp Ser 85 90 95Glu Glu Asp Asp Lys Pro Cys Ser Ser His Thr Arg Lys Leu Thr Gly 100 105 110Pro Arg Thr Ala Glu Ala 11572531DNAHomo sapiensmodified_base(519)n = g, a, c or t 72aaaaaggtaa ttttcagcat tttggcacct aaaagggaaa ctttcatctg cttacacagg 60ccagaagcaa agacaaagat tgcatgttgt tcttacagat gacttaaatc atctctttga 120tgataaaaat atttttaagc cgtgaaagtt atgagatatt ctgggtaagc ctgattatca 180aagaatacca caaatagctt tggagatcgt gtattgtttg tcactgagtc aaagagatct 240gtgggattgt gaggattctt gggtggaggg gtgactaatc ctgcacgtcc ctttgtgaag 300actccagtaa gtactcgcac aacgtacatg tgctttctcc cattgctgtc tggcttggag 360taggtgtcct tggcagaata actggcatcc acagcaaaat aggttccttt tccataggat 420acagcatttt tcccacacaa cttctattaa agccgtgctg attgacatat ggcactgagt 480ctgcatctgt cccatggaag aggagtctct cattattcnt atggtcattc t 531731956DNAHomo sapiens 73attgttatca actctttgat atctgatgat caatgctcca aagaattgga ttaatatttt 60tacacaatat tgttgtagtc agtaactgtt tctatttcca ggcattttta gatgaattca 120ctaactggtc aagaataaat cccaacaagg ccaggattcc catggcagga gatacccaag 180gtgtggtcgg gactgtctct aagccttgtt tcacagcata tgaaatgaaa atcggtgcaa 240ttacttttca ggttgctact ggagatatag ccactgaaca ggtagatgtt attgtaaact 300caacagcaag gacatttaat cggaaatcag gtgtgtcaag agctatttta gaaggtgctg 360gacaagctgt ggaaagtgaa tgtgctgtac tagctgcaca gcctcacaga gattttataa 420ttacaccagg tggatgctta aagtgcaaaa taataattca tgttcctggg ggaaaagatg 480tcaggaaaac ggtcaccagt gttctagaag agtgtgaaca gaggaagtac acatcggttt 540cccttccagc cattggaaca ggaaatgccg gaaaaaaccc tatcacagtt gctgataaca 600taatcgatgc tattgtagac ttctcatcac aacattccac cccatcatta aaaacagtta 660aagttgtcat ttttcaacct gagctgctaa atatattcta cgacagcatg aaaaaaagag 720acctctctgc atcactgaac tttcagtcca cattctccat gactacatgt aatcttcctg 780aacactggac tgacatgaat catcagctgt tttgcatggt ccagctagag ccaggacaat 840cagaatataa taccataaag gacaagttca cccgaacttg ttcttcctac gcaatagaga 900agattgagag gatacagaat gcatttctct ggcagagcta ccaggtaaag aaaaggcaaa 960tggatatcaa gaatgaccat aagaataatg agagactcct cttccatggg acagatgcag 1020actcagtgcc atatgtcaat cagcacggct ttaatagaag ttgtgctggg aaaaatgctg 1080tatcctatgg aaaaggaacc tattttgctg tggatgccag ttattctgcc aaggacacct 1140actccaagcc agacagcaat gggagaaagc acatgtacgt tgtgcgagta cttactggag 1200tcttcacaaa gggacgtgca ggattagtca cccctccacc caagaatcct cacaatccca 1260cagatctctt tgactcagtg acaaacaata cacgatctcc aaagctattt gtggtattct 1320ttgataatca ggcttaccca gaatatctca taactttcac ggcttaaaaa tatttttatc 1380atcaaagaga tgatttaagt catctgtaag aacaacatgc aatctttgtc tttgcttctg 1440gcctgtgtaa gcagatgaaa gtttcccttt taggtgccaa aatgctgaaa attacctttt 1500taaagtgctc tattgctgcg atttgtagca tacctttttt tctcagcaaa ttgatgggtg 1560gaagctgaga aatgtatggt aaatgtcaca gagctacaac cattcacaga caccaaatct 1620ctaggagaat aaaaagcaca ttattctttt tctatcagaa aaaaacaaga tgcatcacct 1680taaaaccaag atgacattgt tcttcttgga acatgttaag acatcgaatg gtggcgggtt 1740aaactgtact gcttaagtgg agcggctacc gttatgcatc tatcacagtt ggggattttg 1800ccttattaag gaaaacttgt caatagttca gctgaaatga ctgaatcaca gaatattaac 1860tctgttatgg aacaaatcat aacagatttt acctgtttac atttcaggta aaaatgtatc 1920gcattgttat ctaatattaa aaaattaccc ccaatt 195674444PRTHomo sapiens 74Met Leu Gln Arg Ile Gly Leu Ile Phe Leu His Asn Ile Val Val Val1 5 10 15Ser Asn Cys Phe Tyr Phe Gln Ala Phe Leu Asp Glu Phe Thr Asn Trp 20 25 30Ser Arg Ile Asn Pro Asn Lys Ala Arg Ile Pro Met Ala Gly Asp Thr 35 40 45Gln Gly Val Val Gly Thr Val Ser Lys Pro Cys Phe Thr Ala Tyr Glu 50 55 60Met Lys Ile Gly Ala Ile Thr Phe Gln Val Ala Thr Gly Asp Ile Ala65 70 75 80Thr Glu Gln Val Asp Val Ile Val Asn Ser Thr Ala Arg Thr Phe Asn 85 90 95Arg Lys Ser Gly Val Ser Arg Ala Ile Leu Glu Gly Ala Gly Gln Ala 100 105 110Val Glu Ser Glu Cys Ala Val Leu Ala Ala Gln Pro His Arg Asp Phe 115 120 125Ile Ile Thr Pro Gly Gly Cys Leu Lys Cys Lys Ile Ile Ile His Val 130 135 140Pro Gly Gly Lys Asp Val Arg Lys Thr Val Thr Ser Val Leu Glu Glu145 150 155 160Cys Glu Gln Arg Lys Tyr Thr Ser Val Ser Leu Pro Ala Ile Gly Thr 165 170 175Gly Asn Ala Gly Lys Asn Pro Ile Thr Val Ala Asp Asn Ile Ile Asp 180 185 190Ala Ile Val Asp Phe Ser Ser Gln His Ser Thr Pro Ser Leu Lys Thr 195 200 205Val Lys Val Val Ile Phe Gln Pro Glu Leu Leu Asn Ile Phe Tyr Asp 210 215 220Ser Met Lys Lys Arg Asp Leu Ser Ala Ser Leu Asn Phe Gln Ser Thr225 230 235 240Phe Ser Met Thr Thr Cys Asn Leu Pro Glu His Trp Thr Asp Met Asn 245 250 255His Gln Leu Phe Cys Met Val Gln Leu Glu Pro Gly Gln Ser Glu Tyr 260 265 270Asn Thr Ile Lys Asp Lys Phe Thr Arg Thr Cys Ser Ser Tyr Ala Ile 275 280 285Glu Lys Ile Glu Arg Ile Gln Asn Ala Phe Leu Trp Gln Ser Tyr Gln 290 295 300Val Lys Lys Arg Gln Met Asp Ile Lys Asn Asp His Lys Asn Asn Glu305 310 315 320Arg Leu Leu Phe His Gly Thr Asp Ala Asp Ser Val Pro Tyr Val Asn 325 330 335Gln His Gly Phe Asn Arg Ser Cys Ala Gly Lys Asn Ala Val Ser Tyr 340 345 350Gly Lys Gly Thr Tyr Phe Ala Val Asp Ala Ser Tyr Ser Ala Lys Asp 355 360 365Thr Tyr Ser Lys Pro Asp Ser Asn Gly Arg Lys His Met Tyr Val Val 370 375 380Arg Val Leu Thr Gly Val Phe Thr Lys Gly Arg Ala Gly Leu Val Thr385 390 395 400Pro Pro Pro Lys Asn Pro His Asn Pro Thr Asp Leu Phe Asp Ser Val 405 410 415Thr Asn Asn Thr Arg Ser Pro Lys Leu Phe Val Val Phe Phe Asp Asn 420 425 430Gln Ala Tyr Pro Glu Tyr Leu Ile Thr Phe Thr Ala 435 44075449DNAHomo sapiens 75cgaggtctga gctcctctgg ttcttctcta gacctgctcc ctctctgaaa tgcaaggccg 60tgcctttaat gggcttttgg cattctgtct ccagacctcc cttctcatct gaagggctct 120caggagaaca gagaaaaaac cagcctgtct ccaaactggc ccgtctcagg gactgggggc 180ctttaccccc agtgaaagat gcagacttta cagcgctgca gtacagtaga gtcaagtgac 240tccttcagat agttggatgg gtctctcgat cattcctgat aataacattt tgcctatgtt 300aagtgctttc cacctatcat gttaccttct aactactccc ttggttggat acaggtatta 360gccccatttc acaattaaga aattgaggct taaaaggatt aaagagtttt ttagaggaga 420aacagctctt ccttacagaa ggatcccaa 4497679PRTHomo sapiens 76Arg Ser His Leu Thr Leu Leu Tyr Cys Ser Ala Val Lys Ser Ala Ser1 5 10 15Phe Thr Gly Gly Lys Gly Pro Gln Ser Leu Arg Arg Ala Ser Leu Glu 20 25 30Thr Gly Trp Phe Phe Leu Cys Ser Pro Glu Ser Pro Ser Asp Glu Lys 35 40 45Gly Gly Leu Glu Thr Glu Cys Gln Lys Pro Ile Lys Gly Thr Ala Leu 50 55 60His Phe Arg Glu Gly Ala Gly Leu Glu Lys Asn Gln Arg Ser Ser65 70 75773067DNAHomo sapiens 77ggcacgagca atgggactta tcgctgctga tgttaacctt gatctcttgg ttcaggtggt 60gcctgccagc tgtctccact gtggagttac tatttttcct tttccccatt ttattcatca 120gaagccagtc actaagcgag gtcaaactcc aggacagggg aattaagtgc caccttctgg 180agagggagca ttcacattta ttacttggga tccttctgta aggaagagct gtttctcctc 240taaaaaactc tttaatcctt ttaagcctca atttcttaat tgtgaaatgg ggctaatacc 300tgtatccaac caagggagta gttagaaggt aacatgatag gtggaaagca cttaacatag 360gcaaaatgtt attatcagga atgatcgaga gacccatcca actatctgaa ggagtcactt 420aactctactg tactgcagcg ctgtaaagtc tgcatctttc

actgggggta aaggccccca 480gtccctgaga cgggccagtt tggagacagg ctggtttttt ctctgttctc ctgagagccc 540ttcagatgag aagggaggtc tggagacaga atgccaaaag cccattaaag gcacggcctt 600gcatttcaga gagggagcag gtctagagaa gaaccagagg agctcagctg agatatggtg 660tatggattgg attttggtag aagatgggaa gaaccaaaca cctgagaaac cactttgaag 720atcggggtca gagtaaggcc taacacatag ttggctccca gtaattattg gttgattgaa 780cagctcaaag agcaactcga ccaagaacac tggactggga gtccagttac ttggatcttg 840cattcctgat ttatttttat tttatatgta ttttttctat ttttttgaga cgaagtctca 900ctcactctgt cgcccaggct ggactacaat ggcacgatct cggctcactg caaactctgc 960ctcccaggtt caagcgattc tcctgcctca gcctctcgag tagctaggat tacaggcatg 1020caccaccacg ctggctaatt tttgtatttt tagtagagac ggggttttgc catgttggcc 1080atgctggtgt ccacctcctg acctcagttg atcttcctgc ctcagccttc caaaatgttg 1140ggattacagg cgtgagccac cgtgcctggc cgtgatttat tttttttgtg tatgtttgtt 1200tttgtcaact tgctgtgtga ccttaagcaa gttacttaac ttctctgggc ttcactttcc 1260atggatgaac attgtaaaga ggctggagag agatgaggac taggtacagg ctttagagga 1320gagccaccgc cccggacttc tccctctgtc accccgcttt ccatgaccct ccttgcctga 1380ctttgtgact ccttgcctcg ctatcaaaac aagtgctgca atctcagtgc tttccaagag 1440ccctgcattg ttagaaactt cccagcacgc agcaaaggct gctgcaatac tcgctctgcc 1500tgcctttgcc ctgcgcttcc tacttaccct ccttttgttt ctcccaaaca tctgtccctg 1560actatgctca tctcatgttt gtcctcagct gctgaaaggg ccacgtttgt tttcattaca 1620aataagacca ccgagtgggc tcctggcgtg ggggcgggag cagccgcgcg cagtcttcag 1680aggcagcccc ccaggctgtc tctggagggt gtgtctctgc ttccctttcc ccgtgtttat 1740tttcagacga agccaagtgg cccgggggga ccctccggac tcccagcctt cagagaggag 1800ggcagctcgg gctttcgccg cagtgcttcc tgcccgtcac gtgtgtgctc ctagccgggg 1860tcgggggagc tggtatcttg gcccttctgg gaggacgcgc acagcccgag gaggcagagc 1920cccagacggg aatgggcttt tcagaggtgg ggtgcgggcg aggggacgat gcattatttt 1980taatatttga tttatttttc caactggact tcttcccggg gctctttctg ggcccagctg 2040cctttgtgat cccgcgcccc ggtcctcggc ctctcacctc cagcgccggg gcgccccctg 2100ctgtcggaag cggctgtgac cgggcagagg tgctatctgg gactctgggt tctcagcccg 2160gggacagcga accgaggggc agatgatcca tcagaaaaga gccggcactg cccagccccg 2220cgcccctgcc cctgcctttt tccgggagcg cgccgcgccg cacccgctac ggccgcttga 2280ccccatcttt gagcccggcc ccaagctctg ggaccgtcgt gcccctcatc aaggaagagc 2340caaggacccc aaggagaagg tcaggagcgg cggtgtggat gtcccttggc tgcaggcccc 2400gccgcgcact cccttcagtc cttcccttct ctagggacca ggtagcatca gtgcctggat 2460ctcggccttg tgtgccctgc tccctgcccc acctactaag aaccaagtct ggttcaccgg 2520ctcccaagag ctggaaccca ttctcagcta gctgggggcc caggccaccc cttccctcca 2580gacctgtgtg ccttctgccc tggctccagg gccccccaca ccgtgaccag ggcgggatcc 2640ctatggggct ggccagtcgg caccgtgcca ggcccacagt gccctgggcg tccatggaag 2700tcgttctgtg tctttaaaat cagaaggaag acattaacct ttaggctgaa gaaaatgttt 2760tagtacacag caataactta tttgtcttta tccaacagcc ataaaatata actttaaata 2820ttctattgat agagaaagga gttcatgaag gcagaaatgc ctggggccca cgaacatccc 2880agtgtggccc tggacgggac atcatgctgg gcaacacagc taaaatgcgg gtgaagacca 2940gatttcttgc acatggcggt gacgggatgc tccctagaga gcttcaagtg gattctttgc 3000tttttatttt ctctcttaat aaaaatgtat gatgtttaca ttgtcagaga aaaaaaaaaa 3060aaaaaaa 306778554DNAHomo sapiensmodified_base(1)..(554)n = g, a, c or t 78aaagcgaatt catactataa cagcagaaac aaaacttcag atttcagaat ttgttattgg 60caaaatttat tctcattata cctgcttcat atgggtatat tactattaaa acagaatacc 120atagagtaat tgcattattt gaaaattctn tcattttaca atgcacttca ccaatgaaac 180agntaatttc cattttgaaa attaaaagaa aacagcacag agaagttaaa tgcggtgtag 240caaagttatg gggtctgctt gagggcacta acctcaacag attattcctc ctctccttag 300aataaccatg aaaatacaaa tttacttagc acatttttgc tttttaagta gctggttcat 360tttctgaatt tcccacattc agagttccag tcattattgt tacatcatgt ttgcagaaac 420cttgtcttat ttagtgtcta tttgcatata accctgaaaa cattattatt tgaaaacttt 480tctatatctc aaattaatat acattttcat aacctacctt tgnattaaga cttgcaattt 540tatcaatcta ttat 554793243DNAHomo sapiens 79ccgcagcctc cgcgggtggc aagcgggctg gggagagccg agggccaaag gaagagaaaa 60tcgcggggag tctctggccg ggagagtcca ggtagcgctc ggcgggcagc agtgcgcagg 120cccctcggct tcaaccgcca caatgctgcc agcagcgcca ggcaaggggc ttgggagccc 180ggaccccgcc ccctgcggcc cagcgccccc aggaaataca aaagatataa taatgatata 240tgaagaagat gctgaggaat gggctctgta cttgacagaa gtatttttac atgttgtgaa 300aagggaagcc atcctgttat atcgcttgga gaatttctct tttcggcatt tggagttgct 360gaacttaacg tcttacaaat gtaaactttt gatattatca aatagcctgc ttagagacct 420aactccaaag aaatgtcagt ttctggaaaa gatacttcat tcaccaaaaa gtgtagttac 480tttgctttgt ggagtgaaga gttcagatca gctctatgaa ttactaaata tctctcaaag 540cagatgggag atctcaactg aacaggaacc tgaagactac atctctgtaa tccagagtat 600catattcaaa gattctgaag actactttga ggtcaacatt ccaacagacc tacgagcaaa 660acattctggg gaaataagtg agagaaagga aattgaagaa ctatcagaag cttcaagaaa 720caccatacca ctagcagtgg tgcttcccac tgaaattcca tgtgagaatc ctggtgaaat 780attcataatt ttgagagatg aagtaattgg tgatactgta gaggttgaat ttacatcaag 840taataagcgc attagaacac ggccagccct ttggaataag aaagtctggt gcatgaaagc 900tttagagttt cctgctggtt cagtccatgt caatgtctac tgtgatggaa tcgttaaagc 960tacaaccaaa attaagtact acccaacagc aaaggcaaag gaatgcctat tcagaatggc 1020agattcagga gagagtttgt gccagaatag cattgaagaa cttgatggtg tccttacatc 1080catattcaaa catgagatac catattatga gttccagtct cttcaaactg aaatttgttc 1140tcaaaacaaa tatactcatt tcaaagaact tccaactctt ctccactgtg cagcaaaatt 1200tggcttaaag aacctggcta ttcatttgct tcaatgttca ggagcaacct gggcatctaa 1260gatgaaaaat atggagggtt cagaccccgc acatattgct gaaaggcatg gtcacaaaga 1320actcaagaaa atcttcgaag acttttcaat ccaagaaatt gacataaata atgagcaaga 1380aaatgattat gaagaggata ttgcctcatt ttccacatat attccttcca cacagaaccc 1440agcatttcat catgaaagca gaaagacata cgggcagagt gcagatggag ctgaggcaaa 1500tgaaatggaa ggggaaggaa aacagaatgg atcaggcatg gagaccaaac acagcccact 1560agaggttggc agtgagagtt ctgaagacca gtatgatgac ttgtatgtgt tcattcctgg 1620tgctgatcca gaaaataatt cacaagagcc actcatgagc agcagacctc ctctcccccc 1680gccgcgacct gtagctaatg ccttccaact ggaaagacct cacttcacct taccagggac 1740aatggtggaa ggccaaatgg aaagaagtca aaactggggt catcctggtg ttagacaaga 1800aacaggagat gaacccaaag gagaaaaaga gaagaaagaa gaggaaaaag agcaggagga 1860ggaagaagac ccatatactt ttgctgagat tgatgacagt gaatatgaca tgatattggc 1920caatctgagt ataaagaaaa aaactgggag tcggtctttc attataaata gacctcctgc 1980ccccacaccc cgacccacaa gtatacctcc aaaagaggaa actacacctt acatagctca 2040agtgtttcaa caaaagacag ccagaagaca atctgatgat gacaagttcc gtggtcttcc 2100taagaaacaa gacagagctc ggatagagag tccagccttt tctactctca ggggctgtct 2160aactgatggt caggaagaac tcatcctcct gcaggagaaa gtaaagaatg ggaaaatgtc 2220tatggatgaa gctctggaga aatttaaaca ctggcagatg ggaaaaagtg gcctggaaat 2280gattcagcag gagaaattac gacaactacg agactgcatt attgggaaaa ggccagaaga 2340agaaaatgtc tataataaac tcaccattgt gcaccatcca ggtggtaagg aaactgccca 2400caatgaaaat aagttttata atgtacactt cagcaataag cttcctgctc gaccccaagt 2460tgaaaaggaa tttggtttct gttgcaagaa agatcattaa agaaggttat tataatgaaa 2520ctcacgaatc tacggacatt ttgctttcag ggtgaagcaa gcttgaattt ggattgcctg 2580ctttctttaa agcgaattca tactataaca gcagaaacaa aacttcagat ttcagaattt 2640gttattggca aaatttattc tcattatacc tgcttcatat gggtatatta ctattaaaac 2700agaataccat agagtaattg cattatttga aaattctctc attttacaat gcacttcacc 2760aatgaaacag ctaatttcca ttttgaaaat taaaagaaaa cagcacagag aagttaaatg 2820cggtgtagca aagttatggg gtctgcttga gggcactaac ctcaacagat tattcctcct 2880ctccttagaa taaccatgaa aatacaaatt tacttagcac atttttgctt tttaagtagc 2940tggttcattt tctgaatttc tcacattcag agttccagtc attattgtta catcatgttt 3000gcagaaacct tgtcttattt agtgtctatt tgcatataac cctgaaaaca ttattatttg 3060aaaacttttc tatatctcaa attaatatac attttcataa cctacctttg tattaagact 3120tgcaatttta tcaatctatt atttcttaga aacaatttac tagcttagaa tagaaagcaa 3180tgttatcgtc atataatttt catgtacaaa tgccacaaat aaattgaatg tttaaagcta 3240aaa 324380755PRTHomo sapiens 80Met Ile Tyr Glu Glu Asp Ala Glu Glu Trp Ala Leu Tyr Leu Thr Glu1 5 10 15Val Phe Leu His Val Val Lys Arg Glu Ala Ile Leu Leu Tyr Arg Leu 20 25 30Glu Asn Phe Ser Phe Arg His Leu Glu Leu Leu Asn Leu Thr Ser Tyr 35 40 45Lys Cys Lys Leu Leu Ile Leu Ser Asn Ser Leu Leu Arg Asp Leu Thr 50 55 60Pro Lys Lys Cys Gln Phe Leu Glu Lys Ile Leu His Ser Pro Lys Ser65 70 75 80Val Val Thr Leu Leu Cys Gly Val Lys Ser Ser Asp Gln Leu Tyr Glu 85 90 95Leu Leu Asn Ile Ser Gln Ser Arg Trp Glu Ile Ser Thr Glu Gln Glu 100 105 110Pro Glu Asp Tyr Ile Ser Val Ile Gln Ser Ile Ile Phe Lys Asp Ser 115 120 125Glu Asp Tyr Phe Glu Val Asn Ile Pro Thr Asp Leu Arg Ala Lys His 130 135 140Ser Gly Glu Ile Ser Glu Arg Lys Glu Ile Glu Glu Leu Ser Glu Ala145 150 155 160Ser Arg Asn Thr Ile Pro Leu Ala Val Val Leu Pro Thr Glu Ile Pro 165 170 175Cys Glu Asn Pro Gly Glu Ile Phe Ile Ile Leu Arg Asp Glu Val Ile 180 185 190Gly Asp Thr Val Glu Val Glu Phe Thr Ser Ser Asn Lys Arg Ile Arg 195 200 205Thr Arg Pro Ala Leu Trp Asn Lys Lys Val Trp Cys Met Lys Ala Leu 210 215 220Glu Phe Pro Ala Gly Ser Val His Val Asn Val Tyr Cys Asp Gly Ile225 230 235 240Val Lys Ala Thr Thr Lys Ile Lys Tyr Tyr Pro Thr Ala Lys Ala Lys 245 250 255Glu Cys Leu Phe Arg Met Ala Asp Ser Gly Glu Ser Leu Cys Gln Asn 260 265 270Ser Ile Glu Glu Leu Asp Gly Val Leu Thr Ser Ile Phe Lys His Glu 275 280 285Ile Pro Tyr Tyr Glu Phe Gln Ser Leu Gln Thr Glu Ile Cys Ser Gln 290 295 300Asn Lys Tyr Thr His Phe Lys Glu Leu Pro Thr Leu Leu His Cys Ala305 310 315 320Ala Lys Phe Gly Leu Lys Asn Leu Ala Ile His Leu Leu Gln Cys Ser 325 330 335Gly Ala Thr Trp Ala Ser Lys Met Lys Asn Met Glu Gly Ser Asp Pro 340 345 350Ala His Ile Ala Glu Arg His Gly His Lys Glu Leu Lys Lys Ile Phe 355 360 365Glu Asp Phe Ser Ile Gln Glu Ile Asp Ile Asn Asn Glu Gln Glu Asn 370 375 380Asp Tyr Glu Glu Asp Ile Ala Ser Phe Ser Thr Tyr Ile Pro Ser Thr385 390 395 400Gln Asn Pro Ala Phe His His Glu Ser Arg Lys Thr Tyr Gly Gln Ser 405 410 415Ala Asp Gly Ala Glu Ala Asn Glu Met Glu Gly Glu Gly Lys Gln Asn 420 425 430Gly Ser Gly Met Glu Thr Lys His Ser Pro Leu Glu Val Gly Ser Glu 435 440 445Ser Ser Glu Asp Gln Tyr Asp Asp Leu Tyr Val Phe Ile Pro Gly Ala 450 455 460Asp Pro Glu Asn Asn Ser Gln Glu Pro Leu Met Ser Ser Arg Pro Pro465 470 475 480Leu Pro Pro Pro Arg Pro Val Ala Asn Ala Phe Gln Leu Glu Arg Pro 485 490 495His Phe Thr Leu Pro Gly Thr Met Val Glu Gly Gln Met Glu Arg Ser 500 505 510Gln Asn Trp Gly His Pro Gly Val Arg Gln Glu Thr Gly Asp Glu Pro 515 520 525Lys Gly Glu Lys Glu Lys Lys Glu Glu Glu Lys Glu Gln Glu Glu Glu 530 535 540Glu Asp Pro Tyr Thr Phe Ala Glu Ile Asp Asp Ser Glu Tyr Asp Met545 550 555 560Ile Leu Ala Asn Leu Ser Ile Lys Lys Lys Thr Gly Ser Arg Ser Phe 565 570 575Ile Ile Asn Arg Pro Pro Ala Pro Thr Pro Arg Pro Thr Ser Ile Pro 580 585 590Pro Lys Glu Glu Thr Thr Pro Tyr Ile Ala Gln Val Phe Gln Gln Lys 595 600 605Thr Ala Arg Arg Gln Ser Asp Asp Asp Lys Phe Arg Gly Leu Pro Lys 610 615 620Lys Gln Asp Arg Ala Arg Ile Glu Ser Pro Ala Phe Ser Thr Leu Arg625 630 635 640Gly Cys Leu Thr Asp Gly Gln Glu Glu Leu Ile Leu Leu Gln Glu Lys 645 650 655Val Lys Asn Gly Lys Met Ser Met Asp Glu Ala Leu Glu Lys Phe Lys 660 665 670His Trp Gln Met Gly Lys Ser Gly Leu Glu Met Ile Gln Gln Glu Lys 675 680 685Leu Arg Gln Leu Arg Asp Cys Ile Ile Gly Lys Arg Pro Glu Glu Glu 690 695 700Asn Val Tyr Asn Lys Leu Thr Ile Val His His Pro Gly Gly Lys Glu705 710 715 720Thr Ala His Asn Glu Asn Lys Phe Tyr Asn Val His Phe Ser Asn Lys 725 730 735Leu Pro Ala Arg Pro Gln Val Glu Lys Glu Phe Gly Phe Cys Cys Lys 740 745 750Lys Asp His 755813195DNAHomo sapiens 81ggaagagaaa atcgcgggga gtctctggcc gggagagtcc aggtagcgct cggcgggcag 60cagtgcgcag gcccctcggc ttcaaccgcc acaatgctgc cagcagcgcc aggcaagggg 120cttgggagcc cggaccccgc cccctgcggc ccagcgcccc caggaaatac aaaagatata 180ataatgatat atgaagaaga tgctgaggaa tgggctctgt acttgacaga agtattttta 240catgttgtga aaagggaagc catcctgtta tatcgcttgg agaatttctc ttttcggcat 300ttggagttgc tgaacttaac gtcttacaaa tgtaaacttt tgatattatc aaatagcctg 360cttagagacc taactccaaa gaaatgtcag tttctggaaa agatacttca ttcaccaaaa 420agtgtagtta ctttgctttg tggagtgaag agttcagatc agctctatga attactaaat 480atctctcaaa gcagatggga gatctcaact gaacaggaac ctgaagacta catctctgta 540atccagagta tcatattcaa agattctgaa gactactttg aggtcaacat tccaacagac 600ctacgagcaa aacattctgg ggaaataagt gagagaaagg aaattgaaga actatcagaa 660gcttcaagaa acaccatacc actagcagtg gtgcttccca ctgaaattcc atgtgagaat 720cctggtgaaa tattcataat tttgagagat gaagtaattg gtgatactgt agaggttgaa 780tttacatcaa gtaataagcg cattagaaca cggccagccc tttggaataa gaaagtctgg 840tgcatgaaag ctttagagtt tcctgctggt tcagtccatg tcaatgtcta ctgtgatgga 900atcgttaaag ctacaaccaa aattaagtac tacccaacag caaaggcaaa ggaatgccta 960ttcagaatgg cagattcagg agagagtttg tgccagaata gcattgaaga acttgatggt 1020gtccttacat ccatattcaa acatgagata ccatattatg agttccagtc tcttcaaact 1080gaaatttgtt ctcaaaacaa atatactcat ttcaaagaac ttccaactct tctccactgt 1140gcagcaaaat ttggcttaaa gaacctggct attcatttgc ttcaatgttc aggagcaacc 1200tgggcatcta agatgaaaaa tatggagggt tcagaccccg cacatattgc tgaaaggcat 1260ggtcacaaag aactcaagaa aatcttcgaa gacttttcaa tccaagaaat tgacataaat 1320aatgagcaag aaaatgatta tgaagaggat attgcctcat tttccacata tattccttcc 1380acacagaacc cagcatttca tcatgaaagc agaaagacat acgggcagag tgcagatgga 1440gctgaggcaa atgaaatgga aggggaagga aaacagaatg gatcaggcat ggagaccaaa 1500cacagcccac tagaggttgg cagtgagagt tctgaagacc agtatgatga cttgtatgtg 1560ttcattcctg gtgctgatcc agaaaataat tcacaagagc cactcatgag cagcagacct 1620cctctccccc cgccgcgacc tgtagctaat gccttccaac tggaaagacc tcacttcacc 1680ttaccaggga caatggtgga aggccaaatg gaaagaagtc aaaactgggg tcatcctggt 1740gttagacaag aaacaggaga tgaacccaaa ggagaaaaag agaagaaaga agaggaaaaa 1800gagcaggagg aggaagaaga cccatatact tttgctgaga ttgatgacag tgaatatgac 1860atgatattgg ccaatctgag tataaagaaa aaaactggga gtcggtcttt cattataaat 1920agacctcctg cccccacacc ccgacccaca agtatacctc caaaagagga aactacacct 1980tacatagctc aagtgtttca acaaaagaca gccagaagac aatctgatga tgacaagttc 2040cgtggtcttc ctaagaaaca agacagagct cggatagaga gtccagcctt ttctactctc 2100aggggctgtc taactgatgg tcaggaagaa ctcatcctcc tgcaggagaa agtaaagaat 2160gggaaaatgt ctatggatga agctctggag aaatttaaac actggcagat gggaaaaagt 2220ggcctggaaa tgattcagca ggagaaatta cgacaactac gagactgcat tattgggaaa 2280aggccagaag aagaaaatgt ctataataaa ctcaccattg tgcaccatcc aggtggtaag 2340gaaactgccc acaatgaaaa taagttttat aatgtacact tcagcaataa gcttcctgct 2400cgaccccaag ttgaaaagga atttggtttc tgttgcaaga aagatcatta aagaaggtta 2460ttataatgaa actcacgaat ctacggacat tttgctttca gggtgaagca agcttgaatt 2520tggattgcct gctttcttta aagcgaattc atactataac agcagaaaca aaacttcaga 2580tttcagaatt tgttattggc aaaatttatt ctcattatac ctgcttcata tgggtatatt 2640actattaaaa cagaatacca tagagtaatt gcattatttg aaaattctct cattttacaa 2700tgcacttcac caatgaaaca gctaatttcc attttgaaaa ttaaaagaaa acagcacaga 2760gaagttaaat gcggtgtagc aaagttatgg ggtctgcttg agggcactaa cctcaacaga 2820ttattcctcc tctccttaga ataaccatga aaatacaaat ttacttagca catttttgct 2880ttttaagtag ctggttcatt ttctgaattt ctcacattca gagttccagt cattattgtt 2940acatcatgtt tgcagaaacc ttgtcttatt tagtgtctat ttgcatataa ccctgaaaac 3000attattattt gaaaactttt ctatatctca aattaatata cattttcata acctaccttt 3060gtattaagac ttgcaatttt atcaatctat tatttcttag aaacaattta ctagcttaga 3120atagaaagca atgttatcgt catataattt tcatgtacaa atgccacaaa taaattgaat 3180gtttaaagct aaaaa 319582816PRTHomo sapiens 82Gly Arg Glu Asn Arg Gly Glu Ser Leu Ala Gly Arg Val Gln Val Ala1 5 10 15Leu Gly Gly Gln Gln Cys Ala Gly Pro Ser Ala Ser Thr Ala Thr Met 20 25 30Leu Pro Ala Ala Pro Gly Lys Gly Leu Gly Ser Pro Asp Pro Ala Pro 35 40 45Cys Gly Pro Ala Pro Pro Gly Asn Thr Lys Asp Ile Ile Met Ile Tyr 50 55 60Glu Glu Asp Ala Glu Glu Trp Ala Leu Tyr Leu Thr Glu Val Phe Leu65 70 75 80His Val Val Lys Arg Glu Ala Ile Leu Leu Tyr Arg Leu Glu Asn Phe

