CN1451045A - IFN-α homologue - Google Patents

Abstract

Alpha interferon homologues (both nucleic acids and polypeptides) are provided. Compositions including these interferon homologue polypeptides and nucleic acids, recombinant cells comprising said homologue polypeptides and nucleic acids, methods of making the new homologues, antibodies to the new homologues, and methods of using the homologues are provided. Integrated systems comprising the sequences of the nucleic acids or polypeptides are also provided.

Description

IFN-α homologue

Copyright Notice

Portions of this patent document contain material that is protected by copyright pursuant to 37 C.F.R.1.71(e). The copyright owner has no objection to the reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or files, and otherwise reserves all copyright rights.

Cross references to related applications

This application is a continuation-in-part of US Patent Application Serial No. 09/145,483, filed October 7, 1999, and claims the benefit and priority of 09/145,483, which is hereby incorporated by reference in its entirety.

field of invention

The present invention relates to the production of novel interferon-alpha homologues.

Background of the invention

Interferon-alpha is a member of the diverse helical bundle superfamily of cytokine genes (Sprang, S.R. et al. (1993) Curr. Opin. Struct. Biol. 3:815-827). Human interferon-α is encoded by a family containing more than 20 tandem-duplicated non-allelic genes that share 85-98% sequence identity at the amino acid level (Henco, K. et al. (1985) J. Molecular Biology. 185:227-260).

It has been proven that interferon-α can inhibit the proliferation of various types of cells, and is especially useful in the treatment of various cell proliferative diseases, which are often associated with cancer, especially blood cancers such as leukemia. These proteins show antiproliferative activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal cell carcinoma, bladder tumors, and ovarian cancer (Bonnem, E.M. et al. (1984) J. Biol. Response Modifiers 3:580; Oldham, R.K (1985) Hospital Practice 20:71).

Interferon-alpha is also useful against various types of viral infections (Finter, N.B et al. (1991) Drugs 42(5):749). Interferon-α also shows anti-human papillomavirus infection, hepatitis B virus and hepatitis C virus infection (Finter, N.B. et al., 1991, literature is the same; Kashima, H et al. (1988) Laryngoscope 98: 334; Dusheiko, G.M. et al. (1986) J Hematology 3(Suppl 2): S199; Davis, GL et al (1989) New England Journal of Medicine. 321:1501). In addition, the role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has been studied (Benoit, P et al. (1993) J. Immunol. 150(3):707).

Although these proteins have therapeutic value for a variety of diseases, they have not been optimized for use as drugs. For example, dose-limiting toxicity, receptor cross-reactivity and short serum half-life significantly impair the clinical utility of many of these cytokines (Dusheiko, G. (1997) Hepatology 26: 112S-121S; Vial, T and Descotes, J. (1994) Drug Experience 10: 115150; Funke, I. et al. (1994) Ann. Hematol. 68: 49-52; Schomburg, A. et al. (1993) J. Cancer Res. Clin. Oncol. 119:745-755). A wide variety of serious side effects associated with interferon administration include flu-like symptoms, fatigue, neurological disorders including hallucinations, fever, elevated liver enzymes and leukopenia (Pontzer, C.H. et al. (1991) Cancer Research, 51:5304 ; Oldham, 1985, op. cit.).

The presence of substantial natural sequence diversity in interferon-α (and thus large sequence space for recombinants), together with the complexity of the interferon-α/receptor interaction, and its multiple therapeutic and prophylactic activities provide a basis for constructing advanced interferon Homologs create opportunities.

Brief description of the invention

The present invention provides a new interferon-α (IFN-α) homologue polypeptide, nucleic acid encoding the polypeptide, and its complementary nucleotide sequence, fragments of the polypeptide and nucleic acid, anti-polypeptide antibodies, and uses thereof , Multiple sets of data containing sequence signatures of interferon-alpha homologues and an automated system for using said signatures.