85 90 95Ser Phe Arg His Leu Glu Leu Leu Asn Leu Thr Ser Tyr Lys Cys Lys 100 105 110Leu Leu Ile Leu Ser Asn Ser Leu Leu Arg Asp Leu Thr Pro Lys Lys 115 120 125Cys Gln Phe Leu Glu Lys Ile Leu His Ser Pro Lys Ser Val Val Thr 130 135 140Leu Leu Cys Gly Val Lys Ser Ser Asp Gln Leu Tyr Glu Leu Leu Asn145 150 155 160Ile Ser Gln Ser Arg Trp Glu Ile Ser Thr Glu Gln Glu Pro Glu Asp 165 170 175Tyr Ile Ser Val Ile Gln Ser Ile Ile Phe Lys Asp Ser Glu Asp Tyr 180 185 190Phe Glu Val Asn Ile Pro Thr Asp Leu Arg Ala Lys His Ser Gly Glu 195 200 205Ile Ser Glu Arg Lys Glu Ile Glu Glu Leu Ser Glu Ala Ser Arg Asn 210 215 220Thr Ile Pro Leu Ala Val Val Leu Pro Thr Glu Ile Pro Cys Glu Asn225 230 235 240Pro Gly Glu Ile Phe Ile Ile Leu Arg Asp Glu Val Ile Gly Asp Thr 245 250 255Val Glu Val Glu Phe Thr Ser Ser Asn Lys Arg Ile Arg Thr Arg Pro 260 265 270Ala Leu Trp Asn Lys Lys Val Trp Cys Met Lys Ala Leu Glu Phe Pro 275 280 285Ala Gly Ser Val His Val Asn Val Tyr Cys Asp Gly Ile Val Lys Ala 290 295 300Thr Thr Lys Ile Lys Tyr Tyr Pro Thr Ala Lys Ala Lys Glu Cys Leu305 310 315 320Phe Arg Met Ala Asp Ser Gly Glu Ser Leu Cys Gln Asn Ser Ile Glu 325 330 335Glu Leu Asp Gly Val Leu Thr Ser Ile Phe Lys His Glu Ile Pro Tyr 340 345 350Tyr Glu Phe Gln Ser Leu Gln Thr Glu Ile Cys Ser Gln Asn Lys Tyr 355 360 365Thr His Phe Lys Glu Leu Pro Thr Leu Leu His Cys Ala Ala Lys Phe 370 375 380Gly Leu Lys Asn Leu Ala Ile His Leu Leu Gln Cys Ser Gly Ala Thr385 390 395 400Trp Ala Ser Lys Met Lys Asn Met Glu Gly Ser Asp Pro Ala His Ile 405 410 415Ala Glu Arg His Gly His Lys Glu Leu Lys Lys Ile Phe Glu Asp Phe 420 425 430Ser Ile Gln Glu Ile Asp Ile Asn Asn Glu Gln Glu Asn Asp Tyr Glu 435 440 445Glu Asp Ile Ala Ser Phe Ser Thr Tyr Ile Pro Ser Thr Gln Asn Pro 450 455 460Ala Phe His His Glu Ser Arg Lys Thr Tyr Gly Gln Ser Ala Asp Gly465 470 475 480Ala Glu Ala Asn Glu Met Glu Gly Glu Gly Lys Gln Asn Gly Ser Gly 485 490 495Met Glu Thr Lys His Ser Pro Leu Glu Val Gly Ser Glu Ser Ser Glu 500 505 510Asp Gln Tyr Asp Asp Leu Tyr Val Phe Ile Pro Gly Ala Asp Pro Glu 515 520 525Asn Asn Ser Gln Glu Pro Leu Met Ser Ser Arg Pro Pro Leu Pro Pro 530 535 540Pro Arg Pro Val Ala Asn Ala Phe Gln Leu Glu Arg Pro His Phe Thr545 550 555 560Leu Pro Gly Thr Met Val Glu Gly Gln Met Glu Arg Ser Gln Asn Trp 565 570 575Gly His Pro Gly Val Arg Gln Glu Thr Gly Asp Glu Pro Lys Gly Glu 580 585 590Lys Glu Lys Lys Glu Glu Glu Lys Glu Gln Glu Glu Glu Glu Asp Pro 595 600 605Tyr Thr Phe Ala Glu Ile Asp Asp Ser Glu Tyr Asp Met Ile Leu Ala 610 615 620Asn Leu Ser Ile Lys Lys Lys Thr Gly Ser Arg Ser Phe Ile Ile Asn625 630 635 640Arg Pro Pro Ala Pro Thr Pro Arg Pro Thr Ser Ile Pro Pro Lys Glu 645 650 655Glu Thr Thr Pro Tyr Ile Ala Gln Val Phe Gln Gln Lys Thr Ala Arg 660 665 670Arg Gln Ser Asp Asp Asp Lys Phe Arg Gly Leu Pro Lys Lys Gln Asp 675 680 685Arg Ala Arg Ile Glu Ser Pro Ala Phe Ser Thr Leu Arg Gly Cys Leu 690 695 700Thr Asp Gly Gln Glu Glu Leu Ile Leu Leu Gln Glu Lys Val Lys Asn705 710 715 720Gly Lys Met Ser Met Asp Glu Ala Leu Glu Lys Phe Lys His Trp Gln 725 730 735Met Gly Lys Ser Gly Leu Glu Met Ile Gln Gln Glu Lys Leu Arg Gln 740 745 750Leu Arg Asp Cys Ile Ile Gly Lys Arg Pro Glu Glu Glu Asn Val Tyr 755 760 765Asn Lys Leu Thr Ile Val His His Pro Gly Gly Lys Glu Thr Ala His 770 775 780Asn Glu Asn Lys Phe Tyr Asn Val His Phe Ser Asn Lys Leu Pro Ala785 790 795 800Arg Pro Gln Val Glu Lys Glu Phe Gly Phe Cys Cys Lys Lys Asp His 805 810 815833544DNAHomo sapiens 83attttggttt ctcttcaaga attaacaaac cacttactct tgaattctct tctagttaac 60acaggcatca ctacttccaa ttgatctcag gatgtgggat cctcatacac attttgaaca 120aaatcctctg tttcagcaag gaattcatat ttgcatatgg tgaagatggt ttctgaagtg 180agatcagaag tagagcttct aatgaccccc agaagcactg agtgaccaag tgacatacct 240gccaggccca ttgtgtccat cgctctcaga gcagctgggg attgtgcttg gctcccagag 300ctatggtgca aaaggcgggg tcgctagggc cactcaggga aagagaaccc agaaacatgg 360catgctgaca aaaggtagtc cctgcttatc cagcttcact ttctgctgat ttagttaccc 420atggtcaact gccatctgaa aataggaaat acaaaagata taataatgat atatgaagaa 480gatgctgagg aatgggctct gtacttgaca gaagtatttt tacatgttgt gaaaagggaa 540gccatcctgt tatatcgctt ggagaatttc tcttttcggc atttggagtt gctgaactta 600acgtcttaca aatgtaaact tttgatatta tcaaatagcc tgcttagaga cctaactcca 660aagaaatgtc agtttctgga aaagatactt cattcaccaa aaagtgtagt tactttgctt 720tgtggagtga agagttcaga tcagctctat gaattactaa atatctctca aagcagatgg 780gagatctcaa ctgaacagga acctgaagac tacatctctg taatccagag tatcatattc 840aaagattctg aagactactt tgaggtcaac attccaacag acctacgagc aaaacattct 900ggggaaataa gtgagagaaa ggaaattgaa gaactatcag aagcttcaag aaacaccata 960ccactagcag tggtgcttcc cactgaaatt ccatgtgagg atcctggtga aatattcata 1020attttgagag atgaagtaat tggtgatact gtagaggttg aatttacatc aagtaataag 1080cgcattagaa cacggccagc cctttggaat aagaaagtct ggtgcatgaa agctttagag 1140tttcctgctg gttcagtcca tgtcaatgtc tactgtgatg gaatcgttaa agctacaacc 1200aaaattaagt actacccaac agcaaaggca aaggaatgcc tattcagaat ggcagattca 1260ggagagagtt tgtgccagaa tagcattgaa gaacttgatg gtgtccttac atccatattc 1320aaacatgaga taccatatta tgagttccag tctcttcaaa ctgaaatttg ttctcaaaac 1380aaatatactc atttcaaaga acttccaact cttctccact gtgcagcaaa atttggctta 1440aagaacctgg ctattcattt gcttcaatgt tcaggagcaa cctgggcatc taagatgaaa 1500aatatggagg gttcagaccc cacacatatt gctgaaaggc atggtcacaa agaactcaag 1560aaaatcttcg aagacttttc aatccaagaa attgacataa ataatgagca agaaaatgat 1620tatgaagagg atattgcctc attttccaca tatattcctt ccacacagaa cccagcattt 1680catcatgaaa gcaggaagac atacgggcag agtgcagatg gagctgaggc aaatgaaatg 1740gaaggggaag gaaaacagaa tggatcaggc atggagacca aacacagccc actagaggtt 1800ggcagtgaga gttctgaaga ccagtatgat gacttgtatg tgttcattcc tggtgctgat 1860ccagaaaata attcacaaga gccactcatg agcagcagac ctcctctccc cccgccgcga 1920cctgtagcta atgccttcca actggaaaga cctcacttca ccttaccagg gacaatggtg 1980gaaggccaaa tggaaagaag tcaaaactgg ggtcatcctg gtgttagaca agaaacagga 2040gatgaaccca aaggagaaaa agagaagaaa gaagaggaaa aagagcagga ggaggaagaa 2100gacccatata cttttgctga gattgatgac agtgaatatg acatgatatt ggccaatctg 2160agtataaaga aaaaaactgg gagtcggtct ttcattataa atagacctcc tgcccccaca 2220ccccgaccca caagtatacc tccaaaagag gaaactacgc cttacatagc tcaagtgttt 2280caacaaaaga cagccagaag acaatctgat gatgacaagt tccgtggtct tcctaagaaa 2340caagacagag ctcggataga gagtccagcc ttttctactc tcaggggctg tctaactgat 2400ggtcaggaag aactcatcct cctgcaggag aaagtaaaga atgggaaaat gtctatggat 2460gaagctctgg agaaatttaa acactggcag atgggaaaaa gtggcctgga aatgattcag 2520caggagaaat tacgacaact acgagactgc attattggga aaaggccaga agaagaaaat 2580gtctataata aactcaccat tgtgcaccat ccaggtggta aggaaactgc ccacaatgaa 2640aataagtttt ataatgtaca cttcagcaat aagcttcctg ctcgacccca agttgaaaag 2700gaatttggtt tctgttgcaa gaaagatcat taaagaaggt tattataatg aaactcacga 2760atctacggac attttgcttt cagggtgaag caagcttgaa tttggattgc ctgctctctt 2820taaagcgaat tcatactatg acagcagaaa caaaacttca gatttcagaa tttgttattg 2880gcaaaattta ttctcattat acctgcttca tatgggtata ttactattaa aacagaatac 2940catagagtaa ttgcattatt tgaaaattct ctcattttac aatgcacttc accaatgaaa 3000cagctaattt ccattttgaa aattaaaaga aaacagcaca gagaagttaa atgcggtgta 3060gcaaagttat ggggtctgct tgagggcact aacctcaaca gattattcct cccctcctta 3120gaataaccat gaaaatacaa atttacttag cacatttctg ctttttaagt agctggttca 3180ttttctgaat ttctcacatt cagagttcca gtcattattg ttacatcatg tttgcagaaa 3240ccttgtctta tttagtgtct atttgcatat aaccctgaaa acattattat ttgaaaactt 3300ttctatatct caaattaata tacattttca taacctacct ttgtattaag acttgcaatt 3360ttatcaatct attatttctt agaaacaatt tactagctta gaatagaaag caatgttatc 3420gtcatataat tttcatgtac aaatgccaca aataaattga atgtttaaag ctatgtctga 3480gtttttaaag taaatttata agaattagcc aataaaattg cttctcggcc ttttggctaa 3540gatc 354484770PRTHomo sapiens 84Met Val Asn Cys His Leu Lys Ile Gly Asn Thr Lys Asp Ile Ile Met1 5 10 15Ile Tyr Glu Glu Asp Ala Glu Glu Trp Ala Leu Tyr Leu Thr Glu Val 20 25 30Phe Leu His Val Val Lys Arg Glu Ala Ile Leu Leu Tyr Arg Leu Glu 35 40 45Asn Phe Ser Phe Arg His Leu Glu Leu Leu Asn Leu Thr Ser Tyr Lys 50 55 60Cys Lys Leu Leu Ile Leu Ser Asn Ser Leu Leu Arg Asp Leu Thr Pro65 70 75 80Lys Lys Cys Gln Phe Leu Glu Lys Ile Leu His Ser Pro Lys Ser Val 85 90 95Val Thr Leu Leu Cys Gly Val Lys Ser Ser Asp Gln Leu Tyr Glu Leu 100 105 110Leu Asn Ile Ser Gln Ser Arg Trp Glu Ile Ser Thr Glu Gln Glu Pro 115 120 125Glu Asp Tyr Ile Ser Val Ile Gln Ser Ile Ile Phe Lys Asp Ser Glu 130 135 140Asp Tyr Phe Glu Val Asn Ile Pro Thr Asp Leu Arg Ala Lys His Ser145 150 155 160Gly Glu Ile Ser Glu Arg Lys Glu Ile Glu Glu Leu Ser Glu Ala Ser 165 170 175Arg Asn Thr Ile Pro Leu Ala Val Val Leu Pro Thr Glu Ile Pro Cys 180 185 190Glu Asp Pro Gly Glu Ile Phe Ile Ile Leu Arg Asp Glu Val Ile Gly 195 200 205Asp Thr Val Glu Val Glu Phe Thr Ser Ser Asn Lys Arg Ile Arg Thr 210 215 220Arg Pro Ala Leu Trp Asn Lys Lys Val Trp Cys Met Lys Ala Leu Glu225 230 235 240Phe Pro Ala Gly Ser Val His Val Asn Val Tyr Cys Asp Gly Ile Val 245 250 255Lys Ala Thr Thr Lys Ile Lys Tyr Tyr Pro Thr Ala Lys Ala Lys Glu 260 265 270Cys Leu Phe Arg Met Ala Asp Ser Gly Glu Ser Leu Cys Gln Asn Ser 275 280 285Ile Glu Glu Leu Asp Gly Val Leu Thr Ser Ile Phe Lys His Glu Ile 290 295 300Pro Tyr Tyr Glu Phe Gln Ser Leu Gln Thr Glu Ile Cys Ser Gln Asn305 310 315 320Lys Tyr Thr His Phe Lys Glu Leu Pro Thr Leu Leu His Cys Ala Ala 325 330 335Lys Phe Gly Leu Lys Asn Leu Ala Ile His Leu Leu Gln Cys Ser Gly 340 345 350Ala Thr Trp Ala Ser Lys Met Lys Asn Met Glu Gly Ser Asp Pro Thr 355 360 365His Ile Ala Glu Arg His Gly His Lys Glu Leu Lys Lys Ile Phe Glu 370 375 380Asp Phe Ser Ile Gln Glu Ile Asp Ile Asn Asn Glu Gln Glu Asn Asp385 390 395 400Tyr Glu Glu Asp Ile Ala Ser Phe Ser Thr Tyr Ile Pro Ser Thr Gln 405 410 415Asn Pro Ala Phe His His Glu Ser Arg Lys Thr Tyr Gly Gln Ser Ala 420 425 430Asp Gly Ala Glu Ala Asn Glu Met Glu Gly Glu Gly Lys Gln Asn Gly 435 440 445Ser Gly Met Glu Thr Lys His Ser Pro Leu Glu Val Gly Ser Glu Ser 450 455 460Ser Glu Asp Gln Tyr Asp Asp Leu Tyr Val Phe Ile Pro Gly Ala Asp465 470 475 480Pro Glu Asn Asn Ser Gln Glu Pro Leu Met Ser Ser Arg Pro Pro Leu 485 490 495Pro Pro Pro Arg Pro Val Ala Asn Ala Phe Gln Leu Glu Arg Pro His 500 505 510Phe Thr Leu Pro Gly Thr Met Val Glu Gly Gln Met Glu Arg Ser Gln 515 520 525Asn Trp Gly His Pro Gly Val Arg Gln Glu Thr Gly Asp Glu Pro Lys 530 535 540Gly Glu Lys Glu Lys Lys Glu Glu Glu Lys Glu Gln Glu Glu Glu Glu545 550 555 560Asp Pro Tyr Thr Phe Ala Glu Ile Asp Asp Ser Glu Tyr Asp Met Ile 565 570 575Leu Ala Asn Leu Ser Ile Lys Lys Lys Thr Gly Ser Arg Ser Phe Ile 580 585 590Ile Asn Arg Pro Pro Ala Pro Thr Pro Arg Pro Thr Ser Ile Pro Pro 595 600 605Lys Glu Glu Thr Thr Pro Tyr Ile Ala Gln Val Phe Gln Gln Lys Thr 610 615 620Ala Arg Arg Gln Ser Asp Asp Asp Lys Phe Arg Gly Leu Pro Lys Lys625 630 635 640Gln Asp Arg Ala Arg Ile Glu Ser Pro Ala Phe Ser Thr Leu Arg Gly 645 650 655Cys Leu Thr Asp Gly Gln Glu Glu Leu Ile Leu Leu Gln Glu Lys Val 660 665 670Lys Asn Gly Lys Met Ser Met Asp Glu Ala Leu Glu Lys Phe Lys His 675 680 685Trp Gln Met Gly Lys Ser Gly Leu Glu Met Ile Gln Gln Glu Lys Leu 690 695 700Arg Gln Leu Arg Asp Cys Ile Ile Gly Lys Arg Pro Glu Glu Glu Asn705 710 715 720Val Tyr Asn Lys Leu Thr Ile Val His His Pro Gly Gly Lys Glu Thr 725 730 735Ala His Asn Glu Asn Lys Phe Tyr Asn Val His Phe Ser Asn Lys Leu 740 745 750Pro Ala Arg Pro Gln Val Glu Lys Glu Phe Gly Phe Cys Cys Lys Lys 755 760 765Asp His 77085564DNAHomo sapiens 85cctctcagaa aactgagcat actagcaaga cagctcttct tgaaaaaaaa aatatgtata 60cacaaatata tacgtatatc tatatatacg tatgtatata cacacatgta tattcttcct 120tgattgtgta gctgtccaaa ataataacat atatagaggg agctgtattc ctttatacaa 180atctgatggc tcctgcagca ctttttcctt ctgaaaatat ttacattttg ctaacctagt 240ttgttacttt aaaaatcagt tttgatgaaa ggagggaaaa gcagatggac ttgaaaaaga 300tccaagctcc tattagaaaa ggtatgaaaa tctttatagt aaaatttttt ataaactaaa 360gttgtacctt ttaatatgta gtaaactctc atttatttgg ggttcgctct tggatctcat 420ccatccattg tgttctcttt aatgctgcct gccttttgag gcattcactg ccctagacaa 480tgccaccaga gatagtgggg gaaatgccag atgaaaccaa ctcttgctct cactagttgt 540cagcttctct ggataagtga ccac 564865024DNAHomo sapiens 86agcggagtgg gtcctgcctg tgacgcgcgg cggcggtcgg tcctgcctgt aacggcggcg 60gcggctgctg ctccagacac ctgcggcggc ggcggcgacc acgcggcggg cgcggagatg 120tggcccctgg tagcggcgct gttgctgggc tcggcgtgct gcggatcagc tcagctacta 180tttaataaaa caaaatctgt agaattcacg ttttgtaatg acactgtcgt cattccatgc 240tttgttacta atatggaggc acaaaacact actgaagtat acgtaaagtg gaaatttaaa 300ggaagagata tttacacctt tgatggagct ctaaacaagt ccactgtccc cactgacttt 360agtagtgcaa aaattgaagt ctcacaatta ctaaaaggag atgcctcttt gaagatggat 420aagagtgatg ctgtctcaca cacaggaaac tacacttgtg aagtaacaga attaaccaga 480gaaggtgaaa cgatcatcga gctaaaatat cgtgttgttt catggttttc tccaaatgaa 540aatattctta ttgttatttt cccaattttt gctatactcc tgttctgggg acagtttggt 600attaaaacac ttaaatatag atccggtggt atggatgaga aaacaattgc tttacttgtt 660gctggactag tgatcactgt cattgtcatt gttggagcca ttcttttcgt cccaggtgaa 720tattcattaa agaatgctac tggccttggt ttaattgtga cttctacagg gatattaata 780ttacttcact actatgtgtt tagtacagcg attggattaa cctccttcgt cattgccata 840ttggttattc aggtgatagc ctatatcctc gctgtggttg gactgagtct ctgtattgcg 900gcgtgtatac caatgcatgg ccctcttctg atttcaggtt tgagtatctt agctctagca 960caattacttg gactagttta tatgaaattt gtggcttcca atcagaagac tatacaacct 1020cctaggaata actgaagtga agtgatggac tccgatttgg agagtagtaa gacgtgaaag 1080gaatacactt gtgtttaagc accatggcct tgatgattca ctgttgggga gaagaaacaa 1140gaaaagtaac tggttgtcac ctatgagacc cttacgtgat tgttagttaa gtttttattc 1200aaagcagctg taatttagtt aataaaataa ttatgatcta tgttgtttgc ccaattgaga 1260tccagttttt tgttgttatt tttaatcaat taggggcaat agtagaatgg acaatttcca 1320agaatgatgc ctttcaggtc ctagggcctc tggcctctag gtaaccagtt taaattggtt 1380cagggtgata actacttagc actgccctgg tgattaccca gagatatcta tgaaaaccag 1440tggcttccat caaacctttg ccaactcagg ttcacagcag ctttgggcag ttatggcagt 1500atggcattag ctgagaggtg tctgccactt ctgggtcaat ggaataataa attaagtaca 1560ggcaggaatt tggttgggag catcttgtat gatctccgta tgatgtgata ttgatggaga 1620tagtggtcct cattcttggg ggttgccatt cccacattcc

cccttcaaca aacagtgtaa 1680caggtccttc ccagatttag ggtactttta ttgatggata tgttttcctt ttattcacat 1740aaccccttga aaccctgtct tgtcctcctg ttacttgctt ctgctgtaca agatgtagca 1800ccttttctcc tctttgaaca tggtctagtg acacggtagc accagttgca ggaaggagcc 1860agacttgttc tcagagcact gtgttcacac ttttcagcaa aaatagctat ggttgtaaca 1920tatgtattcc cttcctctga tttgaaggca aaaatctaca gtgtttcttc acttcttttc 1980tgatctgggg catgaaaaaa gcaagattga aatttgaact atgagtctcc tgcatggcaa 2040caaaatgtgt gtcaccatca ggccaacagg ccagcccttg aatggggatt tattactgtt 2100gtatctatgt tgcatgataa acattcatca ccttcctcct gtagtcctgc ctcgtactcc 2160ccttccccta tgattgaaaa gtaaacaaaa cccacatttc ctatcctggt tagaagaaaa 2220ttaatgttct gacagttgtg atcgcctgga gtacttttag acttttagca ttcgtttttt 2280acctgtttgt ggatgtgtgt ttgtatgtgc atacgtatga gataggcaca tgcatcttct 2340gtatggacaa aggtggggta cctacaggag agcaaaggtt aattttgtgc ttttagtaaa 2400aacatttaaa tacaaagttc tttattgggt ggaattatat ttgatgcaaa tatttgatca 2460cttaaaactt ttaaaacttc taggtaattt gccacgcttt ttgactgctc accaataccc 2520tgtaaaaata cgtaattctt cctgtttgtg taataagata ttcatatttg tagttgcatt 2580aataatagtt atttcttagt ccatcagatg ttcccgtgtg cctcttttat gccaaattga 2640ttgtcatatt tcatgttggg accaagtagt ttgcccatgg caaacctaaa tttatgacct 2700gctgaggcct ctcagaaaac tgagcatact agcaagacag ctcttcttga aaaaaaaaat 2760atgtatacac aaatatatac gtatatctat atatacgtat gtatatacac acatgtatat 2820tcttccttga ttgtgtagct gtccaaaata ataacatata tagagggagc tgtattcctt 2880tatacaaatc tgatggctcc tgcagcactt tttccttctg aaaatattta cattttgcta 2940acctagtttg ttactttaaa aatcagtttt gatgaaagga gggaaaagca gatggacttg 3000aaaaagatcc aagctcctat tagaaaaggt atgaaaatct ttatagtaaa attttttata 3060aactaaagtt gtacctttta atatgtagta aactctcatt tatttggggt tcgctcttgg 3120atctcatcca tccattgtgt tctctttaat gctgcctgcc ttttgaggca ttcactgccc 3180tagacaatgc caccagagat agtgggggaa atgccagatg aaaccaactc ttgctctcac 3240tagttgtcag cttctctgga taagtgacca cagaagcagg agtcctcctg cttgggcatc 3300attgggccag ttccttctct ttaaatcaga tttgtaatgg ctcccaaatt ccatcacatc 3360acatttaaat tgcagacagt gttttgcaca tcatgtatct gttttgtccc ataatatgct 3420ttttactccc tgatcccagt ttctgctgtt gactcttcca ttcagtttta tttattgtgt 3480gttctcacag tgacaccatt tgtccttttc tgcaacaacc tttccagcta cttttgccaa 3540attctatttg tcttctcctt caaaacattc tcctttgcag ttcctcttca tctgtgtagc 3600tgctcttttg tctcttaact taccattcct atagtacttt atgcatctct gcttagttct 3660attagttttt tggccttgct cttctccttg attttaaaat tccttctata gctagagctt 3720ttctttcttt cattctctct tcctgcagtg ttttgcatac atcagaagct aggtacataa 3780gttaaatgat tgagagttgg ctgtatttag atttatcact ttttaatagg gtgagcttga 3840gagttttctt tctttctgtt tttttttttt ttttttttga ctaatttcac atgctctaaa 3900aaccttcaaa ggtgattatt tttctcctgg aaactccagg tccattctgt ttaaatccct 3960aagaatgtca gaattaaaat aacagggcta tcgcgtaatt ggaaatattt cttttttcag 4020gatgctatag tcaatttagt aagtgaccac caaattgtta tttgcactaa caaagctcaa 4080aacacgataa gtttactcct ccatctcagt aataaaaatt aagctgtaat caaccttcta 4140ggtttctctt gtcttaaaat gggtattcaa aaatggggat ctgtggtgta tgtatggaaa 4200cacatactcc ttaatttacc tgttgttgga aactggagaa atgattgtcg ggcaaccgtt 4260tattttttat tgtattttat ttggttgagg gattttttta taaacagttt tacttgtgtc 4320atattttaaa attactaact gccatcacct gctggggtcc tttgttaggt cattttcagt 4380gactaatagg gataatccag gtaactttga agagatgagc agtgagtgac caggcagttt 4440ttctgccttt agctttgaca gttcttaatt aagatcattg aagaccagct ttctcataaa 4500tttctctttt tgaaaaaaag aaagcatttg tactaagctc ctctgtaaga caacatctta 4560aatcttaaaa gtgttgttat catgactggt gagagaagaa aacattttgt ttttattaaa 4620tggagcatta tttacaaaaa gccattgttg agaattagat cccacatcgt ataaatatct 4680attaaccatt ctaaataaag agaactccag tgttgctatg tgcaagatcc tctcttggag 4740cttttttgca tagcaattaa aggtgtgcta tttgtcagta gccatttttt tgcagtgatt 4800tgaagaccaa agttgtttta cagctgtgtt accgttaaag gttttttttt ttatatgtat 4860taaatcaatt tatcactgtt taaagctttg aatatctgca atctttgcca aggtactttt 4920ttatttaaaa aaaaacataa ctttgtaaat attaccctgt aatattatat atacttaata 4980aaacatttta agctaaaaaa aaaaaaacaa aaaaaaaaaa aaaa 502487305PRTHomo sapiens 87Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly1 5 10 15Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp65 70 75 80Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe145 150 155 160Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala225 230 235 240Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Asn 290 295 300Asn305886790DNAHomo sapiens 88ggagatattt tcttgttcaa tttaaggaga ggtaaatttg gtatcaatag aaaaaatgtt 60tctgaaaaat ttaaaccctg gaaatgtatt tatggcatgg agtcagatgt ttcagggaga 120gaagaacaaa tcaagaagca ttgcaagtat gctcatatgg aatgcttaag gcttgtggtt 180aaaaaatata tatatatggc tgtcaatgtc ttaggctcat ggtagcagca gaaatcgtaa 240taattctttt gtcacatggg ttatatccat attggagaga attaactcag gtgaaattaa 300cttgtacact gtttggtttt ataatattta gagggatcac aactgactga tgtccctttg 360aagtaccatt cttcataaat cttttttttt tcagaatggg ccagccaact gtgacatccc 420ttggatcgga gatttagaac tagaaagtat tctttctaca ttattaggga agaaaaggag 480ttacttggcg gttagcaata ttctattttg ttttgttttg tttttagaga cagggtctca 540ttatgttgac caggctggcc tcgagctcct gggctcaagc aatgctccca cctcagcctc 600ccaagtagct gggactacgg gcatgtgcca ctacacctgg cagtgtttat tctgataaat 660acatttatga gctcaaaaat gtaactctaa aaccttatct ctgaacttcc atattaccat 720cagaaattta gatagttgtt tagttctctt tttctttgta gaacatagat ataaggcatg 780gtttcattga agtcagttgt atatacatgt aactatcctg atgttcccaa ataaagctct 840gtatttatgc ttagtttatt ggggaggctg ctaaatgtag tgcatcccaa cccattttac 900cctgttctac tttaaaaaga ggttggcttc ttgtttggat acaaggacca agtcactccc 960ccaggttcct ccacagtaag ggaggcctat ttaaagccgc ccatggcact aacagaaact 1020ggactcctat gagctcagat acataactgg gcctcacagg ggtgggacag tatgtagtct 1080aggaattgga aggatccatt ccatatcaaa gaactgaagc atcgtgttgc cctctcagca 1140gcaagagtaa ggtgatgccc ctgtcagtta tagttcctga gttcctctgt ctttgattct 1200ttgcctatta gccagctagc tcaccctctt gtttatgcca ctgtttttta tcctattcat 1260gccttctcac agacaacttt tcttacctac agctttggac tcatccttgt ctcctttctg 1320tttctttttc actttccctt cccatcacca actttctggg tttttttctg tttcttctta 1380gagtccagtg gcagggagaa acttgtcagt ccagtctgtt gccatttttc ctgtttgaga 1440aagactcacc agcttttggc tggctcacag attggctttc cttgggtcag gacccaccct 1500tttccctgcc agctttggaa gcttgacaga attcgagtgt gcagtggtgg taaataaata 1560gtaaggaaca cagagcagtc ctggaggcgt gcctccatct gctgatgaga aaatccagtg 1620ctgtcatcca gcccaggtcc cagcggaatg ggcctctctg ttcagtagga tccccctcct 1680gctgagtggt tcatggcatg tttctgttca acgcttttcc atctgtagga ttcttattct 1740gtatttattt gtttttttgg gtttttttat tttttgagat ggagtctcgc tctgtcgccc 1800aggctggagt gcagtggcac gaccccagct cgctgcagcc tctgcctccc aggacgaggg 1860agatcctccc acctcagcct tccacgtagc tgggactaca ggcatgcacc acaggcatgc 1920accaccacgc cagctaattt ttgtattttt ggtagagaca gggttgcatc atgttgccca 1980ggctggtctt gaatgcctga gctcaagcaa tctatttgcc ttggcctccc aaagtgctgg 2040gattacaggc atgagccacc acggccagcc ttctcatttg ttttttttat aaggaagcta 2100tctcttcttc cctccccaac tagggtattc tttttccctt tcgtcacttt gctcatgtac 2160tgtattcctt caacttcatt aatgaatcca tttggaagca gtgaaaaagg caactcagaa 2220agctaagaag aaatagatag aggaatactc agagctatct gagtattttc tttagtttgt 2280tagctctttg gagctttgaa actggaaaga cccagggagt gatgtggaga aagagactga 2340gcttgtaaga cacaggagca gtgagctaag ggagatggag tagtggggac aaattctggc 2400acattctgtc tacactctgg gtagatagag gagggaggat ggagcaccca tggtgggggt 2460atgttggtga cagcattttc ccaccagcca gtgtaacaag tggctgattt gggggaaaga 2520tggcataaac aaatgagaga atgtgtttac tatttgatgt agatgggtta tttgcttcat 2580ttttcaaatc agtgtatata atcaagaata ttcagcatgt ttgaatagac tgtcagagct 2640ggaactcttt cattaacatc tctggcacct ttagttttag ccctgaacat tttatcttaa 2700aattaaacat taccaaatgc cttagtttat ttcatttatt aaatttatat tcttatttgt 2760tatttatatc agcttccaat cagaagacta tacaacctcc taggaataac tgaagtgaag 2820tgatggactc cgatttggag agtagtaaga cgtgaaagga atacacttgt gtttaagcac 2880catggccttg atgattcact gttggggaga agaaacaaga aaagtaactg gttgtcacct 2940atgagaccct tacgtgattg ttagttaagt ttttattcaa agcagctgta atttagttaa 3000taaaataatt atgatctatg ttgtttgccc aattgagatc cagttttttg ttgttatttt 3060taatcaatta ggggcaatag tagaatggac aatttccaag aatgatgcct ttcaggtcct 3120agggcctctg gcctctaggt aaccagttta aattggttca gggtgataac tacttagcac 3180tgcccctggt gattacccca gagatatcta tgaaaaccag tggcttccat caaacctttg 3240ccaactcagg ttcacagcag ctttgggcag ttatggcagt atggcattag ctgagaggtg 3300tctgccactt ctgggtcaat ggaataataa attaagtaca ggcaggaatt tggttgggag 3360catcttgtat gatctccgta tgatgtgata ttgatggaga tagtggtcct cattcttggg 3420ggttgccatt cccacattcc cccttcaaca aacagtgtaa caggtccttc ccagatttag 3480ggtactttta ttgatggata tgttttcctt ttattcacat aaccccttga aaccctgtct 3540tgtcctcctg ttacttgctt ctgctgtaca agatgtagca ccttttctcc tctttgaacg 3600tggtctagtg acacggtagc accagttgca ggaaggagcc agacttgttc tcagagcact 3660gtgttcacac ttttcagcaa aaatagctat ggttgtaaca tatgtattcc cttcctctga 3720tttgaaggca aaaatctaca gtgtttcttc acttcttttc tgatctgggg catgaaaaaa 3780gcaagattga aatttgaact atgagtctcc tgcatggcaa caaaatgtgt gtcaccatca 3840ggccaacagg ccagcccttg aatggggatt tattactgtt gtatctatgt tgcatgataa 3900acattcatca ccttcctcct gtagtcctgc ctcgtactcc ccttccccta tgattgaaaa 3960gtaaacaaaa cccacatttc ctatcctggt tagaagaaaa taaatgttct gacagttgtg 4020atcgcctgga gtacttttag acttttagca ttcgtttttt acctgtttgt ggatgtgtgt 4080ttgtatgtgc atacgtatga gataggcaca tgcatcttct gtatggacaa aggtggggta 4140cctacaggag agcaaaggtt aattttgtgc ttttagtaaa aacatttaaa tacaaagttc 4200tttattgggc ggaattatat ttgatgcaaa tatttgatca cttaaaactt ttaaaacttc 4260taggtaattt gccacgcttt ttgactgctc accaataccc tgtaaaaata cgtaattctt 4320cctgtttgtg taataagata ttcatatttg tagttgcatt aataatagtt atttcttagt 4380ccatcagatg ttcccgtgtg cctcttttat gccaaattga ttgtcatatt tcatgttggg 4440accaagtagt ttgcccatgg caaacctaaa tttatgacct gctgaggcct ctcagaaaac 4500tgagcatact agcaagacag ctcttcttga aaaaaaaaat atgtatacac aaatatatac 4560gtatatctat atatacgtat gtatatacac acatgtatat tcttccttga ttgtgtagct 4620gtccaaaata ataacatata tagagggagc tgtattcctt tatacaaatc tgatggctcc 4680tgcagcactt tttccttctg aaaatattta cattttgcta acctagtttg ttactttaaa 4740aatcagtttt gatgaaagga gggaaaagca gatggacttg aaaaagatcc aagctcctat 4800tagaaaaggt atgaaaatct ttatagtaaa attttttata aactaaagtt gtacctttta 4860atatgtagta aactctcatt tatttggggt tcgctcttgg atctcatcca tccattgtgt 4920tctctttaat gctgcctgcc ttttgaggca ttcactgccc tagacaatgc caccagagat 4980agtgggggaa atgccagatg aaaccaactc ttgctctcac tagttgtcag cttctctgga 5040taagtgacca cagaagcagg agtcctcctg cttgggcatc attgggccag ttccttctct 5100ttaaatcaga tttgtaatgg ctcccaaatt ccatcacatc acatttaaat tgcagacagt 5160gttttgcaca tcatgtatct gttttgtccc ataatatgct ttttactccc tgatcccagt 5220ttctgctgtt gactcttcca ttcagtttta ttaattgtgt gttctcacag tgacaccatt 5280tgtccttttc tgcaacaacc tttccagcta cttttgccaa attctatttg tcttctcctt 5340caaaacattc tcctttgcag ttcctcttca tctgtgtagc tgctcttttg tctcttaact 5400taccattcct atagtacttt atgcatctct gcttagttct attagttttt tggccttgct 5460cttctccttg attttaaaat tccttctata gctagagctt ttctttcttt cattctctct 5520tcctgcagtg ttttgcatac atcagaagct aggtacataa gttaaatgat tgagagttgg 5580ctgtatttag atttatcact ttttaatagg gtgagcttga gagttttctt tctttctggt 5640tttttttttt tttttttttt ttttttgact aatttcacat gctctaaaaa ccttcaaagg 5700tgattatttt tctcctggaa actccaggtc cattctgttt aaatccctaa gaatgtcaga 5760attaaaataa cagggctatc ccgtaattgg aaatatttct tttttcagga tgctatagtc 5820aatttagtaa gtggccacca aattgttatt tgcactaaca aagctcaaaa cacgataagt 5880ttactcctcc atctcagtaa taaaaattaa gctgtaatca accttctagg tttctcttgt 5940cttaaaatgg gtattcaaaa atggggatct gtggtgtatg tatggaaaca catactcctt 6000aatttacctg ttgttggaaa ctggagaaat gattgtcggg caaccgttta ttttttattg 6060tattttattt ggttgaggga tttttttata aacagtttta cttgtgtcat attttaaaat 6120tactaactgc catcacctgc tggggtcctt tgttaggtca ttttcagtga ctaataggga 6180taatccaggt aactttgaag agatgagcag ggagtgacca ggcagttttc ttgcctttag 6240ctttgacagt tcttaattaa gatcattgaa gaccagcttt ctcataaatt tctctttttg 6300aaaaaagaaa gcatttgtac taagctcctc tgtaagacaa catcttaaat cttaaaagtg 6360ttgttatcat gactggtgag agaagaaaac gttttgtttt tattaaatgg agcattattt 6420acaaaaagcc attgttgaga attagatccc acatcgtata aatatctatt aaccattcta 6480aataaagaga actccagtgt tgctatgtgc aagatcctct cttggagctt ttttgcatag 6540caattaaagg tgtgctattt gtcagtagcc atttttttgc agtgatttga agaccaaagt 6600tgttttacag ctgtgttacc gttaaaggtt ttttttttta tatgtattaa atcaatttat 6660cactgtttaa agctttgaat atctgcaatc tttgccaagg tactttttta tttaaaaaaa 6720aacataactt tgtaaatatt accctgtaat attatatata cttaataaaa cattttaagc 6780tataaaaaaa 6790892538DNAHomo sapiens 89ttttaaaact tctaggtaat ttgccacgct ttttgactgc tcaccaatac cctgtaaaaa 60tacgtaattc ttcctgtttg tgtaataaga tattcatatt tgtagttgca ttaataatag 120ttatttctta gtccatcaga tgttcccgtg tgcctctttt atgccaaatt gattgtcata 180tttcatgttg ggaccaagta gtttgcccat ggcaaaccta aatttatgac ctgctgaggc 240ctctcagaaa actgagcata ctagcaagac agctcttctt gaaaaaaaaa atatgtatac 300acaaatatat acgtatatct atatatacgt atgtatatac acacatgtat attcttcctt 360gattgtgtag ctgtccaaaa taataacata tatagaggga gctgtattcc tttatacaaa 420tctgatggct cctgcagcac tttttccttc tgaaaatatt tacattttgc taacctagtt 480tgttacttta aaaatcagtt ttgatgaaag gagggaaaag cagatggact tgaaaaagat 540ccaagctcct attagaaaag gtatgaaaat ctttatagta aaattcttta taaactaaag 600ttgtaccttt taatatgtag taaactctca tttatttggg gttcgctctt ggatctcatc 660catccattgt gttctcttta atgctgcctg ccttttgagg cattcactgc cctagacaat 720gccaccagag atagtggggg aaatgccaga tgaaaccaac tcttgctctc actagttgtc 780agcttctctg gataagtgac cacagaagca ggagtcctcc tgcttgggca tcattgggcc 840agttccttct ctttaaatca gatttgtaat ggctcccaaa ttccatcaca tcacatttaa 900attgcagaca gtgttttgca catcatgtat ctgttttgtc ccataatatg ctttttactc 960cctgatccca gtttctgctg ttgactcttc cattcagttt tatttattgt gtgttctcac 1020agtgacacca tttgtccttt tctgcaacaa cctttccagc tacttttgcc aaattctatt 1080tgtcttctcc ttcaaaacat tctcctttgc agttcctctt catctgtgta gctgctcttt 1140tgtctcttaa cttaccattc ctatagtact ttatgcatct ctgcttagtt ctattagttt 1200tttggccttg ctcttctcct tgattttaaa attccttcta tagctagagc ttttctttct 1260ttcattctct cttcctgcag tgttttgcat acatcagaag ctaggtacat aagttaaatg 1320attgagagtt ggctgtattt agatttatca ctttttaata gggtgagctt gagagttttc 1380tttctttctg tttttttttt tttttttttt tttttttttt ttgactaatt tcacatgctc 1440taaaaacctt caaaggtgat tatttttctc ctggaaactc caggtccatt ctgtttaaat 1500ccctaagaat gtcagaatta aaataacagg gctatcccgt aattggaaat atttcttttt 1560tcaggatgct atagtcaatt tagtaagtga ccaccaaatt gttatttgca ctaacaaagc 1620tcaaaacacg ataagtttac tccttcatct cagtaataaa aattaagctg taatcaacct 1680tctaggtttc tcttgtctta aaatgggtat tcaaaaatgg ggatctgtgg tgtatgtatg 1740gaaacacata ctccttaatt tacctgttgt tggaaactgg agaaatgatt gtcgggcaac 1800cgtttatttt ttattgtatt ttatttggtt gagggatttt tttataaaca gttttacttg 1860tgtcatattt taaaattact aactgccatc acctgctggg gtcctttgtt aggtcatttt 1920cagtgactaa tagggataat ccaggtaact ttgaagagat gagcagtgag tgaccaggca 1980gtttttctgc ctttagcttt gacagttctt aattaagatc attgaagacc agctttctca 2040taaatttctc tttttgaaaa aaagaaagca tttgtactaa gctcctctgt aagacaacat 2100cttaaatctt aaaagtgttg ttatcatgac tggtgagaga agaaaacatt ttgtttttat 2160taaatggagc attatttaca aaaagccatt gttgagaatt agatcccaca tcgtataaat 2220atctattaac cattctaaat aaagagaact ccagtgttgc tatgtgcaag atcctctctt 2280ggagcttttt tgcatagcaa ttaaaggtgt gctatttgtc agtagccatt tttttgcagt 2340gatttgaaga ccaaagttgt tttacagctg tgttaccgtt aaaggttttt ttttttatat 2400gtattaaatc aatttatcac tgtttaaagc tttgaatatc tgcaatcttt gccaaggtac 2460ttttttattt aaaaaaaaac ataactttgt aaatattacc ctgtaatatt atatatactt 2520aataaaacat tttaagct 253890550DNAHomo sapiensmodified_base(1)..(550)n = g, a, c or t 90ccatatcatg taccaaaagt tgctgaagtt tctcttctag ctggtaaagt aggagtttgc 60atgacttcac actttttttg cgtagtttct tctgttgtat gatggcgtga gtgtgtgtct 120tgggtaccgc tgtgtactac tgtgtgccta gattccatgc actctcgttg tgtttgaagt 180aaatattgga gaccggaggg taacaggttg gcctgttgat tacagctagt aatcgctgtg 240tcttgttccg ccccctccct gacaccccag cttcccagga tgtggaaagc ctggatctca