In one aspect, the invention encompasses isolated or recombinant interferon-alpha nucleic acid homologues. Including polynucleotide sequences selected from SEQ ID NO: 1 to SEQ ID NO: 35, or to SEQ ID NO: 72 to SEQ ID NO: 78 and complementary polynucleotide sequences thereof. Polynucleotide sequences encoding polypeptides selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 81 or SEQ ID NO: 79 to SEQ ID NO: 85 and their complementary polynucleotide sequences are also a feature of the invention. Similarly, polynucleotide sequences that hybridize under highly stringent conditions to substantially the full length of any of the polynucleotide sequences described above are also a feature of the invention. In addition, a polynucleotide sequence comprising a nucleotide fragment of any of the polynucleotide sequences described above, wherein said nucleotide fragment encodes a polypeptide that proliferates in cells based on the human Daudi cell line, is also a feature of the present invention. Antiproliferative activity in the test. Similarly, a polynucleotide sequence comprising a nucleotide fragment of any of the above and following polynucleotide sequences of the present invention is also a feature of the present invention, wherein said nucleotide fragment encodes a polypeptide based on Antiviral activity in murine cell line/EMCV assays.

The present invention also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising the following amino acid sequence: CDLPQTHSLG-X ₁₁ -X ₁₂ -RA-X ₁₅ -X ₁₆ -LL-X ₁₉ - QM-X ₂₂ -RX ₂₄ -SX ₂₆ -FSCLKDR-X ₃₄ -DFG-X ₃₈ -PX ₄₀ -EEFD-X ₄₅ -X 46 -X _{47 -FQ} -X ₅₀ -X ₅₁ -QAI-X ₅₅ -X ₅₆ -X ₅₇ -HE-X ₆₀ -X ₆₁ -QTFN-X ₆₇ -FSTK-X ₇₂ -SS-X ₇₅ -X ₇₆ -WX ₇₈ -X ₇₉ -X ₈₀ -LL-X ₈₃ -KX ₈₅ -X ₈₆ - TX ₈₈ -LX ₉₀ -QQLN-X ₉₅ -LEACV-X ₁₀₁ -QX ₁₀₃ -VX ₁₀₅ -X ₁₀₆ -X ₁₀₇ -X ₁₀₈ -TPLMN-X ₁₁₄ -DX ₁₁₆ -ILAV-X ₁₂₁ -KY-X ₁₂₄ -QRITLYL -X ₁₃₂ -EX ₁₃₄ -KYSPC-X ₁₄₀ -WEVVRAEIMRSFSFSTNLQKRLRRKE, or conservatively substituted variants thereof, wherein X ₁₁ is N or D; X ₁₂ is R, S or K; X ₁₅ is L or M; X ₁₆ is I , M or V; X ₁₉ is A or G; X ₂₂ is G or R; X ₂₄ is I or T; X ₂₆ is P or H; X ₃₄ is H, Y or Q; X ₃₈ is F or L; X ₄₀ is Q or R; X ₄₅ is G or S; X ₄₆ is N or H; X ₄₇ is Q or R; X ₅₀ is K or R; X ₅₁ is A or T; X ₅₅ is S or F; X ₅₆ X ₅₇ is L or F; X ₆₀ is M or I; X ₆₁ is I or M; X ₆₇ is L or F; X ₇₂ is D or N; X ₇₅ is A or V; X ₇₆ is A or T; X ₇₈ is E or D; X ₇₉ is Q or E; X ₈₀ is S, R, T or N; X ₈₃ is E or D; X ₈₅ is F or L; X ₈₆ is S or Y; X ₈₈ is E or G; X ₉₀ is Y, H, N; X ₉₅ is D, E or N; X ₁₀₁ is I, M or V; X ₁₀₃ is E or G; X ₁₀₅ is G or W; X ₁₀₆ is V or M; X ₁₀₇ is E, G or K; X ₁₀₈ is E or G; X ₁₁₄ is V, E or G; X ₁₁₆ is S or P; X ₁₂₁ is K or R; X ₁₂₄ is F or L ; X ₁₃₂ is T, I or M; X ₁₃₄ is K or R; and X ₁₄₀ is A or S. According to the general knowledge of those skilled in the art, each single letter in the amino acid sequence represents a specific amino acid residue.