300gctccttgcc ccatatccct tctgtaattt gtacctaaag agtgtgatta tcctaattca 360agagtcacta aaactcatca cattatcatt gcatatcagc aaagggtaaa gtcctagcac 420caattgcttc acataccagc atgttccatt tccaatttag aattagccac ataataaaat 480cttagaatct tccttgagaa agagctgcct gagatgtagt tttgntatat ggntccccac 540cgaccatttt 550911209DNAHomo sapiens 91ccatatcatg taccaaaagt tgctgaagtt tctcttctag ctggtaaagt aggagtttgc 60atgacttcac actttttttg cgtagtttct tctgttgtat gatggcgtga gtgtgtgtct 120tgggtaccgc tgtgtactac tgtgtgccta gattccatgc actctcgttg tgtttgaagt 180aaatattgga gaccggaggg taacaggttg gcctgttgat tacagctagt aatcgctgtg 240tcttgttccg ccccctccct gacaccccag cttcccagga tgtggaaagc ctggatctca 300gctccttgcc ccatatccct tctgtaattt gtacctaaag agtgtgatta tcctaattca 360agagtcacta aaactcatca cattatcatt gcatatcagc aaagggtaaa gtcctagcac 420caattgcttc acataccagc atgttccatt tccaatttag aattagccac ataataaaat 480cttagaatct tccttgagaa agagctgcct gagatgtagt tttgttatat ggttccccac 540cgaccatttt tgtgcttttt tcttgttttg ttttgttttg actgcactgt gagttttgta 600gtgtcctctt cttgccaaaa caaacgcgag atgaactgga cttatgtaga caaatcgtga 660tgccagtgta tccttccttt cttcagttcc agcaataatg aatggtcaac ttttttaaaa 720tctagatctc tctcattcat ttcaatgtat ttttacttta agatgaacca aaattattag 780acttatttaa gatgtacagg catcagaaaa aagaagcaca taatgctttt ggtgcgatgg 840cactcactgt gaacatgtgt aaccacatat taatatgcaa tattgtttcc aatactttct 900aatacagttt tttataatgt tgtgtgtggt gattgttcag gtcgaatctg ttgtatccag 960tacagcttta ggtcttcagc tgcccttctg gcgagtacat gcacaggatt gtaaatgaga 1020aatgcagtca tatttccagt ctgcctctat gatgatgtta aattattgct gtttagctgt 1080gaacaaggga tgtaccactg gaggaataga gtatcctttt gtacacattt tgaaatgctt 1140cttctgtagt gatagaacaa ataaatgcaa cgaatactct gtcaaaaaaa aaaaaaaaaa 1200aaaaaaaaa 1209921661DNAHomo sapiens 92ccatatcatg taccaaaagt tgctgaagtt tctcttctag ctggtaaagt aggagtttgc 60atgacttcac actttttttg cgtagtttct tctgttgtat gatggcgtga gtgtgtgtct 120tgggtaccgc tgtgtactac tgtgtgccta gattccatgc actctcgttg tgtttgaagt 180aaatattgga gaccggaggg taacaggttg gcctgttgat tacagctagt aatcgctgtg 240tcttgttccg ccccctccct gacaccccag cttcccagga tgtggaaagc ctggatctca 300gctccttgcc ccatatccct tctgtaattt gtacctaaag agtgtgatta tcctaattca 360agagtcacta aaactcatca cattatcatt gcatatcagc aaagggtaaa gtcctagcac 420caattgcttc acataccagc atgttccatt tccaatttag aattagccac ataataaaat 480cttagaatct tccttgagaa agagctgcct gagatgtagt tttgttatat ggttccccac 540cgaccatttt tgtgcttttt tcttgttttg ttttgttttg actgcactgt gagttttgta 600gtgtcctctt cttgccaaaa caaacgcgag atgaactgga cttatgtaga caaatcgtga 660tgccagtgta tccttccttt cttcagttcc agcaataatg aatggtcaac ttttttaaaa 720tctagatctc tctcattcat ttcaatgtat ttttacttta agatgaacca aaattattag 780acttatttaa gatgtacagg catcagaaaa aagaagcaca taatgctttt ggtgcgatgg 840cactcactgt gaacatgtgt aaccacatat taatatgcaa tattgtttcc aatactttct 900aatacagttt tttataatgt tgtgtgtggt gattgttcag gtcgaatctg ttgtatccag 960tacagcttta ggtcttcagc tgcccttctg gcgagtacat gcacaggatt gtaaatgaga 1020aatgcagtca tatttccagt ctgcctctat gatgatgtta aattattgct gtttagctgt 1080gaacaaggga tgtaccactg gaggaataga gtatcctttt gtacacattt tgaaatgctt 1140cttctgtagt gatagaacaa ataaatgcaa cgaatactct gtctgcccta tcccgtgaag 1200tccacactgg cgtaagagaa ggcccagcag agcaggaatc tgcctagact ttctcccaat 1260gagatcccaa tatgagaggg agaagagatg ggcctcagga cagctgcaat accacttggg 1320aacacatgtg gtgtcttgat gtggccagcg cagcagttca gcacaacgta cctcccatct 1380acaacagtgc tggacgtggg aattctaagt cccagtcttg agggtgggtg gagatggagg 1440gcaacaagag atacatttcc agttctccac tgcagcatgc ttcagtcatt ctgtgagtgg 1500ccgggcccag ggccctcaca atttcactac cttgtcttta catagtcata agaattatcc 1560tcaacatagc cttttgacgc ttgtaaatct tgagtattca atttaaccct tttctgaatc 1620tccctggaaa caggtgcctg cctggattgc cttcttcttc c 1661936400DNAHomo sapiens 93gaattccggc gtcgcggacg catcccagtc tgggcgggac gctcggccgc ggcgaggcgg 60gcaagcctgg cagggcagag ggagccccgg ctccgaggtt gctcttcgcc cccgaggatc 120agtcttggcc ccaaagcgcg acgcacaaat ccacataacc tgaggaccat ggatgctgat 180gagggtcaag acatgtccca agtttcaggg aaggaaagcc cccctgtaag cgatactcca 240gatgagggcg atgagcccat gccgatcccc gaggacctct ccaccacctc gggaggacag 300caaagctcca agagtgacag agtcgtggcc agtaatgtta aagtagagac tcagagtgat 360gaagagaatg ggcgtgcctg tgaaatgaat ggggaagaat gtgcggagga tttacgaatg 420cttgatgcct cgggagagaa aatgaatggc tcccacaggg accaaggcag ctcggctttg 480tcgggagttg gaggcattcg acttcctaac ggaaaactaa agtgtgatat ctgtgggatc 540atttgcatcg ggcccaatgt gctcatggtt cacaaaagaa gccacactgg agaacggccc 600ttccagtgca atcagtgcgg ggcctcattc acccagaagg gcaacctgct ccggcacatc 660aagctgcatt ccggggagaa gcccttcaaa tgccacctct gcaactacgc ctgccgccgg 720agggacgccc tcactggcca cctgaggacg cactccgttg gtaaacctca caaatgtgga 780tattgtggcc gaagctataa acagcgaagc tctttagagg aacataaaga gcgctgccac 840aactacttgg aaagcatggg ccttccgggc acactgtacc cagtcattaa agaagaaact 900aatcacagtg aaatggcaga agacctgtgc aagataggat cagagagatc tctcgtgctg 960gacagactag caagtaacgt cgccaaacgt aagagctcta tgcctcagaa atttcttggg 1020gacaagggcc tgtccgacac gccctacgac agcagcgcca gctacgagaa ggagaacgaa 1080atgatgaagt cccacgtgat ggaccaagcc atcaacaacg ccatcaacta cctgggggcc 1140gagtccctgc gcccgctggt gcagacgccc ccgggcggtt ccgaggtggt cccggtcatc 1200agcccgatgt accagctgca caagccgctc gcggagggca ccccgcgctc caaccactcg 1260gcccaggaca gcgccgtgga gaacctgctg ctgctctcca aggccaagtt ggtgccctcg 1320gagcgcgagg cgtccccgag caacagctgt caagactcca cggacaccga gagcaacaac 1380gaggagcagc gcagcggtct catctacctg accaaccaca tcgccccgca cgcgcgcaac 1440ggcttgtcgc tcaaggagga gcaccgcgcc tacgacctgc tgcgcgccgc ctccgagaac 1500tcgcaggacg cgctccgcgt ggtcagcacc agcggggagc agatgaaggt gtacaagtgc 1560gaacactgcc gggtgctctt cctggatcac gtcatgtaca ccatccacat gggctgccac 1620ggcttccgtg atccttttga gtgcaacatg tgcggctacc acagccagga ccggtacgag 1680ttctcgtcgc acataacgcg aggggagcac cgcttccaca tgagctaaag ccctcccgcg 1740cccccacccc agaccccgag ccaccccagg aaaagcacaa ggactgccgc cttctcgctc 1800ccgccagcag catagactgg actggaccag acaatgttgt gtttggattt gtaactgttt 1860tttgtttttt gtttgagttg gttgattggg gtttgatttg cttttgaaaa gatttttatt 1920tttagaggca gggctgcatt gggagcatcc agaactgcta ccttcctaga tgtttcccca 1980gaccgctggc tgagattccc tcacctgtcg cttcctagaa tccccttctc caaacgatta 2040gtctaaattt tcagagagaa atagataaaa cacgccacag cctgggaagg agcgtgctct 2100accctgtgct aagcacgggg ttcgcgcacc aggtgtcttt ttccagtccc cagaagcaga 2160gagcacagcc cctgctgtgt gggtctgcag gtgagcagac aggacaggtg tgccgccacc 2220caagtgccaa gacacagcag ggccaacaac ctgtgcccag gccagcttcg agctacatgc 2280atctagggcg gagaggctgc acttgtgaga gaaaatactt atttcaagtc atattctgcg 2340gtaggaaaat gattgggttg gggaaagtcg gtgtctgtca gactgccctg ggtggaggga 2400gacgccgggt tagagccttt gggatcgtcc tggattcact ggcttggggg aggctgttca 2460gatggcctga gcctcccgag gcttgctgcc ccgtaggagg agactgtctt cccgtgggca 2520tatctgggga gccctgttcc ccgctttttc actcccatac ctttaatggc ccccaaaatc 2580tgtcactaca atttaaacac cagtcccgaa atttggatct tctttctttt tgaatctctc 2640aaacggcaac attcctcaga aaccaaagct ttatttcaaa tctcttcctt ccctggctgg 2700ttccatctag taccagaggc ctcttttcct gaagaaatcc aatcctagcc ctcattttaa 2760ttatgtacat ctgcttgtag ccacaagcct gaatttctca gtgttggtaa gtttctttac 2820ctaccctcac tatatattat tctcgtttta aaacccataa aggagtgatt tagaacagtc 2880attaattttc caactcaatg aaaatatgtg aagcccagca tctctgttgc taacacacag 2940agctcacctg tttgaaacca agctttcaaa catgttgaag ctctttactg taaaggcaag 3000ccagcatgtg tgtccacaca tacataggat ggctggctct gcacctgtag gatattggaa 3060tgcacagggc aattgaggga ctgagccaga ccttcggaga gtaatgccac cagatcccct 3120aggaaagagg aggcaaatgg cactgcaggt gagaaccccg cccatccgtg ctatgacatg 3180gaggcactga agcccgagga aggtgtgtgg agattctaat cccaacaagc aagggtctcc 3240ttcaagatta atgctatcaa tcattaaggt cattactctc aaccacctag gcaatgaaga 3300atataccatt tcaaatattt acagtacttg tcttcaccaa cactgtccca aggtgaaatg 3360aagcaacaga gaggaaattg tacataagta cctcagcatt taatccaaac aggggttctt 3420agtctcagca ctatgacatt ttgggctgac tacttatttg ttaggcggga gctctcctgt 3480gcattgtagg ataattagca gtatccctgg tggctaccca atagacgcca gtagcacccc 3540gaattgacaa cccaaactct ccagacatca ccaactgtcc cctgcgagga gaaatcactc 3600ctgggggaga accactgacc caaatgaatt ctaaaccaat caaatgtctg ggaagccctc 3660caagaaaaaa aatagaaaag cacttgaaga atattcccaa tattcccggt cagcagtatc 3720aaggctgact tgtgttcatg tggagtcatt ataaattcta taaatcaatt attccccttc 3780ggtcttcaaa aatatatttc ctcataaaca tttgagtttt gttgaaaaga tggagtttac 3840aaagatacca ttcttgagtc atggatttct ctgctcacag aagggtgtgg catttggaaa 3900cgggaataaa caaaattgct gcaccaatgc actgagtgaa ggaagagaga cagaggatca 3960agggctttag acagcactcc ttcaatatgc aatcacagag aaagatgcgc cttatccaag 4020ttaatatctc taaggtgaga gccttcttag agtcagtttg ttgcaaattt cacctactct 4080gttcttttcc atccatcccc ctgagtcagt tggttgaagg gagttatttt ttcaagtgga 4140attcaaacaa agctcaaacc agaactgtaa atagtgattg caggaattct tttctaaact 4200gctttgccct ttcctctcac tgccttttat agccaatata aatgtctctt tgcacacctt 4260ttgttgtggt tttatattgt aacaccattt ttctttgaaa ctattgtatt taaagtaagg 4320tttcatatta tgtcagcaag taattaactt atgtttaaaa ggtggccata tcatgtacca 4380aaagttgctg aagtttctct tctagctggt aaagtaggag tttgcatgac ttcacacttt 4440ttttgcgtag tttcttctgt tgtatgatgg cgtgagtgtg tgtcttgggt accgctgtgt 4500actactgtgt gcctagattc catgcactct cgttgtgttt gaagtaaata ttggagaccg 4560gagggtaaca ggttggcctg ttgattacag ctagtaatcg ctgtgtcttg ttccgccccc 4620tccctgacac cccagcttcc caggatgtgg aaagcctgga tctcagctcc ttgccccata 4680tcccttctgt aatttgtacc taaagagtgt gattatccta attcaagagt cactaaaact 4740catcacatta tcattgcata tcagcaaagg gtaaagtcct agcaccaatt gcttcacata 4800ccagcatgtt ccatttccaa tttagaatta gccacataat aaaatcttag aatcttcctt 4860gagaaagagc tgcctgagat gtagttttgt tatatggttc cccaccgacc atttttgtgc 4920ttttttcttg ttttgttttg ttttgactgc actgtgagtt ttgtagtgtc ctcttcttgc 4980caaaacaaac gcgagatgaa ctggacttat gtagacaaat cgtgatgcca gtgtatcctt 5040cctttcttca gttccagcaa taatgaatgg tcaacttttt taaaatctag atcattggag 5100accggagggt aacaggttgg cctgttgatt acagctagta atcgctgtgt cttgttccgc 5160cccctccctg acaccccagc ttcccaggat gtggaaagcc tggatctcag ctccttgccc 5220catatccctt ctgtaatttg tacctaaaga gtgtgattat cctaattgat ctctctcatt 5280catttcaatg tatttttact ttaagatgaa ccaaaattat tagacttatt taagatgtac 5340aggcatcaga aaaaagaagc acataatgct tttggtgcga tggcactcac tgtgaacatg 5400tgtaaccaca tattaatatg caatattgtt tccaatactt tctaatacag ttttttataa 5460tgttgtgtgt ggtgattgtt caggtcgaat ctgttgtatc cagtacagct ttaggtcttc 5520agctgccctt ctggcgagta catgcacagg attgtaaatg agaaatgcag tcatatttcc 5580agtctgcctc tatgatgatg ttaaattatt gctgtttagc tgtgaacaag ggatgtacca 5640ctggaggaat agagtatcct tttgtacaca ttttgaaatg cttcttctgt agtgatagaa 5700caaataaatg caacgaatac tctgtctgcc ctatcccgtg aagtccacac tggcgtaaga 5760gaaggcccag cagagcagga atctgcctag actttctccc aatgagatcc caatatgaga 5820gggagaagag atgggcctca ggacagctgc aataccactt gggaacacat gtggtgtctt 5880gatgtggcca gcgcacgagt tcagcacaac gtacctccca tctacaacag tgctggacgt 5940gggaattcta agtcccagtc ttgagggtgg gtggagatgg agggcaacaa gagatacatt 6000tccagttctc cactgcagca tgcttcagtc attctgtgag tggccgggcc cagggccctc 6060acaatttcac taccttgtct tttacatagt cataagaatt atcctcaaca tagccttttg 6120acgctgtaaa tcttgagtat tcatttaccc ttttctgatc tcctggaaac agctgcctgc 6180ctgcattgca cttctcttcc cgaggagtgg ggtaaattta aaagtcaagt tatagtttgg 6240atgttagtat agaattttga aattgggaat taaaaatcag gactggggac tgggagacca 6300aaaatttctg atcccatttc tgatggatgt gtcacacctt ttctgtcaaa ataaaatgtc 6360ttggaggtta tgactccttg gtgaaaaaaa aaaaaaaaaa 6400941364DNAHomo sapiens 94aatcaaaggt gggaggattt tccctaaact gacttagcag gactcttgtt acaattggac 60taggcaggct gaagacagga tgcaaggaca aagcttgttg aaaagaggcc tcagaggagc 120tcatctaaaa tttggtcaag gggagggtct ttcttggtcc ctcctcttgt tcaagggaaa 180aagagacatt cttctttcct ttgaacaata taagtcaatt tctcattggt ggcctttttt 240tcattaagga caagctgagc cccctgctga aacttggtag cagggcagcc agttgagaag 300atttctagat gtcaaggcat ctttagatga tggggtgagg actgcagtgg ccatcccaga 360tcatggattt tctggtttgc agtttgaatg tccttggtga tggcatagac atcagtgtca 420cagtcatggt tatttttccg agcagagtgt agaagtgtcc aacttcatct tgaagggctt 480ctttagrcag tcacaataaa gatctgggaa gttaggtttt agttctcagt gatgccaaat 540caggacagtg ggagaaaaat taaaaacctc agtttggaga gtggtagcca gatagtaaag 600ggaactagaa gaactgagaa tttggtaagg actgacaagc tgtgcatgat gacaggatcc 660cgttcaattt acaagtagat aacaaaacct gaaagacaag tacaggacca gaataataac 720ccataagaag gtgctatagt ttttataaaa tatctttcta cagtcatccc ccttttttga 780tccaaattaa ccaaagtaag attattcttg tttacaaaat aagtcttgtc tcattatatt 840tgacttactt atttgcataa ttgcagcaag aatggcaact gaccaggtag gcttatttaa 900gtttgcattg ctggaacttt ttacaagtaa tctcagatta tgctttcaag agttcttgaa 960gctataaagc caagtcaagc accaccaggc cttatctgca atgcctagag attccagatg 1020ggttcttctc ttcttgaggt cctaaaaaca tcctgagttt ctttggcctg ccagaaagtc 1080accttcctga ctcacctgta aggctgggaa ctccataatc caggtaccag gcagactttc 1140cgggagggct tcatatgcat tggctccata aagttaacct tagttcctca aaactgtctg 1200ttcatatgtg attttatgtc ttattctcag ttggaaatgc agaaatcacc tgtcttctgc 1260gtcgatcagg ctgggagctg cagaccggag ctgttcctat tcggccatct tggaatggac 1320ccccatgtct tattctcaaa taaaacattt tggtcaaaaa aaaa 136495411DNAHomo sapiens 95cctaatatga cattatttca aagcttatta taaaggaaca gtaatcaaac tagtgcaatt 60ttggcataaa gttagaaaaa cagatcaatg aagcagaaga gagagtccag aaacagaact 120gcacatttat gtgttggtga atgccaggga ttcagcttag gtctgattgc tcaccacaca 180gaaagccaat cactgagaca acaagtactg ccaggaagaa aggctttatt gctggtgatg 240ccagccagga tatgggagac aagtctaaaa tctgtctctg taaccaataa agttaggagt 300ttatgtagga gttgctcaac aggcagtagg tagttgaatc agggttctgg caccttgctg 360ttaggatgca gcgatctgga aatcttcagc tttctgatac tatctgggag g 411961632DNAHomo sapiensmodified_base(1)..(1632)n = g, a, c or t 96gtgccagtta taaaatatct tatattttct tataatgcct ccatagtttt attatatatt 60cactcaatac atcatttttc tatgtggtat gaggtaagaa tctaactttt actgatttta 120tctttatgca ttttttttaa tttaaaatgt ggggtaggga tctaactttt ttcaaacaca 180tataaatgtg cactactatt tatttaaata gtctgttctt ttccctttta ttattatgct 240atcttatctc acttgaattc aacctaagcc tgttttagac tccaactaat actacagatc 300ttcctaccac tcttcccctt gcataattaa cttcaagcac attagcctcc gggttcctca 360agcacaccaa atttagtccc agctcaggaa ctctgtactt tctatttcca tgctttaatg 420ttctttctct tgatatcctt gttttcttat ttccttcatt tgcatttctg ctttgatttt 480ctgtttctgg tccatggaca tttttatttt ctttttatag aacaaacaca gcttttttac 540attttgtatt tttcctgcca ttgctatgtg cttggagctc agggagggcc tcaaaggatg 600aaattggagt atggtgtgat cagaagtttg aacttctttg tattgtatga tcatcccttt 660accttaatac tcacatgaaa tgctatctat ggcttcttac attccacttc ttcttaatca 720atttctttct tcatgaactt aaacgttccc atcatttttg atagggtctg tgagtttatt 780tgtccaaaaa gcccaaaagc agaatttaag attgatagca tagctttgtg ctcaacagtt 840gtaatatttt tttccatggt cgtctagctt cttctgtttt ctttgagaaa tctatgtaat 900tgttgtttct ttataattaa tctatctttt ctctccagtt gcctttaaga ctttttatat 960ttgatattat gcaatttcac tatgatttgt ctaaatgtgc atttattttg ctagagatta 1020ataactcaag tctaaggtat catgtctttt ttcaagttta gaaaatattt ggctattatc 1080tctttattat catgctgcta cagcattatt tgaattcttt ccctcagaaa tttatattag 1140aagtttgcta gacttcattc tagtctcatg actcttaatt agtcttgcaa aattttcatt 1200tcattatcac ttattgcatt ttaggtaatt tcttaatctc tgtcttccag tttactggtt 1260ctttcttcag ctgtatctat tttattgttt aacctattta ttttctattt caatgattac 1320attttttgag attttattag caaaatggtt aaaagcatgg tttcagagga ttgtctggat 1380ttacattttg cctccattat ttactagctc tccagttttg gttaaattaa ttaacctttt 1440gtgcttcttg gtgtgtaaaa ttgaagtaac aattgtatat aaatatagtg ttttttggta 1500attaattaaa attatttgca taaaatgctt aagacagggc ctgaaatgac attgagtcct 1560caaaaaataa attattatta tcattccttc aaaaaaaaaa aaaaaaaaaa aaaaaaaana 1620aaaaaaanac ca 1632972378DNAHomo sapiens 97tctaaaagct gcggaattcc tcgagcactg ttggcctttg gtagatgccc ctctgggaga 60gatccccagg ggtgacagcc atgggccctg gaagggcctg ggctagggac agggaccaga 120gccagtccag ggagaggaca gagccaatgg actggggtgt actgtaacag ccctgctggc 180gagagggacc agggcaccgt cctccaggga gcccatgctg caagtcgggc cagaggtgcc 240cctgaacctg aaggccaatg agacccaaga caggccaagt gggttgtgag acccctgagg 300agctgggccc tggtcccagg cagcgctggc ccctgctgct gctgggtctg gccatggtcg 360cccatggcct gctgcgccca atggttgcac cgcaaagcgg ggacccagac cctggagcct 420cagttggaag cagccgatcc agcctgcgga gcctgtgggg caggtaaggg gcaagagata 480tgtgggggtc ctgcagcaga gctgggaaag ggtgaccaag gggggacaag ccagaggagt 540gaggaggaag gttaacccct aagaggggcc tgggctgaca ctggctttag taatgggttg 600atattttgtc catcacagat ttgtttgatt tactgttttt aatatcatat tacgatatta 660tttttcttca tttctgagtt ttctggcgcc acttaaattt tcaccagggt cagtgcctca 720atcacctagt cctagtcctc tgggtaggga aggaacagag gcagggacag gacatccaca 780gggggtggtg gccactgtcc ccacagggtg cccaggcctg ttcctccccc tcctcctctc 840tgcccatgtg cctcctgccc agtgagggca ggggccactc cctggagaag gcagcaaggg 900cttggtttgg tctcccccaa ggctgtctgt tcaccaactt gcacataaat acttactggg 960gccaggctca aggacacagg gagggtggga tgaaccgagg ggagctgtcc agtcattgga 1020acaggcccac ggcccatgtt tgcagcaatg aagggagagg gcatctccct ctgggatgat 1080gcccaggctg gtctcacaga tcgaggggca ctggctggtg atgggtgccc ccaaaagaca 1140gagcagtgtc agaggagagg agagcacagg atgaggctgg gagctcctgg gtgactggga 1200aggggaggca agaagaccat agggtccgtg caccattccc agtccaggac gagtccttgg 1260atggatttag gtagattgat tatcagagtc agatttgtgt ttttggaaaa atcagcaccg 1320gattggaggc tgatgcgacg cccaattaga ggagggagga gagggggtga tggccaagtc 1380cagggtaggt ggggatcctg gaggaagccg tgccttgggg atggggagga cactcagatt 1440cagagcaccc aggggcccag tttcctatga aatgggagca tgaggttgaa gtgagggctg 1500agcagagggg agcagacacg ctcggggact gtctatgggc attgaaaatg tataaccatt 1560ttagcaacag gcggcgagtc aaaacccaag gtgtgtttat ctaaactggg caattcctct 1620tctaggaatt tatcctaagg gttggttggg ggaataatca aagctgaaac caaatcttta 1680taacaagggt ggttaggtca gcattcttag tgatgggaga aaactggaaa aaatccaaat

1740atctaccaga aagggtgtga aaaaacacaa ttgtatttgg gggactgttg ttgattttgt 1800tttgaaacag tcttgatctg ttgctcaggc tggagtacag tggcgtggcc acagctcact 1860gcagcctcaa cctccagggc tcaaaagatc ctccagcctc agcctcctga gtagttagga 1920ctacagatgc aggccactac acctggctaa ttttgattag gattatcatt agtttagaga 1980cagagcctcg ctatattgct caggcctgtc tcaaattcct aagctcaagc aatctttctg 2040cctcagtttc ccacgtgctg gaattacagg cgtgagccac tgcacctgac ccaactgtgt 2100ttttaaagta tatatgcatt ttcaaaaacc tgtcagaaaa tatagaaaaa tgtcaatggt 2160gtgtctggct ggctgatggg atttcaccta attttaatgt ggctttataa ttttctggtt 2220ttgtgaagtt gttcacaaaa agagacattt cttctaatat aatttttaat acaacagtaa 2280tgtactcatg tgcattactc tttttgtaat gagtatatta caaaatgtaa tgacttttgt 2340acattactct tttttcttgc caaaaaaaaa aaaaaaaa 237898313DNAHomo sapiensmodified_base(1)..(313)n = g, a, c or t 98ccacaaaata aggtctaatt caataaatta tagtaaatta atgtaatata atattacatg 60ccactaaaaa gaataaggta gctgtatatt tcctggtatg gaaaaaacat attaatatgt 120tataaactat taggttggtg caaaactaat tgtggttttt gccattgaaa tggcattgaa 180ataaaagtgt aaagaaatct ataccagatg tagtaacagt ggtttggttc tgggaggttg 240gattacaggg agcatttgat ttctatgttg ngtatttcta tantgtttga attgtttaga 300atgaatctgt ntt 31399317DNAHomo sapiens 99ccagtatgga atccagaagg accgagtgga taagagcgct gtcggcttca atgaaatgga 60ggccccgacc acagcttata agaagacgac gcccatagaa gccgcttcta gtggtgcccg 120tgggctgaag gcgaaatttg agtccatggc tgaggagaag aggaagcgag aggaagagga 180gaaggcacag caggtggcca ggaggcaaca ggagcgaaag gctgtgacaa agaggagccc 240tgaggctcca cagccagtga tagctatgga agagccagca gtaccggccc cactgcccaa 300gaaaatctcc tcagagg 3171001968DNAHomo sapiens 100aattccgccg ggcgcttaga acagaggctt gcacaggtgg agatgtggaa gtctgtagtg 60ggccatgatg tgtctgtttc cgtggagacc cagggtgatg attgggacac agatcctgac 120tttgtgaatg acatctctga aaaggagcaa cgatggggag ccaagaccat cgaggggtct 180ggacgcacag aacacatcaa catccaccag ctgaggaaca aagtatcaga ggagcatgat 240gttctcagga agaaagagat ggagtcaggg cccaaagcat cccatggcta tggaggtcgg 300tttggagtag aaagagaccg aatggacaag agtgcagtgg gccatgagta tgttgccgag 360gtggagaagc actcttctca gacggatgct gccaaaggct ttgggggcaa gtacggagtt 420gagagggaca gggcagacaa gtcagcagtc ggctttgatt ataaaggaga agtggagaag 480catacatctc agaaagatta ctctcgtggc tttggtggcc ggtacggggt ggagaaggat 540aaatgggaca aagcagctct gggatatgac tacaagggag agacggagaa acacgagtcc 600cagagagatt atgccaaggg ctttggtggc cagtatggaa tccagaagga ccgagtggat 660aagagcgctg tcggcttcaa tgaaatggag gccccgacca cagcttataa gaagacgacg 720cccatagaag ccgcttctag tggtgcccgt gggctgaagg cgaaatttga gtccatggct 780gaggagaaga ggaagcgaga ggaagaggag aaggcacagc aggtggccag gaggcaacag 840gagcgaaagg ctgtgacaaa gaggagccct gaggctccac agccagtgat agctatggaa 900gagccagcag taccggcccc actgcccaag aaaatctcct cagaggcctg gcctccagtt 960gggactcctc catcatcaga gtctgagcct gtgagaacca gcagggaaca cccagtgccc 1020ttgctgccca ttaggcagac tctcccggag gacaatgagg agcccccagc tctgccccct 1080aggactctgg aaggcctcca ggtggaggaa gagccagtgt acgaagcaga gcctgagcct 1140gagcccgagc ctgagcccga gcctgagaat gactatgagg acgttgagga gatggacagg 1200catgagcagg aggatgaacc agagggggac tatgaggagg tgctcgagcc tgaagattct 1260tctttttctt ctgctctggc tggatcatca ggctgcccgg ctggggctgg ggctggggct 1320gtggctctgg ggatctcagc tgtggctcta tatgattacc aaggagaggg aagtgatgag 1380ctttcctttg atccggacga cgtaatcact gacattgaga tggtggacga gggctggtgg 1440cggggacgtt gccatggcca ctttggactc ttccctgcaa attatgtcaa gcttctggag 1500tgactagagc tcactgtcta ctgcaactgt gatttcccat gtccaaagtg gctctgctcc 1560accccctccc tattcctgat gcaaatgtct aaccagatga gtttctggac agacttccct 1620ctcctgcttc attaagggct tggggcagag acagcatggg gaaggaggtc cccttcccca 1680agagtcctct ctatcctgga tgagctcatg aacatttctc ttgtgttcct gactccttcc 1740caatgaacac ctctctgcca ccccaagctc tgctctcctc ctctgtgagc tctgggcttc 1800ccagtttgtt tacccgggaa agtacgtcta gattgtgtgg tttgcctcat tgtgctattt 1860gcccactttc cttccctgaa gaaatatctg tgaaccttct ttctgttcag tcctaaaatt 1920cgaaataaag tgagactatg gttcacctgt aaaaaaaaaa aaggaatt 1968101486PRTHomo sapiens 101Met Trp Lys Ser Val Val Gly His Asp Val Ser Val Ser Val Glu Thr1 5 10 15Gln Gly Asp Asp Trp Asp Thr Asp Pro Asp Phe Val Asn Asp Ile Ser 20 25 30Glu Lys Glu Gln Arg Trp Gly Ala Lys Thr Ile Glu Gly Ser Gly Arg 35 40 45Thr Glu His Ile Asn Ile His Gln Leu Arg Asn Lys Val Ser Glu Glu 50 55 60His Asp Val Leu Arg Lys Lys Glu Met Glu Ser Gly Pro Lys Ala Ser65 70 75 80His Gly Tyr Gly Gly Arg Phe Gly Val Glu Arg Asp Arg Met Asp Lys 85 90 95Ser Ala Val Gly His Glu Tyr Val Ala Glu Val Glu Lys His Ser Ser 100 105 110Gln Thr Asp Ala Ala Lys Gly Phe Gly Gly Lys Tyr Gly Val Glu Arg 115 120 125Asp Arg Ala Asp Lys Ser Ala Val Gly Phe Asp Tyr Lys Gly Glu Val 130 135 140Glu Lys His Thr Ser Gln Lys Asp Tyr Ser Arg Gly Phe Gly Gly Arg145 150 155 160Tyr Gly Val Glu Lys Asp Lys Trp Asp Lys Ala Ala Leu Gly Tyr Asp 165 170 175Tyr Lys Gly Glu Thr Glu Lys His Glu Ser Gln Arg Asp Tyr Ala Lys 180 185 190Gly Phe Gly Gly Gln Tyr Gly Ile Gln Lys Asp Arg Val Asp Lys Ser 195 200 205Ala Val Gly Phe Asn Glu Met Glu Ala Pro Thr Thr Ala Tyr Lys Lys 210 215 220Thr Thr Pro Ile Glu Ala Ala Ser Ser Gly Ala Arg Gly Leu Lys Ala225 230 235 240Lys Phe Glu Ser Met Ala Glu Glu Lys Arg Lys Arg Glu Glu Glu Glu 245 250 255Lys Ala Gln Gln Val Ala Arg Arg Gln Gln Glu Arg Lys Ala Val Thr 260 265 270Lys Arg Ser Pro Glu Ala Pro Gln Pro Val Ile Ala Met Glu Glu Pro 275 280 285Ala Val Pro Ala Pro Leu Pro Lys Lys Ile Ser Ser Glu Ala Trp Pro 290 295 300Pro Val Gly Thr Pro Pro Ser Ser Glu Ser Glu Pro Val Arg Thr Ser305 310 315 320Arg Glu His Pro Val Pro Leu Leu Pro Ile Arg Gln Thr Leu Pro Glu 325 330 335Asp Asn Glu Glu Pro Pro Ala Leu Pro Pro Arg Thr Leu Glu Gly Leu 340 345 350Gln Val Glu Glu Glu Pro Val Tyr Glu Ala Glu Pro Glu Pro Glu Pro 355 360 365Glu Pro Glu Pro Glu Pro Glu Asn Asp Tyr Glu Asp Val Glu Glu Met 370 375 380Asp Arg His Glu Gln Glu Asp Glu Pro Glu Gly Asp Tyr Glu Glu Val385 390 395 400Leu Glu Pro Glu Asp Ser Ser Phe Ser Ser Ala Leu Ala Gly Ser Ser 405 410 415Gly Cys Pro Ala Gly Ala Gly Ala Gly Ala Val Ala Leu Gly Ile Ser 420 425 430Ala Val Ala Leu Tyr Asp Tyr Gln Gly Glu Gly Ser Asp Glu Leu Ser 435 440 445Phe Asp Pro Asp Asp Val Ile Thr Asp Ile Glu Met Val Asp Glu Gly 450 455 460Trp Trp Arg Gly Arg Cys His Gly His Phe Gly Leu Phe Pro Ala Asn465 470 475 480Tyr Val Lys Leu Leu Glu 48510296DNAHomo sapiens 102ctgacagcat ctggctttca gttcctcagt caccactact ttgtaccaaa ttcactgttt 60tggctctgaa atctaatttt gagtttagca aggatg 96103349DNAHomo sapiens 103ccagagtgca ggatacatca ttggcaccaa gggtcttttt caattcttgg tcaatcctct 60gcagcaagca cccccggatg acgtcctcat agatgccctc agtggtcaga gcctggctgc 120ccacggcaag gacatccccc tcgaactcag gcagctcctt tttgcagcct ggctcgagtt 180ggctcagcac aaaaggtaaa aagatgcaga gaccccagcc tcggatgaac ctcctctgcg 240ccaacccgct gtccgatttg aatttcttca gcacgcgccc cctgactctc tccagcctct 300gggcagcctg gtcacagttg agggccgtcg tcagacactg gtcagccag 349104116PRTHomo sapiens 104Leu Ala Asp Gln Cys Leu Thr Thr Ala Leu Asn Cys Asp Gln Ala Ala1 5 10 15Gln Arg Leu Glu Arg Val Arg Gly Arg Val Leu Lys Lys Phe Lys Ser 20 25 30Asp Ser Gly Leu Ala Gln Arg Arg Phe Ile Arg Gly Trp Gly Leu Cys 35 40 45Ile Phe Leu Pro Phe Val Leu Ser Gln Leu Glu Pro Gly Cys Lys Lys 50 55 60Glu Leu Pro Glu Phe Glu Gly Asp Val Leu Ala Val Gly Ser Gln Ala65 70 75 80Leu Thr Thr Glu Gly Ile Tyr Glu Asp Val Ile Arg Gly Cys Leu Leu 85 90 95Gln Arg Ile Asp Gln Glu Leu Lys Lys Thr Leu Gly Ala Asn Asp Val 100 105 110Ser Cys Thr Leu 115105311DNAHomo sapiensmodified_base(1)..(311)n = g, a, c or t 105ctgcaagaca gcagagaanc tgccaatatc cagttagcag atgactttgc tggcaagcag 60aggaagncgg taaaagcttg tctcccagcc aggaaacttg acaccaagnt aagatttgga 120gctaggaaac aaacccaaaa ggctcacagc aagcggagaa aaaaacccca aaatctgtaa 180cctgtatcac aaagcgttca tatccttcag atataaagag ttattagata tcaataagaa 240aaatgcaaac actcctgaaa agtagaaaaa agctatgaac aggcaattca ctgaaattaa 300aaaaaaaaaa a 3111065107DNAHomo sapiens 106cgcaggcggt ggtcgtgggg aagggaagag gagccccggg agacgacagc agcatgggtg 60ggcggccttc gagccctctg gacaagcagc agcggcagca cctaaggggt caggtggaca 120ccctgctgag gaacttcctg ccttgctacc gtgggcagct ggcagcgtct gtcctgcggc 180agatctctcg agagctgggc cctcaggagc cgaccggaag ccagttgcta cgcagcaaaa 240agctgccccg agtccgtgag caccgaggac ccctgaccca gcttcggggc cacccacccc 300ggtggcagcc gatcttctgt gttctgcgtg gggacggccg cctagagtgg ttcagccaca 360aggaggaata tgaaaacggg ggccactgcc ttggctcaac agccctgaca ggatacacgc 420tcctgacttc ccagcgagaa tatctccgcc ttttggatgc tctctgccct gaatccttgg 480gagaccatac tcaggaagag cctgactccc tcttggaagt gcctgtgagc ttcccgctgt 540tcctgcagca ccccttccgc cggcacctct gcttctctgc agccaccagg gaggcacagc 600atgcctggag gctggccctg cagggtggca tccggcttca gggcacagtc ctgcagcgaa 660gccaggcccc tgctgcccgg gccttcctgg acgccgtccg actctaccgg cagcaccaag 720gccactttgg cgacgacgac gtgaccctag gctcagacgc cgaggtgctg accgcggtgc 780tgatgcggga gcaacttccc gcgctgcgag cccagaccct tcctggcctg cggggggcag 840gccgcgcccg cgcctgggcc tggaccgagc ttctagacgc cgttcacgca gctgtcctgg 900ccggggcctc cgccgggctc tgcgccttcc agcccgaaaa ggacgagctg cttgcgtcgc 960tggagaagac gatccgcccg gacgtggacc agctgctgcg gcagcgggcg cgtgtggcgg 1020ggcggctgag gacggatatc aggggaccgc tcgagtcgtg cctgcgccgg gaggtggacc 1080cgcagctgcc ccgggtcgtg cagaccctgc tgcgcaccgt ggaagcctcg ctcgaggcgg 1140tgcggaccct cctggctcaa ggcatggacc gactgtccca ccgcctgcgc cagagcccct 1200cgggcacgcg gctgcgcagg gaggtttact catttgggga gatgccgtgg gacttggcgc 1260tgatgcagac atgctaccgt gaggccgagc ggagccgggg gcgcttgggg cagctggcag 1320caccgtttgg ctttctgggg atgcagagcc tcgtgtttgg ggcccaagat cttgcacagc 1380agctcatggc tgacgccgtg gccaccttcc tgcagctggc tgaccagtgt ctgacgacgg 1440ccctcaactg tgaccaggct gcccagaggc tggagagagt cagggggcgc gtgctgaaga 1500aattcaaatc ggacagcggg ttggcgcaga ggaggttcat ccgaggctgg ggtctctgca 1560tctttttacc ttttgtgctg agccaactcg agccaggctg caaaaagacg gagtctcgct 1620ctgtcgccca ggctgtagtg cagtggtgtg atcttggctc gctgcggcct ccacctccta 1680ggttcaagcg atcctcccat ctcggcctcc caagtagctg ggattacagg cacccgctat 1740agggaccagc cccacagggt cggtgggtct ctccctgtgt gcagagacaa gagagtgtag 1800aaataaagac acaagacaaa gagataaaag aaaagacagc tgggcccggg ggaccactac 1860taccaagttg cggagaccgg tagtggcccc gaatgtctgg ctgcgctgtt atttattgga 1920tacaaagcaa aaggggcagg gtaaagagtg tgagtcatct ccaatgatag gtaaggtcac 1980gtgggtcatg tgtccactgg acagggggcc cctccctgcc tggcagctga ggcagagaga 2040gagaggagac aaagagaaag acagcttaag ccattatttc tgcatatcag agacttttag 2100tactttcact aactgactac tgctatctag aaggcagagc caggtgtaca ggatggaaca 2160cgaaggcgga ctaggagcga gaccactgaa gcacagcatc acagggagac ggttaggtct 2220ctggataact gtgggcaagc ctgactgata tcaggccctc cacaagaggt ggaggagcag 2280agtcttctct aaactccccc ggagaaaagg agactccctt tcccggtctg ctaagtagcc 2340ggtgtttttc cttgacactt ttcgctaccg ctagaccacg gtctgcctgg caacaggcat 2400cttcccagac gctggcgtca ccgctagacc aaggagccct tctgctggcc ctgtccgggc 2460ataacagaag gctcgcactc ttgtcttctg gtcatacctc actatgcccc ctcagctcct 2520atctctgtat ggcctggttt ttcctaggtt atgattgtag agtgaggatt attataatat 2580tggaataaag agtaactgct accaactaat cattaatgat attcatatat aatcatatct 2640aatatctata tctggtataa ctattcttgt tttatatttt gttatactgg aacagctcat 2700gtcctcggtc tcttgcctca gcacctgggt ggcttgccgc ccacaacccg ccaccacgcc 2760cagctaattt ttgtactttt ggtagagacg gtggtttcac catgttggtc aggctggtct 2820tgaactcctg acctcatgat ccgcccacct cagccaacca aagtgctggg attacaggca 2880tgagccaccg cacccggcct gtttatttta aaataaaaat atttaaaaat aaagataagg 2940aaactaaggc ccaagccccg ccccccaacc ccacagctaa tcaggcccag ggctagggca 3000gaagcctgtg ttgtaggcct ctagaggggc cctcctctcc atccgagccc ctaacccgcc 3060atggttccag gagctgcctg agttcgaggg ggatgtcctt gccgtgggca gccaggctct 3120gaccactgag ggcatctatg aggacgtcat ccgggggtgc ttgctgcaga ggattgacca 3180agacccttgg tgccaatgat gtatcctgca ctctggacgg ctgcttggag gtcccatggg 3240aacaggaggg agcagatgag gaaactgagg ctgagcggga aggaggggct tgtcccaggc 3300agccagactc tggtgcccag atccagccac tctgcccacc gccttctcca ggaacattcc 3360ggagctgaat cttcacccac atctatcttg tttctattgg ataaatgtct acaagtggaa 3420tttctgggcc aaaacggatg tgccatcttt aggcttttgt aacccctgca acttcagaaa 3480actgtaccat tttatactcc aagcagcagc atttatttgt gtattttccc caaggctttc 3540tttattttaa tttttttttt tttttttgag actgggtctt gctctgtcac ccgggctggg 3600gtgcagtggc aggatctcgg ctcactgcga cctccgcctc ccgggttcaa gcgattctcc 3660tgcctcagcc tcccgagtag ctgggatttc aggcacccgc caccatgcct ggttaattgt 3720gtttttggta gagatggggt ttcgccgtgt tggccaggct ggtctcgaac tcctgtcctt 3780aggtggtctg cccgcctcag cctcccggag tgctgggatt gcaggtgtga gccaccacac 3840gtggcctaat tttttttttt taaataatag agacaaggtc tcgctatgct gcccaggctg 3900atctcaaact cctggactca agcaatcctc ctgccttggc ctcccaaagt gctaggatta 3960taggagtgat ccactatgtc cagcctccaa atcctttcta aacactagga cttttcatga 4020aaagaaaaaa gctatgccag ttagacacac acagaaatct catgatttta ttttgaattt 4080ctttgactaa attgaactta caaataagtt tattatggcc gggcgtggcg gtgcacacct 4140gtggtcccgg cactttggga ggctgaggcg ggcagatcac ttgagctcag gagttcggga 4200ccagcctggc ggacgtggtg ggacctcatc tctacaaaaa atacaaaatt agcggccggg 4260agtggtggct cacgcctgtc atcccagcac tttgggaggc tgagacaggt ggattgcttg 4320agccaaggag ttttgaggcc agcttgggca atgtggtgaa acctgtctct actaaaaaat 4380aaaataaata aataaataaa taaataaata aataaataaa taaaatttaa aagaagctgg 4440gctgagatgg gagatttgcc tgagcctggg aactcaaggc tgcagtgagt ggtgattgca 4500ccactgcact ccagcctggg tgatgggagt gagaccctgt ctcaaaaaac aaaatccaaa 4560tatgttgatt agccatttac atgtttgtag tttttttttt tttaatttca gtgaattgcc 4620tgttcatagc ttttttctac ttttcaggag tgtttgcatt tttcttattg atatctaata 4680actctttata tctgaaggat atgaacgctt tgtgatacag gttacagatt ttggggtttt 4740tttctccgct tgctgtgagc cttttgggtt tgtttcctag ctccaaatct taacttggtg 4800tcaagtttcc tggctgggag acaagctttt accgacttcc tctgcttgcc agcaaagtca 4860tctgctaact ggatattggc agcttctctg ctgtcttgca gctgcttccg gagtgggttc 4920cacagggatt cccgtgtgtt cttggttcag cttgcagagg gactttcaca ctccctggag 4980accgtttcct cccattctgt ctggagtttt cggcctaccc caagacaatg agatattcct 5040gacctttcca cctatttccc tccaacccca ccttccaaaa tacatttgct caatacattt 5100gcacttc 5107107579PRTHomo sapiens 107Gln Ala Val Val Val Gly Lys Gly Arg Gly Ala Pro Gly Asp Asp Ser1 5 10 15Ser Met Gly Gly Arg Pro Ser Ser Pro Leu Asp Lys Gln Gln Arg Gln 20 25 30His Leu Arg Gly Gln Val Asp Thr Leu Leu Arg Asn Phe Leu Pro Cys 35 40 45Tyr Arg Gly Gln Leu Ala Ala Ser Val Leu Arg Gln Ile Ser Arg Glu 50 55 60Leu Gly Pro Gln Glu Pro Thr Gly Ser Gln Leu Leu Arg Ser Lys Lys65 70 75 80Leu Pro Arg Val Arg Glu His Arg Gly Pro Leu Thr Gln Leu Arg Gly 85 90 95His Pro Pro Arg Trp Gln Pro Ile Phe Cys Val Leu Arg Gly Asp Gly 100 105 110Arg Leu Glu Trp Phe Ser His Lys Glu Glu Tyr Glu Asn Gly Gly His 115 120 125Cys Leu Gly Ser Thr Ala Leu Thr Gly Tyr Thr Leu Leu Thr Ser Gln 130 135 140Arg Glu Tyr Leu Arg Leu Leu Asp Ala Leu Cys Pro Glu Ser Leu Gly145 150 155 160Asp His Thr Gln Glu Glu Pro Asp Ser Leu Leu Glu Val Pro Val Ser 165 170 175Phe Pro Leu Phe Leu Gln His Pro Phe Arg Arg His Leu Cys Phe Ser 180 185 190Ala Ala Thr Arg Glu Ala Gln His Ala Trp Arg Leu Ala Leu Gln Gly 195 200 205Gly Ile Arg Leu Gln Gly Thr Val Leu Gln Arg Ser Gln Ala Pro Ala 210 215 220Ala Arg Ala Phe Leu Asp Ala Val Arg Leu Tyr Arg Gln His Gln Gly225 230 235 240His Phe Gly Asp Asp Asp Val Thr Leu Gly Ser Asp Ala Glu Val Leu 245 250 255Thr Ala Val Leu Met Arg Glu Gln Leu Pro Ala Leu Arg Ala Gln Thr 260 265 270Leu Pro Gly Leu Arg Gly Ala Gly Arg Ala Arg Ala Trp Ala Trp Thr 275 280 285Glu Leu