The present invention also relates to a polypeptide having any of the above sequences, such as SEQ ID NO: 36 to SEQ ID NO: 54.

In other embodiments, the encoded polypeptide comprises an amino acid sequence selected from SEQ ID NO: 36 to SEQ ID NO: 54; the nucleic acid comprises a polynucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 19.

The present invention also provides a polypeptide fragment of any one of SEQ ID NO: 36-70 and SEQ ID NO: 72-79. In one aspect of the present invention, said polypeptide fragment exhibits antiproliferative activity in a cell proliferation assay based on human Daudi cell line, or exhibits antiviral activity in an assay based on murine cell line/EMCV, or both. Two kinds of activity. The cell proliferation assay based on the human Daudi cell line and the antiviral activity in the assay based on the murine cell line/EMCV will be described in detail below. In another aspect, the present invention provides a polynucleotide sequence comprising a nucleotide segment of any one of the nucleic acids of the invention described above and below, wherein said nucleotide segment encodes a polypeptide segment, as described in detail below, said Said polypeptide fragments exhibit antiproliferative activity in a cell proliferation assay based on the human Daudi cell line, or antiviral activity in an assay based on a murine cell line/EMCV, or both.

The invention also includes isolated or recombinant nucleic acids comprising a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence comprising at least 20 contiguous amino acids of any one of SEQ ID NO: 36-70. In other embodiments, a polypeptide of the invention comprises an amino acid sequence comprising one or more of the following amino acid residues: (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166 , wherein the numbering of the amino acid residues corresponds to the numbering of the residues in the amino acid sequence of SEQ ID NO:36. In various embodiments, the encoded polypeptide of the present invention comprises at least 30, at least 50, at least 70, at least 75, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 155, at least 160, or at least 165 contiguous amino acid residues. In other embodiments, the encoded polypeptide is at least 150, at least 155, at least 160, at least 163, or at least 165 amino acids in length. In another embodiment, the encoded polypeptide is about 166 amino acids in length. In other embodiments, the encoded polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 42, SEQ ID NO: Amino acid sequences of ID NO: 45 and SEQ ID NO: 46.

In other embodiments, the invention provides a nucleic acid comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 , SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

In other embodiments, the polypeptide encoded by any nucleic acid of the invention described herein, or a fragment thereof, has antiproliferative activity in an assay based on the human Daudi cell line, or has antiviral activity in an assay based on human WISH cells/EMCV active. In other embodiments, the encoded polypeptide has at least about 8.3 x ¹⁰⁶ units/mg (1 unit is the amount in milligrams (mg) required to induce 50% antiproliferative activity) in an assay based on the human Daudi cell line. Antiproliferative activity in the amount of protein expressed in 100 Å), or at least about 2.1 x ¹⁰⁷ units/mg in a human WISH cell/EMCV-based assay (1 unit is the amount in mg required to induce 50% of the antiviral activity protein amount) antiviral activity. In other embodiments, the encoded polypeptide is capable of binding a type I interferon receptor, preferably a human type I interferon receptor, more preferably a human (eg, type I) interferon-alpha receptor.

The invention also includes cells comprising any nucleic acid of the invention described herein, or expressing any polypeptide of the invention described herein. In one embodiment, the cell expresses a polypeptide encoded by a nucleic acid of the invention described herein.