Leu Asp Ala Val His Ala Ala Val Leu Ala Gly Ala Ser Ala 290 295 300Gly Leu Cys Ala Phe Gln Pro Glu Lys Asp Glu Leu Leu Ala Ser Leu305 310 315 320Glu Lys Thr Ile Arg Pro Asp Val Asp Gln Leu Leu Arg Gln Arg Ala 325 330 335Arg Val Ala Gly Arg Leu Arg Thr Asp Ile Arg Gly Pro Leu Glu Ser 340 345 350Cys Leu Arg Arg Glu Val Asp Pro Gln Leu Pro Arg Val Val Gln Thr 355 360 365Leu Leu Arg Thr Val Glu Ala Ser Leu Glu Ala Val Arg Thr Leu Leu 370 375 380Ala Gln Gly Met Asp Arg Leu Ser His Arg Leu Arg Gln Ser Pro Ser385 390 395 400Gly Thr Arg Leu Arg Arg Glu Val Tyr Ser Phe Gly Glu Met Pro Trp 405 410 415Asp Leu Ala Leu Met Gln Thr Cys Tyr Arg Glu Ala Glu Arg Ser Arg 420 425 430Gly Arg Leu Gly Gln Leu Ala Ala Pro Phe Gly Phe Leu Gly Met Gln 435 440 445Ser Leu Val Phe Gly Ala Gln Asp Leu Ala Gln Gln Leu Met Ala Asp 450 455 460Ala Val Ala Thr Phe Leu Gln Leu Ala Asp Gln Cys Leu Thr Thr Ala465 470 475 480Leu Asn Cys Asp Gln Ala Ala Gln Arg Leu Glu Arg Val Arg Gly Arg 485 490 495Val Leu Lys Lys Phe Lys Ser Asp Ser Gly Leu Ala Gln Arg Arg Phe 500 505 510Ile Arg Gly Trp Gly Leu Cys Ile Phe Leu Pro Phe Val Leu Ser Gln 515 520 525Leu Glu Pro Gly Cys Lys Lys Thr Glu Ser Arg Ser Val Ala Gln Ala 530 535 540Val Val Gln Trp Cys Asp Leu Gly Ser Leu Arg Pro Pro Pro Pro Arg545 550 555 560Phe Lys Arg Ser Ser His Leu Gly Leu Pro Ser Ser Trp Asp Tyr Arg 565 570 575His Pro Leu1082917DNAHomo sapiens 108ctagaatgct aattgcactt aggcctcatg gttctagtaa acggcagctg tgggcccttt 60tgcctcttcc cctgttcttg gcctcacatc tccagctgag ctgccggtct tggcttcctg 120gtcgcctctg tcccagagat ggtcccaggg agccatccta gggcaggtag cactgaggct 180cctgtggaaa caggagccac ctgctcagga gacccctttc ctgaggaagt ccttacctct 240ccccttgaga tgtaaaaatg gtccagcaga gacaagctcc cgtggaaaac agacaggagc 300atgggggcag ctgtcatggc tgtggcgggc acttttcctc agagtttctg ccttgcgctg 360gtccaggagc cattttgcac caaggacttg gtaggcagag gcagccccac tgtaaagaag 420ggtcagatta aaacaaaaaa ctgccaaaag catcccctct gccccccatg tggcactggc 480atcattctct gcttccctgg gaggaatttt ttcaccatgt tattgaaggg gatggttcat 540taaggactcc acccctcaga gctcactcag accccaagga cagaggtgac tggggcttgg 600tgacttgttc actccttttt tcccaggtat actgaagggg tgacagagag aggtcttcat 660ggcagaccag gccttcacag ctaatgggga gaggaactca tgttacctct gcaggcctgg 720ggtcctgagg gggtcttttg gcttcagcct gttcccccag aggcttgatc atcccacatt 780gtcccttcag ctcagctgct cttctccccc acccaccctg ggatgtgggt gctctgggct 840gaaccaaggc tatgacttct ggagagaggc tcaggggttg gtctgagagg cctgccatcc 900acccctcagg gagctaggtt ttctcagagg ctcagctgga cagcactttt tagaaaagtt 960tgtagcatta agctggttta aaatatgaag ttggttttgt tggatggctc ctgagctgac 1020tgactgatgt ctgaagtttg agacgaggga ttatttcagg gtggggccca atgtgatcta 1080atgcccagct ggggacaatt gtgcctcatc atttgctcaa attcctgggc ccccaagtta 1140gccccctccc aggagtggtc agcgggtcac agctgccccc actctataag cagggctaat 1200tgtgtaccct ttgcagaaat gcttttggtc tcctacccaa atactcacaa gggtcttatc 1260agacgcccgt cttaaagtcc agcatgctca gggaccctgt gtaggatctc gtttgtggtg 1320agtgggctgc tctgaggtct ccactgggct gccatttagc catgtgccat ctctgaagtc 1380agaggtgttt gactcccatt ccttgggctc tggagctttc cccaagaatt acatcagaga 1440aaaggaagaa ggggcctgca ggacccattg ggaatgagtt taatactgaa gtctggaatg 1500taagctcatg ccctagaggc ctctccatat ggctggtcag gggagctgcc ttcaggcttg 1560tgccccgtgt gctcagcagc tgcctctgtc cccctctact gtccctttca caccttgcct 1620ggccaagggg ctagacctcc caggctaagc ctcagattca gtgcaggaca caagctcatg 1680cccccgtctt gccagtgaca cttgaagcct cccgacttcc acagagtgct tcaggacaca 1740ttttgagtgg tattttcttt tctttttttc ttcttttttt tttttttgag atggagtctc 1800gctctgttgc ccaggctgga gtgcagtggc ctgatctcgg ctcactgcaa cctctgcctc 1860ccaggttcaa gcgattcttc tgcctcagcc tccagagtag ctgggactat agacatgcac 1920caccacgccc ggctaatttt gtatttttgg tcgagacggg gttttgccat gttagtcagg 1980ctggtcttga actcctgacc tcaagtgatc caccacctcg gcctcccaaa gtgttgagat 2040gacaggcacg agccaccagg cccagcctga gtggtatttt ctttagggac caggtagact 2100ttaaaacgag ggtaagagaa aagccagtgt ctttctgagg taaataattt ctgccaggaa 2160acttcccagc cccaccagca gcccccctaa aaaaatcact cgtgtcccca gggacttcta 2220aagcttgggg ctccaggaaa tcatccagta gagttggaga ttcagagatt tcttgaagcc 2280agggacatgc tcctaactcc tttcccatta aaggtgttag aatagaccag agggtgtccc 2340ttttccacag taatgggatc ggctggtgtg ccttcaggga ggaagaggga ggtggtcaag 2400cttgaaaaac tggctttagg atggttctga ctttgttctc cctccccaag tgttctcaac 2460ctccattctg cagtgttcag agttttaggg aaagggtttg ggtgccccag catccaggtg 2520ttgtgtggct tagcgcatgt gaagtgaaaa ccttctgggg ttgtttggaa gcagctttct 2580ggttcttgtg attgtatcct gaggtcccag aaccctattc tcccacgagg atcctcagtg 2640accatggtgg ccacacgcct ggccagcctg ctggctcctg ggtgagctga agaaccttgc 2700ctgtggcact tttcgagggt gagctggaac cgagagaaca tggtccccgt gctgggactc 2760atgcgggtca tttcctgccg gcctggtttc gcctggtcgt gtctttatga gcaccatgta 2820agcctccttg tattgagata attgggcatt aaacattaaa ctgcagctct gggaaaaaaa 2880aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaac 291710983PRTHomo sapiens 109Met Glu Ser Arg Ser Val Ala Gln Ala Gly Val Gln Trp Pro Asp Leu1 5 10 15Gly Ser Leu Gln Pro Leu Pro Pro Arg Phe Lys Arg Phe Phe Cys Leu 20 25 30Ser Leu Gln Ser Ser Trp Asp Tyr Arg His Ala Pro Pro Arg Pro Ala 35 40 45Asn Phe Val Phe Leu Val Glu Thr Gly Phe Cys His Val Ser Gln Ala 50 55 60Gly Leu Glu Leu Leu Thr Ser Ser Asp Pro Pro Pro Arg Pro Pro Lys65 70 75 80Val Leu Arg110509DNAHomo sapiensmodified_base(467)n = g, a, c or t 110aaaccttaag aaccacataa tactatataa tgcttttctg tacaaatctc aagaaacact 60ttcattcatt aaaacatcat gaaaatcctt aaatgtgtta aatggaaaaa aatgaaacca 120tgaacaaaaa agctatacat gtaggtgcat atttatctcc tcctgagttg ggagaaatct 180ttctaagcat agaaacaatg gtagcaaaag agaagaatag atttggctgg attaacaata 240aaaaatttct gccagaaata tgaaaattca atttagacaa aattcaatat aaacaaaatt 300aatatagaca aaggtggtaa acaggtggtt ctcagagaag ataaatacat gattatttaa 360cataaaaaga aatgttcaat gtttctagaa gacaaataat tacaaaccta aacaaattgt 420atatttgtta gattggcata aattataata atccaacatt gagttangtg gaatataaat 480tggtaaaata tttctggaag acaatttgg 509111525DNAHomo sapiens 111agaagtgatt atgggattaa aagaatacat aattacagtg ttttgggatt gggctctttt 60ttttcttaat agaaaagcag aaacttcata aataatagct gtgctttaga taccagataa 120caaatattgt ttcccctgaa gatatgacct actagaacta ctcacatata tagtccaata 180attgctgact taataggtat ggtaaaatag ctgataataa gtcagactct caagagtttc 240tgtaccttga ttattgacaa attcattgtt ttacatccta ctaaagaaca tgtgtgtggg 300gagggggtgg ggaactggtt cacaacataa tctgaaggag atcaaacatc tgtaaggaca 360ggtacccagt gatgataata tatctgaaaa cacaagccat ttttattctt tatcccaatt 420aacttgaggt actctaatga tgaagcactc gattgcacta tgacctcctt gagtgatggg 480cagcttggtt cctctctcac tttttgtttc tttttaatat gcaaa 525112183DNAHomo sapiensmodified_base(1)..(183)n = g, a, c or t 112aaaaaaagac aatttgagca ggacgaccct ctccaatctg ggtagcatgg ttagcctgtg 60cagtaacaac gtaggcttgg aggatgggtn caatgaaaat gattctgatt cggaaacgtt 120ttgactttgg actgtanaag cttttctttg atcacctgtg ntggaggaaa ggaaagaagc 180ctt 1831131750DNAHomo sapiens 113cagctctctg tcagaatggc caccatggta ccatccgtgt tgtggcccag ggcctgctgg 60actctgctgg tctgctgtct gctgacccca ggtgtccagg ggcaggagtt ccttttgcgg 120gtggagcccc agaaccctgt gctctctgct ggagggtccc tgtttgtgaa ctgcagtact 180gattgtccca gctctgagaa aatcgccttg gagacgtccc tatcaaagga gctggtggcc 240agtggcatgg gctgggcagc cttcaatctc agcaacgtga ctggcaacag tcggatcctc 300tgctcagtgt actgcaatgg ctcccagata acaggctcct ctaacatcac cgtgtacggg 360ctcccggagc gtgtggagct ggcacccctg cctccttggc agccggtggg ccagaacttc 420accctgcgct gccaagtgga gggtgggtcg ccccggacca gcctcacggt ggtgctgctt 480cgctgggagg aggagctgag ccggcagccc gcagtggagg agccagcgga ggtcactgcc 540actgtgctgg ccagcagaga cgaccacgga gcccctttct catgccgcac agaactggac 600atgcagcccc aggggctggg actgttcgtg aacacctcag ccccccgcca gctccgaacc 660tttgtcctgc ccgtgacccc cccgcgcctc gtggcccccc ggttcttgga ggtggaaacg 720tcgtggccgg tggactgcac cctagacggg ctttttccag cctcagaggc ccaggtctac 780ctggcgctgg gggaccagat gctgaatgcg acagtcatga accacgggga cacgctaacg 840gccacagcca cagccacggc gcgcgcggat caggagggtg cccgggagat cgtctgcaac 900gtgaccctag ggggcgagag acgggaggcc cgggagaact tgacggtctt tagcttccta 960ggacccattg tgaacctcag cgagcccacc gcccatgagg ggtccacagt gaccgtgagt 1020tgcatggctg gggctcgagt ccaggtcacg ctggacggag ttccggccgc ggccccgggg 1080cagccagctc aacttcagct aaatgctacc gagagtgacg acggacgcag cttcttctgc 1140agtgccactc tcgaggtgga cggcgagttc ttgcacagga acagtagcgt ccagctgcga 1200gtcctgtatg gtcccaaaat tgaccgagcc acatgccccc agcacttgaa atggaaagat 1260aaaacgagac acgtcctgca gtgccaagcc aggggcaacc cgtaccccga gctgcggtgt 1320ttgaaggaag gctccagccg ggaggtgccg gtggggatcc cgttcttcgt caacgtaaca 1380cataatggta cttatcagtg ccaagcgtcc agctcacgag gcaaatacac cctggtcgtg 1440gtgatggaca ttgaggctgg gagctcccac tttgtccccg tcttcgtggc ggtgttactg 1500accctgggcg tggtgactat cgtactggcc ttaatgtacg tcttcaggga gcaccaacgg 1560agcggcagtt accatgttag ggaggagagc acctatctgc ccctcacgtc tatgcagccg 1620acagaagcaa tgggggaaga accgtccaga gctgagtgac gctgggatcc gggatcaaag 1680ttggcggggg cttggctgtg ccctcagatt ccgcaccaat aaagccttca aactccctaa 1740aaaaaaaaaa 1750114547PRTHomo sapiens 114Met Ala Thr Met Val Pro Ser Val Leu Trp Pro Arg Ala Cys Trp Thr1 5 10 15Leu Leu Val Cys Cys Leu Leu Thr Pro Gly Val Gln Gly Gln Glu Phe 20 25 30Leu Leu Arg Val Glu Pro Gln Asn Pro Val Leu Ser Ala Gly Gly Ser 35 40 45Leu Phe Val Asn Cys Ser Thr Asp Cys Pro Ser Ser Glu Lys Ile Ala 50 55 60Leu Glu Thr Ser Leu Ser Lys Glu Leu Val Ala Ser Gly Met Gly Trp65 70 75 80Ala Ala Phe Asn Leu Ser Asn Val Thr Gly Asn Ser Arg Ile Leu Cys 85 90 95Ser Val Tyr Cys Asn Gly Ser Gln Ile Thr Gly Ser Ser Asn Ile Thr 100 105 110Val Tyr Gly Leu Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Pro Trp 115 120 125Gln Pro Val Gly Gln Asn Phe Thr Leu Arg Cys Gln Val Glu Gly Gly 130 135 140Ser Pro Arg Thr Ser Leu Thr Val Val Leu Leu Arg Trp Glu Glu Glu145 150 155 160Leu Ser Arg Gln Pro Ala Val Glu Glu Pro Ala Glu Val Thr Ala Thr 165 170 175Val Leu Ala Ser Arg Asp Asp His Gly Ala Pro Phe Ser Cys Arg Thr 180 185 190Glu Leu Asp Met Gln Pro Gln Gly Leu Gly Leu Phe Val Asn Thr Ser 195 200 205Ala Pro Arg Gln Leu Arg Thr Phe Val Leu Pro Val Thr Pro Pro Arg 210 215 220Leu Val Ala Pro Arg Phe Leu Glu Val Glu Thr Ser Trp Pro Val Asp225 230 235 240Cys Thr Leu Asp Gly Leu Phe Pro Ala Ser Glu Ala Gln Val Tyr Leu 245 250 255Ala Leu Gly Asp Gln Met Leu Asn Ala Thr Val Met Asn His Gly Asp 260 265 270Thr Leu Thr Ala Thr Ala Thr Ala Thr Ala Arg Ala Asp Gln Glu Gly 275 280 285Ala Arg Glu Ile Val Cys Asn Val Thr Leu Gly Gly Glu Arg Arg Glu 290 295 300Ala Arg Glu Asn Leu Thr Val Phe Ser Phe Leu Gly Pro Ile Val Asn305 310 315 320Leu Ser Glu Pro Thr Ala His Glu Gly Ser Thr Val Thr Val Ser Cys 325 330 335Met Ala Gly Ala Arg Val Gln Val Thr Leu Asp Gly Val Pro Ala Ala 340 345 350Ala Pro Gly Gln Pro Ala Gln Leu Gln Leu Asn Ala Thr Glu Ser Asp 355 360 365Asp Gly Arg Ser Phe Phe Cys Ser Ala Thr Leu Glu Val Asp Gly Glu 370 375 380Phe Leu His Arg Asn Ser Ser Val Gln Leu Arg Val Leu Tyr Gly Pro385 390 395 400Lys Ile Asp Arg Ala Thr Cys Pro Gln His Leu Lys Trp Lys Asp Lys 405 410 415Thr Arg His Val Leu Gln Cys Gln Ala Arg Gly Asn Pro Tyr Pro Glu 420 425 430Leu Arg Cys Leu Lys Glu Gly Ser Ser Arg Glu Val Pro Val Gly Ile 435 440 445Pro Phe Phe Val Asn Val Thr His Asn Gly Thr Tyr Gln Cys Gln Ala 450 455 460Ser Ser Ser Arg Gly Lys Tyr Thr Leu Val Val Val Met Asp Ile Glu465 470 475 480Ala Gly Ser Ser His Phe Val Pro Val Phe Val Ala Val Leu Leu Thr 485 490 495Leu Gly Val Val Thr Ile Val Leu Ala Leu Met Tyr Val Phe Arg Glu 500 505 510His Gln Arg Ser Gly Ser Tyr His Val Arg Glu Glu Ser Thr Tyr Leu 515 520 525Pro Leu Thr Ser Met Gln Pro Thr Glu Ala Met Gly Glu Glu Pro Ser 530 535 540Arg Ala Glu545115275DNAHomo sapiens 115cctgatgccc gaatttcagt ttggcactta cagcgaatct gagaggaaaa ccgaggagta 60cgatactcag gccatgaagt acttgtcata cctgctgtac cctctctgtg tcgggggtgc 120tgtctattca ctcctgaata tcaaatataa gagctggtac tcctggttaa tcaacagctt 180cgtcaacggg gtctatgcct ttggtttcct cttcatgctg ccccagctct ttgtgaacta 240caagttgaag tcagtggcac atctgccctg gaagg 2751162040DNAHomo sapiens 116cagctccttc accagcttgg tggtgggcgt gttcgtggtc tacgtggtgc acacctgctg 60ggtcatgtac ggcatcgtct acacccgccc gtgctccggc gacgccaact gcatccagcc 120ctacctggcg cggcggccca agctgcagct gagcgtgtac accacgacga ggtcccacct 180gggtgctgag aacaacatcg acctggtctt gaatgtggaa gactttgatg tggagtccaa 240atttgaaagg acagttaatg tttctgtacc aaagaaaacg agaaacaatg ggacgctgta 300tgcctacatc ttcctccatc acgctggggt cctgccgtgg cacgacggga agcaggtgca 360cctggtcagt cctctgacca cctacatggt ccccaagcca gaagaaatca acctgctcac 420cggggagtct gatacacagc agatcgaggc ggagaagaag ccgacgagtg ccctggatga 480gccagtgtcc cactggcgac cgcggctggc gctgaacgtg atggcggaca actttgtctt 540tgacgggtcc tccctgcctg ccgatgtgca tcggtacatg aagatgatcc agctggggaa 600aaccgtgcat tacctgccca tcctgttcat cgaccagctc agcaaccgcg tgaaggacct 660gatggtcata aaccgctcca ccaccgagct gcccctcacc gtgtcctacg acaaggtctc 720actggggcgg ctgcgcttct ggatccacat gcaggacgcc gtgtactccc tgcagcagtt 780cgggttttca gagaaagatg ctgatgaggt gaaaggaatt tttgtagata ccaacttata 840cttcctggcg ctgaccttct ttgtcgcagc gttccatctt ctctttgatt tcctggcctt 900taaaaatgac atcagtttct ggaagaagaa gaagagcatg atcggcatgt ccaccaaggc 960agtgctctgg cgctgcttca gcaccgtggt catctttctg ttcctgctgg acgagcagac 1020gagcctgctg gtgctggtcc cggcgggtgt tggagccgcc attgagctgt ggaaagtgaa 1080gaaggcattg aagatgacta ttttttggag aggcctgatg cccgaatttc agtttggcac 1140ttacagcgaa tctgagagga aaaccgagga gtacgatact caggccatga agtacttgtc 1200atacctgctg taccctctct gtgtcggggg tgctgtctat tcactcctga atatcaaata 1260taagagctgg tactcctggt taatcaacag cttcgtcaac ggggtctatg cctttggttt 1320cctcttcatg ctgccccagc tctttgtgaa ctacaagttg aagtcagtgg cacatctgcc 1380ctggaaggcc ttcacctaca aggctttcaa caccttcatt gatgacgtct ttgccttcat 1440catcaccatg cccacgtctc accggctggc ctgcttccgg gacgacgtgg tgtttctggt 1500ctacctgtac cagcggtggc tttatcctgt ggataaacgc agagtgaacg agtttgggga 1560gtcctacgag gagaaggcca cgcgggcgcc ccacacggac tgaaggccgc ccgggctgcc 1620gccagccaag tgcaacttga attgtcaatg agtatttttg gaagcatttg gaggaattcc 1680tagacattgc gttttctgtg ttgccaaaat cccttcggac atttctcaga catctcccaa 1740gttcccatca cgtcagattt ggagctggta gcgcttacga tgcccccacg tgtgaacatc 1800tgtcttggtc acagagctgg gtgctgccgg tcaccttgag ctgtggtggc tcccggcaca 1860cgagtgtccg gggttcggcc atgtcctcac gcgggcaggg gtgggagccc tcacaggcaa 1920gggggctgtt ggatttccat ttcaggtggt tttctaagtg ctccttatgt gaatttcaaa 1980cacgtatgga attcattccg catggactct gggatcaaag gctctttcct cttttgtttg 2040117538PRTHomo sapiens 117Met Trp Ser Gly Arg Ser Ser Phe Thr Ser Leu Val Val Gly Val Phe1 5 10 15Val Val Tyr Val Val His Thr Cys Trp Val Met Tyr Gly Ile Val Tyr 20 25 30Thr Arg Pro Cys Ser Gly Asp Ala Asn Cys Ile Gln Pro Tyr Leu Ala 35 40 45Arg Arg Pro Lys Leu Gln Leu Ser Val Tyr Thr Thr Thr Arg Ser His 50 55 60Leu Gly Ala Glu Asn Asn Ile Asp Leu Val Leu Asn Val Glu Asp Phe65 70 75 80Asp Val Glu Ser Lys Phe Glu Arg Thr Val Asn Val Ser Val Pro Lys 85 90 95Lys Thr Arg Asn Asn Gly Thr Leu Tyr Ala Tyr Ile Phe Leu His His 100 105 110Ala Gly Val Leu Pro Trp His Asp Gly Lys Gln Val His Leu Val Ser 115 120 125Pro Leu Thr Thr Tyr Met Val Pro Lys Pro Glu Glu Ile Asn

Leu Leu 130 135 140Thr Gly Glu Ser Asp Thr Gln Gln Ile Glu Ala Glu Lys Lys Pro Thr145 150 155 160Ser Ala Leu Asp Glu Pro Val Ser His Trp Arg Pro Arg Leu Ala Leu 165 170 175Asn Val Met Ala Asp Asn Phe Val Phe Asp Gly Ser Ser Leu Pro Ala 180 185 190Asp Val His Arg Tyr Met Lys Met Ile Gln Leu Gly Lys Thr Val His 195 200 205Tyr Leu Pro Ile Leu Phe Ile Asp Gln Leu Ser Asn Arg Val Lys Asp 210 215 220Leu Met Val Ile Asn Arg Ser Thr Thr Glu Leu Pro Leu Thr Val Ser225 230 235 240Tyr Asp Lys Val Ser Leu Gly Arg Leu Arg Phe Trp Ile His Met Gln 245 250 255Asp Ala Val Tyr Ser Leu Gln Gln Phe Gly Phe Ser Glu Lys Asp Ala 260 265 270Asp Glu Val Lys Gly Ile Phe Val Asp Thr Asn Leu Tyr Phe Leu Ala 275 280 285Leu Thr Phe Phe Val Ala Ala Phe His Leu Leu Phe Asp Phe Leu Ala 290 295 300Phe Lys Asn Asp Ile Ser Phe Trp Lys Lys Lys Lys Ser Met Ile Gly305 310 315 320Met Ser Thr Lys Ala Val Leu Trp Arg Cys Phe Ser Thr Val Val Ile 325 330 335Phe Leu Phe Leu Leu Asp Glu Gln Thr Ser Leu Leu Val Leu Val Pro 340 345 350Ala Gly Val Gly Ala Ala Ile Glu Leu Trp Lys Val Lys Lys Ala Leu 355 360 365Lys Met Thr Ile Phe Trp Arg Gly Leu Met Pro Glu Phe Gln Phe Gly 370 375 380Thr Tyr Ser Glu Ser Glu Arg Lys Thr Glu Glu Tyr Asp Thr Gln Ala385 390 395 400Met Lys Tyr Leu Ser Tyr Leu Leu Tyr Pro Leu Cys Val Gly Gly Ala 405 410 415Val Tyr Ser Leu Leu Asn Ile Lys Tyr Lys Ser Trp Tyr Ser Trp Leu 420 425 430Ile Asn Ser Phe Val Asn Gly Val Tyr Ala Phe Gly Phe Leu Phe Met 435 440 445Leu Pro Gln Leu Phe Val Asn Tyr Lys Leu Lys Ser Val Ala His Leu 450 455 460Pro Trp Lys Ala Phe Thr Tyr Lys Ala Phe Asn Thr Phe Ile Asp Asp465 470 475 480Val Phe Ala Phe Ile Ile Thr Met Pro Thr Ser His Arg Leu Ala Cys 485 490 495Phe Arg Asp Asp Val Val Phe Leu Val Tyr Leu Tyr Gln Arg Trp Leu 500 505 510Tyr Pro Val Asp Lys Arg Arg Val Asn Glu Phe Gly Glu Ser Tyr Glu 515 520 525Glu Lys Ala Thr Arg Ala Pro His Thr Asp 530 5351184217DNAHomo sapiens 118cttcccggcc ccagccaagg ctgtcgttta cgtgtcggac attcaggagc tgtacatccg 60tgtggttgac aaggtggaga ttgggaagac agtgaaggca tacgtccgcg tgctggactt 120gcacaagaag cccttccttg ccaaatactt cccctttatg gacctgaagc tccgagcagc 180ctccccgatc attacattgg tggcccttga tgaagccctt gacaactaca ccatcacatt 240cctcatccgc ggtgtggcca tcggccagac cagtctaact gcaagtgtga ccaataaagc 300tggacagaga atcaactcag ccccacaaca gattgaagtc tttcccccgt tcaggctgat 360gcccaggaag gtgacactgc ttatcggggc cacgatgcag gtcacctccg agggcggccc 420ccagcctcag tccaacatcc ttttctccat cagcaatgag agcgttgcgc tggtgagcgc 480tgctgggctg gtacagggcc tcgccatcgg gaacggcact gtgtctgggc tcgtgcaggc 540agtggatgca gagaccggca aggtggtcat catctctcag gacctcgtgc aggtggaggt 600gctgctgcta agggccgtga ggatccgcgc ccccatcatg cggatgagga cgggcaccca 660gatgcccatc tatgtcaccg gcatcaccaa ccaccagaac cctttctcct ttggcaatgc 720cgtgccaggc ctgaccttcc actggtctgt caccaagcgg gacgtcctgg acctccgagg 780gcggcaccac gaggcgtcga tccgactccc gtcacagtac aactttgcca tgaacgtgct 840cggccgggta aaaggccgga ccgggctgag ggtggtggtc aaggctgtgg accccacatc 900ggggcagctg tatggcctgg ccagagaact ctcggatgag atccaagtcc aggtgtttga 960gaagctgcag ctgctcaacc ctgaaataga agcagaacaa atattaatgt cgcccaactc 1020atatataaag ctgcagacaa acagggatgg tgcagcctct ctgagctacc gcgtcctgga 1080tggacccgaa aaggttccag ttgtgcatgt tgatgagaaa ggctttctag catcagggtc 1140tatgatcggg acatccacca tcgaagtgat tgcacaagag ccctttgggg ccaaccaaac 1200catcattgtt gctgtaaagg tatcccctgt ttcctacctg agggtttcca tgagccctgt 1260cctgcacacc cagaacaagg aggccctggt ggccgtgcct ttgggaatga ccgtgacctt 1320cactgtccac ttccacgaca actctggaga tgtcttccat gctcacagtt cggtcctcaa 1380ctttgccact aacagagacg actttgtgca gatcgggaag ggccccacca acaacacctg 1440cgttgtccgc acagtcagcg tgggcctgac actgctccgt gtgtgggacg cagagcaccc 1500gggcctctcg gacttcatgc ccctgcctgt cctacaggcc atctccccag agctgtctgg 1560ggccatggtg gtgggggacg tgctctgtct ggccactgtt ctgaccagcc tggaaggcct 1620ctcaggaacc tggagctcct cagccaacag catcctccac atcgacccca agacgggtgt 1680ggctgtggcc cgggccgtgg gatccgtgac ggtttactat gaggtcgctg ggcacctgag 1740gacctacaag gaggtggtgg tcagcgtccc tcagaggatc atggcccgtc acctccaccc 1800catccagaca agcttccagg aggctacagc ctccaaagtg attgttgccg tgggagacag 1860aagctctaac ctgagaggcg agtgcacccc cacccagagg gaagtcatcc aggccttgca 1920cccagagacc ctcatcagct gccagtccca gttcaagccg gccgtctttg atttcccatc 1980tcaagatgtg ttcaccgtgg agccacagtt tgacactgct ctcggccagt acttctgctc 2040aatcacaatg cacaggctga cggacaagca gcggaagcac ctgagcatga agaagacagc 2100tctggtggtc agtgcctccc tctccagcag ccacttctcc acagagcagg tgggggccga 2160ggtgcccttc agcccaggtc tcttcgccga ccaggctgaa atccttttga gcaaccacta 2220caccagttcc gagatcaggg tctttggtgc cccggaggtt ctggagaact tggaggtgaa 2280atccgggtcc ccggccgtgc tggcattcgc aaaggagaag tcttttgggt ggcccagctt 2340catcacatac acggtcggcg tctcggaccc cgcggctggc agccaagggc ctctgtccac 2400taccctgacc ttctccagcc ccgtgaccaa ccaagccatt gccatcccag tgacagtggc 2460ttttgtgatg gatcgccgtg ggcccggtcc ttatggagcc agcctcttcc agcacttcct 2520ggattcctac caggtcatgt tcttcacgct cttcgccctg ttggctggga cagcggtcat 2580gatcatagcc taccacactg tctgcacgcc ccgggatctt gctgtgcctg cagccctcac 2640gcctcgagcc agccctggac acagccccca ctatttcgct gcctcatcac ccacatctcc 2700caatgcattg cctcctgctc gcaaagccag ccctccctca gggctgtgga gcccagccta 2760tgcctcccac taggccgcgt gaaggttccc ggaggatggg tctcagccga gcctcgtgca 2820cccccaagat ggaacatccc tgctgcattc acactggaac aagcccctcc agatgagtgc 2880cccggcccca ggccagcttc actgccgtct cttcacacag agctgtagtt tcggctctgc 2940ccattagctc attttatgta ggagttttaa atgtgtgttt ttttcctttc aagtcttaca 3000aagctaagac tttttggctc attccttttt gcatggttgt ctagggtttc tggacaatgt 3060gctgttgcat ttttattttc ctagccttgc taaaatcttt cccttctcaa gactttgagc 3120agttagaagt gctctttaga agttgtctgt gggtgatgtt actgtagtgg tctcagggaa 3180aggattgtcc agttacttta gggggttttt ggtggggttt ttccccctgt gaaaacttac 3240tttgccccta gtctggctgc tgctaggact tctgaggagc aatgggacat gagtgtccct 3300gtatctgcgc cactgccgca agggaagcct caggaaccag cacctggagg ccaggatagc 3360caagccctgg gtgagcgaga ggctggagaa cacaggagct cacccagggc tgctgcccaa 3420ccatgggcca ctgtgaacag acttcagtcc tctgtttttg tttcataagc cgttgagaca 3480tctgatggac ttggcttagg ccctgctggg acatcccacg tgtgatccct ttcactccat 3540caggacacca ggactgtcct taggaaaatg tccttgagat ggcagcagga gtcatatttt 3600ctgtgtgtgt gtttcggaaa gccgctgtgt cctgcctcag cacaaagacc cagtgtcatt 3660tgctcctcct gttcctgtgc cactccagaa cctcagcaga tctgagccac cgcctgccag 3720tgtgagaggc ggccactttc atggcagctt atcaggcgca gggccccaga cagcttccca 3780gccggcccta gagcccggcc tgggccaatg atggagggcg gccaccagcc cagggcctgc 3840ccatccagaa gggactcccc agggcctggg ggaggagacc cttggaaaag tcctctcttc 3900ccagctcctg attctggatc tgagattctc agatcacagg cccctgtgct ccaggccgag 3960gctgggccac cctcagggag atccagagac tcatgcccat ggccatccat gcgtggacgc 4020tgtgtggaga gtccaggatg acgggatccc gcacaagctc ccttcagtcc ttcagggctg 4080ggccatgtgg ttgatttttc taaagctgga gaaaggaaga attgtgcctt gcatattact 4140tgagcttaaa ctgacaacct ggatgtaaat aggagccttt ctactggttt atttaataaa 4200gttctatgtg atttttt 4217119923PRTHomo sapiens 119Phe Pro Ala Pro Ala Lys Ala Val Val Tyr Val Ser Asp Ile Gln Glu1 5 10 15Leu Tyr Ile Arg Val Val Asp Lys Val Glu Ile Gly Lys Thr Val Lys 20 25 30Ala Tyr Val Arg Val Leu Asp Leu His Lys Lys Pro Phe Leu Ala Lys 35 40 45Tyr Phe Pro Phe Met Asp Leu Lys Leu Arg Ala Ala Ser Pro Ile Ile 50 55 60Thr Leu Val Ala Leu Asp Glu Ala Leu Asp Asn Tyr Thr Ile Thr Phe65 70 75 80Leu Ile Arg Gly Val Ala Ile Gly Gln Thr Ser Leu Thr Ala Ser Val 85 90 95Thr Asn Lys Ala Gly Gln Arg Ile Asn Ser Ala Pro Gln Gln Ile Glu 100 105 110Val Phe Pro Pro Phe Arg Leu Met Pro Arg Lys Val Thr Leu Leu Ile 115 120 125Gly Ala Thr Met Gln Val Thr Ser Glu Gly Gly Pro Gln Pro Gln Ser 130 135 140Asn Ile Leu Phe Ser Ile Ser Asn Glu Ser Val Ala Leu Val Ser Ala145 150 155 160Ala Gly Leu Val Gln Gly Leu Ala Ile Gly Asn Gly Thr Val Ser Gly 165 170 175Leu Val Gln Ala Val Asp Ala Glu Thr Gly Lys Val Val Ile Ile Ser 180 185 190Gln Asp Leu Val Gln Val Glu Val Leu Leu Leu Arg Ala Val Arg Ile 195 200 205Arg Ala Pro Ile Met Arg Met Arg Thr Gly Thr Gln Met Pro Ile Tyr 210 215 220Val Thr Gly Ile Thr Asn His Gln Asn Pro Phe Ser Phe Gly Asn Ala225 230 235 240Val Pro Gly Leu Thr Phe His Trp Ser Val Thr Lys Arg Asp Val Leu 245 250 255Asp Leu Arg Gly Arg His His Glu Ala Ser Ile Arg Leu Pro Ser Gln 260 265 270Tyr Asn Phe Ala Met Asn Val Leu Gly Arg Val Lys Gly Arg Thr Gly 275 280 285Leu Arg Val Val Val Lys Ala Val Asp Pro Thr Ser Gly Gln Leu Tyr 290 295 300Gly Leu Ala Arg Glu Leu Ser Asp Glu Ile Gln Val Gln Val Phe Glu305 310 315 320Lys Leu Gln Leu Leu Asn Pro Glu Ile Glu Ala Glu Gln Ile Leu Met 325 330 335Ser Pro Asn Ser Tyr Ile Lys Leu Gln Thr Asn Arg Asp Gly Ala Ala 340 345 350Ser Leu Ser Tyr Arg Val Leu Asp Gly Pro Glu Lys Val Pro Val Val 355 360 365His Val Asp Glu Lys Gly Phe Leu Ala Ser Gly Ser Met Ile Gly Thr 370 375 380Ser Thr Ile Glu Val Ile Ala Gln Glu Pro Phe Gly Ala Asn Gln Thr385 390 395 400Ile Ile Val Ala Val Lys Val Ser Pro Val Ser Tyr Leu Arg Val Ser 405 410 415Met Ser Pro Val Leu His Thr Gln Asn Lys Glu Ala Leu Val Ala Val 420 425 430Pro Leu Gly Met Thr Val Thr Phe Thr Val His Phe His Asp Asn Ser 435 440 445Gly Asp Val Phe His Ala His Ser Ser Val Leu Asn Phe Ala Thr Asn 450 455 460Arg Asp Asp Phe Val Gln Ile Gly Lys Gly Pro Thr Asn Asn Thr Cys465 470 475 480Val Val Arg Thr Val Ser Val Gly Leu Thr Leu Leu Arg Val Trp Asp 485 490 495Ala Glu His Pro Gly Leu Ser Asp Phe Met Pro Leu Pro Val Leu Gln 500 505 510Ala Ile Ser Pro Glu Leu Ser Gly Ala Met Val Val Gly Asp Val Leu 515 520 525Cys Leu Ala Thr Val Leu Thr Ser Leu Glu Gly Leu Ser Gly Thr Trp 530 535 540Ser Ser Ser Ala Asn Ser Ile Leu His Ile Asp Pro Lys Thr Gly Val545 550 555 560Ala Val Ala Arg Ala Val Gly Ser Val Thr Val Tyr Tyr Glu Val Ala 565 570 575Gly His Leu Arg Thr Tyr Lys Glu Val Val Val Ser Val Pro Gln Arg 580 585 590Ile Met Ala Arg His Leu His Pro Ile Gln Thr Ser Phe Gln Glu Ala 595 600 605Thr Ala Ser Lys Val Ile Val Ala Val Gly Asp Arg Ser Ser Asn Leu 610 615 620Arg Gly Glu Cys Thr Pro Thr Gln Arg Glu Val Ile Gln Ala Leu His625 630 635 640Pro Glu Thr Leu Ile Ser Cys Gln Ser Gln Phe Lys Pro Ala Val Phe 645 650 655Asp Phe Pro Ser Gln Asp Val Phe Thr Val Glu Pro Gln Phe Asp Thr 660 665 670Ala Leu Gly Gln Tyr Phe Cys Ser Ile Thr Met His Arg Leu Thr Asp 675 680 685Lys Gln Arg Lys His Leu Ser Met Lys Lys Thr Ala Leu Val Val Ser 690 695 700Ala Ser Leu Ser Ser Ser His Phe Ser Thr Glu Gln Val Gly Ala Glu705 710 715 720Val Pro Phe Ser Pro Gly Leu Phe Ala Asp Gln Ala Glu Ile Leu Leu 725 730 735Ser Asn His Tyr Thr Ser Ser Glu Ile Arg Val Phe Gly Ala Pro Glu 740 745 750Val Leu Glu Asn Leu Glu Val Lys Ser Gly Ser Pro Ala Val Leu Ala 755 760 765Phe Ala Lys Glu Lys Ser Phe Gly Trp Pro Ser Phe Ile Thr Tyr Thr 770 775 780Val Gly Val Ser Asp Pro Ala Ala Gly Ser Gln Gly Pro Leu Ser Thr785 790 795 800Thr Leu Thr Phe Ser Ser Pro Val Thr Asn Gln Ala Ile Ala Ile Pro 805 810 815Val Thr Val Ala Phe Val Met Asp Arg Arg Gly Pro Gly Pro Tyr Gly 820 825 830Ala Ser Leu Phe Gln His Phe Leu Asp Ser Tyr Gln Val Met Phe Phe 835 840 845Thr Leu Phe Ala Leu Leu Ala Gly Thr Ala Val Met Ile Ile Ala Tyr 850 855 860His Thr Val Cys Thr Pro Arg Asp Leu Ala Val Pro Ala Ala Leu Thr865 870 875 880Pro Arg Ala Ser Pro Gly His Ser Pro His Tyr Phe Ala Ala Ser Ser 885 890 895Pro Thr Ser Pro Asn Ala Leu Pro Pro Ala Arg Lys Ala Ser Pro Pro 900 905 910Ser Gly Leu Trp Ser Pro Ala Tyr Ala Ser His 915 9201201270PRTHomo sapiens 120Arg Asp Phe Gln Ser Glu Val Leu Leu Ser Ala Met Glu Leu Phe His1 5 10 15Met Thr Ser Gly Gly Asp Ala Ala Met Phe Arg Asp Gly Lys Glu Pro 20 25 30Gln Pro Ser Ala Glu Ala Ala Ala Ala Pro Ser Leu Ala Asn Ile Ser 35 40 45Cys Phe Thr Gln Lys Leu Val Glu Lys Leu Tyr Ser Gly Met Phe Ser 50 55 60Ala Asp Pro Arg His Ile Leu Leu Phe Ile Leu Glu His Ile Met Val65 70 75 80Val Ile Glu Thr Ala Ser Ser Gln Arg Asp Thr Val Leu Ser Thr Leu 85 90 95Tyr Ser Ser Leu Asn Lys Val Ile Leu Tyr Cys Leu Ser Lys Pro Gln 100 105 110Gln Ser Leu Ser Glu Cys Leu Gly Leu Leu Ser Ile Leu Gly Phe Leu 115 120 125Gln Glu His Trp Asp Val Val Phe Ala Thr Tyr Asn Ser Asn Ile Ser 130 135 140Phe Leu Leu Cys Leu Met His Cys Leu Leu Leu Leu Asn Glu Arg Ser145 150 155 160Tyr Pro Glu Gly Phe Gly Leu Glu Pro Lys Pro Arg Met Ser Thr Tyr 165 170 175His Gln Val Phe Leu Ser Pro Asn Glu Asp Val Lys Glu Lys Arg Glu 180 185 190Asp Leu Pro Ser Leu Ser Asp Val Gln His Asn Ile Gln Lys Thr Val 195 200 205Gln Thr Leu Trp Gln Gln Leu Val Ala Gln Arg Gln Gln Thr Leu Glu 210 215 220Asp Ala Phe Lys Ile Asp Leu Ser Val Lys Pro Gly Glu Arg Glu Val225 230 235 240Lys Ile Glu Glu Val Thr Pro Leu Trp Glu Glu Thr Met Leu Lys Ala 245 250 255Trp Gln His Tyr Leu Ala Ser Glu Lys Lys Ser Leu Ala Ser Arg Ser 260 265 270Asn Val Ala His His Ser Lys Val Thr Leu Trp Ser Gly Ser Leu Ser 275 280 285Ser Ala Met Lys Leu Met Pro Gly Arg Gln Ala Lys Asp Pro Glu Cys 290 295 300Lys Thr Glu Asp Phe Val Ser Cys Ile Glu Asn Tyr Arg Arg Arg Gly305 310 315 320Gln Glu Leu Tyr Ala Ser Leu Tyr Lys Asp His Val Gln Arg Arg Lys 325 330 335Cys Gly Asn Ile Lys Ala Ala Asn Ala Trp Ala Arg Ile Gln Glu Gln 340 345 350Leu Phe Gly Glu Leu Gly Leu Trp Ser Gln Gly Glu Glu Thr Lys Pro 355 360 365Cys Ser Pro Trp Glu Leu Asp Trp Arg Glu Gly Pro Ala Arg Met Arg 370 375 380Lys Arg Ile Lys Arg Leu Ser Pro Leu Glu Ala Leu Ser Ser Gly Arg385 390 395 400His Lys Glu Ser Gln Asp Lys Asn Asp His Ile Ser Gln Thr Asn Ala 405 410 415Glu Asn Gln Asp Glu Leu Thr Leu Arg Glu Ala Glu Gly Glu Pro Asp 420 425 430Glu Val Gly Val Asp Cys Thr Gln Leu Thr Phe Phe Pro Ala