The present invention also includes vectors comprising any of the nucleic acids of the above and following inventions. The vectors include plasmids, cosmids, phages, or viruses; the vectors can be, for example, expression vectors, cloning vectors, packaging vectors, integration vectors and the like. The present invention also includes cells transduced with the vectors of the present invention. The invention also includes compositions comprising any of the nucleic acids of the above and below inventions and an excipient (preferably a pharmaceutically acceptable excipient). The present invention also includes cells and transgenic animals produced by, for example, transduction vectors, comprising any of the polypeptides or nucleic acids of the above and below-described inventions.

The present invention also includes compositions produced by digesting one or more nucleic acids in the above or following inventions with restriction endonucleases, RNases or DNases; A composition produced by incubating one or more nucleic acids of the above or following invention in the presence of a polymerase, such as a thermostable polymerase.

The present invention also includes compositions comprising two or more nucleic acids as described above or as described below. The composition can include a library of nucleic acids, wherein the library contains at least about 5, 10, 20 or 50 nucleic acids.

In another aspect, the invention includes isolated or recombinant polypeptides encoded by any of the nucleic acids described above or described below. In one embodiment, the polypeptide may comprise a sequence selected from SEQ ID NO: 36 to SEQ ID NO: 70, or SEQ ID NO: 79 to SEQ ID NO: 85.

The invention also includes polypeptides comprising at least 50 contiguous amino acids of a protein encoded by a polynucleotide sequence selected from: (a) SEQ ID NO: 1 to SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85; and (c) The complement of a polynucleotide sequence that hybridizes to substantially the full-length polynucleotide sequence (a) or (b) under highly stringent conditions. In various embodiments, the polypeptide comprises at least about 70, 100, 120, 130, 140, 150, 155, 160, 165, or 166 contiguous amino acids of the encoded protein.

The present invention also includes isolated or recombinant polypeptides comprising an amino acid sequence comprising at least 50 contiguous amino acid residues of any one of SEQ ID NO: 36-70, and one or more of the following amino acids: Ala19, ( Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein the numbering of amino acids corresponds to the numbering in SEQ ID NO:36. In various embodiments, the polypeptide comprises at least about 50, 70, 75, 100, 110, 120, 130, 140, 150, 155, 160, 163, 165 or 166 of any one of SEQ ID NO: 36-70 consecutive amino acids. In a more preferred embodiment, the polypeptide comprises SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45 Or at least about 50, 70, 75, 100, 110, 120, 130, 140, 150, 155, 160, 163, 165 or 166 contiguous amino acid residues of any one of SEQ ID NO:46. In other embodiments, the polypeptides of the invention are at least about 50, 70, 75, 100, 110, 120, 130, 140, 150, 155, 160, 163, 165, or 166 amino acid residues in length, or preferably It is 166 amino acids. Longer polypeptides, such as those containing purification tags and the like, can also be used. The polypeptide may exhibit antiproliferative activity in an assay based on the human Daudi cell line, and/or antiviral activity in an assay based on human WISH cells/EMCV.

The present invention also includes a polypeptide that specifically binds to a polyclonal antiserum against at least one antigen comprising a protein selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: The amino acid sequence shown in 85 or the polypeptide sequence of its fragment. In particular, the invention provides polypeptides that bind to polyclonal antiserum raised against at least one antigen, wherein said at least one antigen comprises SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 70 At least one amino acid sequence shown in ID NO: 85, or a fragment of any of the above amino acid sequences, wherein polyclonal antiserum has been subtracted with one or more known interferon-alpha polypeptides or proteins, Said polypeptides or proteins include, for example, polypeptides or proteins encoded by nucleic acids having or corresponding to one or more of the following GenBank ^™ accession numbers and other similar or homologous interferon-alpha nucleic acid sequences described in GenBank, said GenBank ^TM accession numbers are: J00210(α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545( IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α -10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1).

Any of the above or below polypeptides optionally has antiproliferative activity in an assay based on the human Daudi cell line, and/or has antiviral activity in an assay based on human WISH cells/EMCV. Any of the above or following polypeptides has an antiproliferative activity of at least about 8.3 x ¹⁰⁶ units/mg in an assay based on the human Daudi cell line, or at least about 2.1 x 106 units/mg in an assay based on human WISH cells/EMCV Antiviral activity of ⁷ units/mg. In other embodiments, any polypeptide described above or below is capable of binding a type I interferon receptor, preferably a human type I interferon receptor, more preferably a human interferon-alpha receptor.

In other embodiments, any of the above or below polypeptides may further include secretion/localization sequences, such as signal sequences, organelle targeting sequences, membrane localization sequences and the like. Any of the polypeptides described herein may further include sequences to facilitate purification, such as epitope tags (eg, FLAG epitopes), polyhistidine tags, GST fusions, and the like. Optionally, the N-terminus of the polypeptide may contain a methionine. Any polypeptide in the invention described herein optionally includes one or more modified amino acids, such as glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, carboxylated amino acids, Phosphorylated amino acids, acylated amino acids, etc.

The invention also includes compositions comprising any of the polypeptides described herein together with an excipient, preferably a pharmaceutically acceptable excipient.

The present invention also includes antibodies or antisera produced by administering to a mammal one or more polypeptides of the invention described herein, wherein the antibodies or antisera do not specifically bind to known alpha-interferon polypeptides or proteins , said polypeptide or protein includes, for example, any polypeptide or protein encoded by nucleic acids having or corresponding to one or more of the following GenBank ^™ accession numbers and other similar or homologous interferon-alpha sequences described in GenBank, The above GenBank ^TM accession numbers are: J00210(α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545 (IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961( α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1).

The present invention also includes antibodies capable of specifically binding to a polypeptide comprising a sequence selected from SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85. The antibodies are, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, Fab fragments, fragments produced by a Fab expression library, and the like.

The invention also includes methods of producing the polypeptides of the invention. One method comprises introducing into a population of cells any of the nucleic acids described herein operably linked to regulatory sequences effective for production of the encoded polypeptide, culturing the cells in culture medium to produce the polypeptide, and optionally extracting the polypeptide from the cells or culture medium isolate peptides. A nucleic acid can be part of a vector, such as a recombinant expression vector.

The invention also includes a method of inhibiting the growth of a tumor cell comprising contacting the tumor cell with a polypeptide of the invention described herein, thereby inhibiting the growth of the tumor cell. In one embodiment, the invention includes a method of inhibiting the growth of a tumor cell population comprising: contacting a tumor cell population with an effective amount of a polypeptide of the invention sufficient to inhibit the growth of tumor cells in said tumor cell population, thereby inhibiting Tumor cell growth in the cell population. In various embodiments, the tumor cells can be human cancer cells, human leukemia cells, human T-lymphoma cells, human melanoma cells, other human cancer cells described herein, and the like. Tumor cells can be in vivo, ex vivo or in vitro (eg, cultured cells).

The present invention also includes a method of inhibiting viral replication in one or more cells infected by a virus, said method comprising: contacting one or more infected cells with an effective amount of the polypeptides of the above and below inventions, whereby Inhibiting viral replication in the one or more cells, wherein the amount is sufficient to inhibit viral replication in the one or more infected cells. In various embodiments, the virus can be an RNA virus, such as human immunodeficiency virus or hepatitis C virus, or a DNA virus, such as hepatitis B virus. The infected cells can be in vivo, ex vivo or in vitro (eg, cultured cells).

The present invention also includes a method of treating an autoimmune disease in a subject in need thereof, the method comprising: administering to the subject an effective amount of a polypeptide of the invention described herein sufficient to treat the autoimmune disease. In various embodiments, the autoimmune disease can be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, and the like. The invention also includes an improvement in the method of treating a disease treatable by administering interferon-alpha to a subject, the improvement comprising: administering to the subject an effective amount of the invention described herein, sufficient to treat the disease of polypeptides. Diseases that can be treated by administering interferon-α to a subject may be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, AIDS or AIDS-related syndromes, and the like.