Leu His 435 440 445Glu Ser Leu His Ser Glu Asp Phe Leu Glu Leu Cys Arg Glu Arg Gln 450 455 460Val Ile Leu Gln Glu Leu Leu Asp Lys Glu Lys Val Thr Gln Lys Phe465 470 475 480Ser Leu Val Ile Val Gln Gly His Leu Val Ser Glu Gly Val Leu Leu 485 490 495Phe Gly His Gln His Phe Tyr Ile Cys Glu Asn Phe Thr Leu Ser Pro 500 505 510Thr Gly Asp Val Tyr Cys Thr Arg His Cys Leu Ser Asn Ile Ser Asp 515 520 525Pro Phe Ile Phe Asn Leu Cys Ser Lys Asp Arg Ser Thr Asp His Tyr 530 535 540Ser Cys Gln Cys His Ser Tyr Ala Asp Met Arg Glu Leu Arg Gln Ala545 550 555 560Arg Phe Leu Leu Gln Asp Ile Ala Leu Glu Ile Phe Phe His Asn Gly 565 570 575Tyr Ser Lys Phe Leu Val Phe Tyr Asn Asn Asp Arg Ser Lys Ala Phe 580 585 590Lys Ser Phe Cys Ser Phe Gln Pro Ser Leu Lys Gly Lys Ala Thr Ser 595 600 605Glu Asp Thr Leu Asn Leu Arg Arg Tyr Pro Gly Ser Asp Arg Ile Met 610 615 620Leu Gln Lys Trp Gln Lys Arg Asp Ile Ser Asn Phe Glu Tyr Leu Met625 630 635 640Tyr Leu Asn Thr Ala Ala Gly Arg Thr Cys Asn Asp Tyr Met Gln Tyr 645 650 655Pro Val Phe Pro Trp Val Leu Ala Asp Tyr Thr Ser Glu Thr Leu Asn 660 665 670Leu Ala Asn Pro Lys Ile Phe Arg Asp Leu Ser Lys Pro Met Gly Ala 675 680 685Gln Thr Lys Glu Arg Lys Leu Lys Phe Ile Gln Arg Phe Lys Glu Val 690 695 700Glu Lys Thr Glu Gly Asp Met Thr Val Gln Cys His Tyr Tyr Thr His705 710 715 720Tyr Ser Ser Ala Ile Ile Val Ala Ser Tyr Leu Val Arg Met Pro Pro 725 730 735Phe Thr Gln Ala Phe Cys Ala Leu Gln Gly Gly Ser Phe Asp Val Ala 740 745 750Asp Arg Met Phe His Ser Val Lys Ser Thr Trp Glu Ser Ala Ser Arg 755 760 765Glu Asn Met Ser Asp Val Arg Glu Leu Thr Pro Glu Phe Phe Tyr Leu 770 775 780Pro Glu Phe Leu Thr Asn Cys Asn Gly Val Glu Phe Gly Cys Met Gln785 790 795 800Asp Gly Thr Val Leu Gly Asp Val Gln Leu Pro Pro Trp Ala Asp Gly 805 810 815Asp Pro Arg Lys Phe Ile Ser Leu His Arg Lys Ala Leu Glu Ser Asp 820 825 830Phe Val Ser Ala Asn Leu His His Trp Ile Asp Leu Ile Phe Gly Tyr 835 840 845Lys Gln Gln Gly Pro Ala Ala Val Asp Ala Val Asn Ile Phe His Pro 850 855 860Tyr Phe Tyr Gly Asp Arg Met Asp Leu Ser Ser Ile Thr Asp Pro Leu865 870 875 880Ile Lys Ser Thr Ile Leu Gly Phe Val Ser Asn Phe Gly Gln Val Pro 885 890 895Lys Gln Leu Phe Thr Lys Pro His Pro Ala Arg Thr Ala Ala Gly Lys 900 905 910Pro Leu Pro Gly Lys Asp Val Ser Thr Pro Val Ser Leu Pro Gly His 915 920 925Pro Gln Pro Phe Phe Tyr Ser Leu Gln Ser Leu Arg Pro Ser Gln Val 930 935 940Thr Val Lys Asp Met Tyr Leu Phe Ser Leu Gly Ser Glu Ser Pro Lys945 950 955 960Gly Ala Ile Gly His Ile Val Ser Thr Glu Lys Thr Ile Leu Ala Val 965 970 975Glu Arg Asn Lys Val Leu Leu Pro Pro Leu Trp Asn Arg Thr Phe Ser 980 985 990Trp Gly Phe Asp Asp Phe Ser Cys Cys Leu Gly Ser Tyr Gly Ser Asp 995 1000 1005Lys Val Leu Met Thr Phe Glu Asn Leu Ala Ala Trp Gly Arg Cys Leu 1010 1015 1020Cys Ala Val Cys Pro Ser Pro Thr Thr Ile Val Thr Ser Gly Thr Ser1025 1030 1035 1040Thr Val Val Cys Val Trp Glu Leu Ser Met Thr Lys Gly Arg Pro Arg 1045 1050 1055Gly Leu Arg Leu Arg Gln Ala Leu Tyr Gly His Thr Gln Ala Val Thr 1060 1065 1070Cys Leu Ala Ala Ser Val Thr Phe Ser Leu Leu Val Ser Gly Ser Gln 1075 1080 1085Asp Cys Thr Cys Ile Leu Trp Asp Leu Asp His Leu Thr His Val Thr 1090 1095 1100Arg Leu Pro Ala His Arg Glu Gly Ile Ser Ala Ile Thr Ile Ser Asp1105 1110 1115 1120Val Ser Gly Thr Ile Val Ser Cys Ala Gly Ala His Leu Ser Leu Trp 1125 1130 1135Asn Val Asn Gly Gln Pro Leu Ala Ser Ile Thr Thr Ala Trp Gly Pro 1140 1145 1150Glu Gly Ala Ile Thr Cys Cys Cys Leu Met Glu Gly Pro Ala Trp Asp 1155 1160 1165Thr Ser Gln Ile Ile Ile Thr Gly Ser Gln Asp Gly Met Val Arg Val 1170 1175 1180Trp Lys Thr Glu Asp Val Lys Met Ser Val Pro Gly Arg Pro Ala Gly1185 1190 1195 1200Glu Glu Pro Leu Ala Gln Pro Pro Ser Pro Arg Gly His Lys Trp Glu 1205 1210 1215Lys Asn Leu Ala Leu Ser Arg Glu Leu Asp Val Ser Ile Ala Leu Thr 1220 1225 1230Gly Lys Pro Ser Lys Thr Ser Pro Ala Val Thr Ala Leu Ala Val Ser 1235 1240 1245Arg Asn His Thr Lys Leu Leu Val Gly Asp Glu Arg Gly Arg Ile Phe 1250 1255 1260Cys Trp Ser Ala Asp Gly1265 1270121647PRTHomo sapiens 121Met Leu Gln Lys Trp Gln Lys Arg Asp Ile Ser Asn Phe Glu Tyr Leu1 5 10 15Met Tyr Leu Asn Thr Ala Ala Gly Arg Thr Cys Asn Asp Tyr Met Gln 20 25 30Tyr Pro Val Phe Pro Trp Val Leu Ala Asp Tyr Thr Ser Glu Thr Leu 35 40 45Asn Leu Ala Asn Pro Lys Ile Phe Arg Asp Leu Ser Lys Pro Met Gly 50 55 60Ala Gln Thr Lys Glu Arg Lys Leu Lys Phe Ile Gln Arg Phe Lys Glu65 70 75 80Val Glu Lys Thr Glu Gly Asp Met Thr Val Gln Cys His Tyr Tyr Thr 85 90 95His Tyr Ser Ser Ala Ile Ile Val Ala Ser Tyr Leu Val Arg Met Pro 100 105 110Pro Phe Thr Gln Ala Phe Cys Ala Leu Gln Gly Gly Ser Phe Asp Val 115 120 125Ala Asp Arg Met Phe His Ser Val Lys Ser Thr Trp Glu Ser Ala Ser 130 135 140Arg Glu Asn Met Ser Asp Val Arg Glu Leu Thr Pro Glu Phe Phe Tyr145 150 155 160Leu Pro Glu Phe Leu Thr Asn Cys Asn Gly Val Glu Phe Gly Cys Met 165 170 175Gln Asp Gly Thr Val Leu Gly Asp Val Gln Leu Pro Pro Trp Ala Asp 180 185 190Gly Asp Pro Arg Lys Phe Ile Ser Leu His Arg Lys Ala Leu Glu Ser 195 200 205Asp Phe Val Ser Ala Asn Leu His His Trp Ile Asp Leu Ile Phe Gly 210 215 220Tyr Lys Gln Gln Gly Pro Ala Ala Val Asp Ala Val Asn Ile Phe His225 230 235 240Pro Tyr Phe Tyr Gly Asp Arg Met Asp Leu Ser Ser Ile Thr Asp Pro 245 250 255Leu Ile Lys Ser Thr Ile Leu Gly Phe Val Ser Asn Phe Gly Gln Val 260 265 270Pro Lys Gln Leu Phe Thr Lys Pro His Pro Ala Arg Thr Ala Ala Gly 275 280 285Lys Pro Leu Pro Gly Lys Asp Val Ser Thr Pro Val Ser Leu Pro Gly 290 295 300His Pro Gln Pro Phe Phe Tyr Ser Leu Gln Ser Leu Arg Pro Ser Gln305 310 315 320Val Thr Val Lys Asp Met Tyr Leu Phe Ser Leu Gly Ser Glu Ser Pro 325 330 335Lys Gly Ala Ile Gly His Ile Val Ser Thr Glu Lys Thr Ile Leu Ala 340 345 350Val Glu Arg Asn Lys Val Leu Leu Pro Pro Leu Trp Asn Arg Thr Phe 355 360 365Ser Trp Gly Phe Asp Asp Phe Ser Cys Cys Leu Gly Ser Tyr Gly Ser 370 375 380Asp Lys Val Leu Met Thr Phe Glu Asn Leu Ala Ala Trp Gly Arg Cys385 390 395 400Leu Cys Ala Val Cys Pro Ser Pro Thr Thr Ile Val Thr Ser Gly Thr 405 410 415Ser Thr Val Val Cys Val Trp Glu Leu Ser Met Thr Lys Gly Arg Pro 420 425 430Arg Gly Leu Arg Leu Arg Gln Ala Leu Tyr Gly His Thr Gln Ala Val 435 440 445Thr Cys Leu Ala Ala Ser Val Thr Phe Ser Leu Leu Val Ser Gly Ser 450 455 460Gln Asp Cys Thr Cys Ile Leu Trp Asp Leu Asp His Leu Thr His Val465 470 475 480Thr Arg Leu Pro Ala His Arg Glu Gly Ile Ser Ala Ile Thr Ile Ser 485 490 495Asp Val Ser Gly Thr Ile Val Ser Cys Ala Gly Ala His Leu Ser Leu 500 505 510Trp Asn Val Asn Gly Gln Pro Leu Ala Ser Ile Thr Thr Ala Trp Gly 515 520 525Pro Glu Gly Ala Ile Thr Cys Cys Cys Leu Met Glu Gly Pro Ala Trp 530 535 540Asp Thr Ser Gln Ile Ile Ile Thr Gly Ser Gln Asp Gly Met Val Arg545 550 555 560Val Trp Lys Thr Glu Asp Val Lys Met Ser Val Pro Gly Arg Pro Ala 565 570 575Gly Glu Glu Pro Leu Ala Gln Pro Pro Ser Pro Arg Gly His Lys Trp 580 585 590Glu Lys Asn Leu Ala Leu Ser Arg Glu Leu Asp Val Ser Ile Ala Leu 595 600 605Thr Gly Lys Pro Ser Lys Thr Ser Pro Ala Val Thr Ala Leu Ala Val 610 615 620Ser Arg Asn His Thr Lys Leu Leu Val Gly Asp Glu Arg Gly Arg Ile625 630 635 640Phe Cys Trp Ser Ala Asp Gly 64512232DNAArtificial SequenceDescription of Artificial SequencePCR amplification primer PDM-797 122gtgtcacaat ctacagtcag gcaggattct cc 3212335DNAArtificial SequenceDescription of Artificial SequencePCR amplification primer PDM-799 123gttatgtagc ggccgcttat catgttgctg cagag 35124980DNAHomo sapiens 124gccgctgccg ctccaggaga caggttccca tgcaggaatg aaagacatgg aagggaagag 60gggggccagc tccctgagtc ctgtgtccac cagctgctgc taaatacctc tgagaaactc 120tgcttctatc taaggggacc tacttctctc gggaatctca atacttggaa caagaacctc 180ctagacggac cctttggcat aatgaattgg accaactgta ggttccagga ctagagagcc 240agcaatgcct ccatgaacaa tctcacccaa ttactctgct caggaaacga ggtaactgat 300ggacagccga ggcagcccct taggcggctt aggcctcccc tgtggagcat ccctgaggcg 360gactccggcc agcccgagtg atgcgatcca aagagcactc ccgggtagga aattgccccg 420gtggaatgcc tcaccagagc agcgtgtagc agttccctgt ggaggattaa cacagtggct 480gaacaccggg aaggaactgg cacttggagt ccggacatct gaaacttgta gactgggagc 540tgtacatgga tgggagcagc ttcaccaacc cctgcaaagt gactctgaag aagacgacaa 600gccctgctcc agtcacaccc ggaagctgac tggtccacgc acagctgaag catgaggaaa 660ctcatcgcgg gactaatttt ccttaaaatt tagacttgca cagtaaggac ttcaactgac 720cttcctcaga ctgagaactg tttccagtat atacatcaag tcactgaggt aggacaaaag 780attgctacat tcctattatt ttaaggttac atttttgggg acccctcttt cttctgttct 840agctattacc tttcttgtgt cacctagaaa aggaccagtc cttaattgta ttttaaaaac 900tgtgatcatg ggaagcttta aattggttca ataacacgca tcaagttggt tatttcctgg 960gctacatacc ttggatagat 980

Claims

1-54: (canceled)

55. A method for treating multiple myeloma in a mammalian subject wherein a B-cell from said subject overexpresses SEQ ID NO:4, comprising administering to said subject an effective amount of an isolated monoclonal antibody that specifically binds to a polypeptide comprising the sequence set forth in SEQ ID NO: 4, wherein said monoclonal antibody has a binding constant for SEQ ID NO:4 that exceeds 10.sup.3 L/mol.

56. The method of claim 55, wherein said antibody is a humanized antibody.

57. The method of claim 55, wherein said antibody is a chimeric antibody.

58. The method of claim 55, wherein said antibody is a Fab fragment.

59. The method of claim 55, wherein said antibody is a Fv fragment.

60. The method of claim 55, wherein said antibody is a scFv.

61. The method of claim 55, wherein said antibody further comprises a therapeutic moiety.

62. The method of claim 61, wherein the therapeutic moiety is a radionuclide.

63. The method of claim 62, wherein the radionuclide is chosen from: .sup.90Y, .sup.123I, .sup.125I, .sup.131I, .sup.186Re, .sup.211At, and .sup.212Bi.

64. The method of claim 55, wherein the mammalian subject is a human.

65. The method of claim 55, wherein administration is intravenous.