In general, the invention also includes nucleic acids and proteins obtained by mutating the sequences herein. Similarly, the invention also includes those nucleic acids and proteins produced by diversity generation or repeat sequence recombination (RSR) methods such as DNA shuffling. The invention also includes mutagenesis and recombination methods using the nucleic acids described herein. For example, one method of the invention involves iteratively recombining one or more nucleic acid sequences of the above and below inventions with one or more other nucleic acids (including but not limited to those described herein), one or more Each of the other nucleic acid sequences encodes an interferon-alpha homologue or an amino acid subsequence thereof. The recombination step is optionally performed in vivo, ex vivo, in silico or in vitro. The repeated recombination can generate at least one nucleic acid library of recombinant interferon-alpha homologues. The present invention also includes recombinant interferon-alpha homolog nucleic acid produced by this method, cells containing said recombinant interferon-alpha homolog nucleic acid, nucleic acid library produced by this repeated recombination method, containing two or more of said Compositions of recombinant interferon-alpha nucleic acids, and cell populations containing said recombinant interferon-alpha nucleic acids or containing said libraries. In one embodiment, said library contains at least 10 of said recombinant nucleic acids.

The invention also provides a method of producing a modified or recombinant interferon-alpha homolog nucleic acid comprising mutating the nucleic acid of the invention described herein.

The present invention also provides nucleic acid encoding interferon-alpha homologs, respectively relative to human interferon-alpha 2a or other known interferon-alpha growth inhibitory activity on cell populations, cytostatic activity or cytotoxic activity In other words, the homologue has increased growth inhibitory, cytostatic or cytotoxic activity on a cell population (eg, cancer cells).

These and other objects and features of the present invention will become more apparent when taken in conjunction with the accompanying drawings and the following detailed description.

Brief description of the drawings

Figures 1A-1E show the sequence alignment of the exemplary mature interferon homologue polypeptide sequences (SEQ ID NO: 36-70 and 79-85) of the present invention.

Figure 2 shows: relative to two control compounds, human interferon alpha-2a ("IFN-alpha-2a" or "2a") and consensus human interferon ("IFN-Conl" or "Con1") In terms of respective antiproliferative activity and antiviral activity, the antiproliferative activity of the interferon homologs exemplified by the present invention in the test based on human Daudi cell line, and the antiviral activity in the test based on human WISH cells/EMCV .

Figure 3A, 3B and 3C illustrate IFN-alpha homologue 3DA11 (SEQ ID NO:40) and control interferon, human interferon alpha-2a ("2a") and consensus human interferon alpha ("Con1") pair one Activity profiles of a series of tumor cell lines. Figure 3A shows the total cellular growth inhibitory activity of the IFN-α homologue 3DA11 and each control IFN on individual cell lines, as reflected in the GI50 value required to inhibit the growth of a particular cell line by 50% Concentration (μg/ml) of interferon alpha homolog or control IFN alpha, determined by a 50% reduction in net protein/polypeptide increase in the interferon alpha homolog or control IFN alpha experimental group at the end of the incubation period .

Figure 3B shows the quiescent activity of the IFN-[alpha] homologue 3DA11 and each control IFN against each of a series of cell lines. Quiescent activity refers to activity that can inhibit cell growth and reproduction. Static activity is assessed as the complete inhibition of growth and/or proliferation of cells of a particular cell line such that the amount of cellular protein at the end of the incubation period is equal to the amount of cellular protein at the beginning of the incubation period ("total growth inhibition" or "TGI") Reflection of desired interferon alpha homologue or control IFN alpha concentration ([mu]g/ml).

Figure 3C illustrates the cytotoxic activity of the IFN-[alpha] homologue 3DA11 and each control IFN on the respective cell lines. The cytotoxicity of an agent (such as an IFN homologue or IFN compound) is the degree to which the agent has a specific damaging effect on certain cells, or refers to having said damaging effect. The term generally refers to agents that cause cell death, and is used in particular to refer to immunological phenomena that result in the lysis of cells, and to compounds that selectively kill dividing cells. In Figure 3C, cytotoxicity is illustrated in terms of LC50, looking at the amount of protein required to reduce the net protein increase in control cells (control IFNα) by 50% at the end of the incubation period compared to the net protein amount of the cells at the beginning of the incubation period. The concentration ([mu]g/ml) of the IFN-[alpha] homologue 3DA11 represents the net loss of cells after addition of the specific interferon. Cytotoxicity can be assessed as the concentration of IFN-[alpha] homologue 3DA11 required to destroy or kill 50% of the total number of cells (ie, the total population) of a particular cell line relative to control cells.

Figures 4A, 4B, 4C, and 4D show, respectively, the effects on leukemia cell lines ( Cell quiescence of RPMI-8226) (Figure 4A), lung cancer cell line (NCI-H23) (Figure 4B), kidney cancer cell line (ACHN) (Figure 4C) and ovarian cancer cell line (OVCAR-3) (Figure 4D) In terms of activity, the corresponding cytostatic activity of selected interferon-alpha homologues of the invention. Cell quiescence activity is determined by the TGI value of a specific interferon-alpha (the concentration of interferon-alpha required for global inhibition of cell growth in a cell line where the amount of cellular protein at the end of the incubation period is equal to the amount of cellular protein at the beginning of the incubation period) to reflect.

Figure 5 shows the dosage of two exemplified interferon-α homologues of the present invention (referred to as "IFN-CH2.2" and "IFN-CH2.3") at doses of 2 μg, 10 μg and 50 μg, respectively. Comparison of the number of surviving mice (total number of mice: 6) after doses of 2 μg, 10 μg and 50 μg of murine IFN-α-4 and doses of 2 μg, 10 μg and 50 μg of human IFN-α-2a. The results shown in Figure 5 demonstrate that the improved in vitro antiviral activity of the two exemplified IFN-[alpha] homologues can be maintained and extended in vivo in a murine model system. Phosphate-buffered saline (PBS) was used as a control.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

Unless otherwise defined by context in the rest of the specification, 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.

Depending on the context, a "polynucleotide sequence" is a nucleic acid (a polymer of nucleotides (A, C, T, U, G, etc., or natural or artificial nucleotide analogs)) or expresses a characteristic strand of nucleic acid. A given nucleic acid or complementary nucleic acid can be identified from any particular polynucleotide sequence.

Similarly, an "amino acid sequence" is, depending on the context, a polymer of amino acids (protein, polypeptide, etc.), or a characteristic chain expressing a polymer of amino acids. A given nucleic acid or complementary nucleic acid can be identified from any particular polynucleotide sequence.

When a nucleic acid, protein, peptide, polypeptide or other component is associated with it in nature (other peptides, polypeptides, proteins (including complexes such as polymerases and ribosomes associated with the native sequence), nucleic acids, cells, synthetic reagents, cellular impurities, cellular components, etc.) are considered "isolated" when they are partially or completely separated from other components with which they are normally associated, for example, in the cell of origin. When nucleic acids, polypeptides or other components are partially or completely separated from or recovered from other components in their natural environment such that they are present as the predominant species in a composition, mixture or collection of components (i.e. They are also said to be isolated when they are present in greater abundance than any other individual species in the composition on a molar basis). In preferred embodiments, the preparation consists of more than 70%, generally more than 80%, or preferably more than 90% of the isolated species.

In one aspect, "substantially pure" or "isolated" nucleic acid (e.g., RNA or DNA), polypeptide, protein or component also refers to the species of interest (e.g., nucleic acid or polypeptide) at least about by weight of all macromolecular species present 50, 60 or 70% (on a molar basis). Substantially pure or isolated components can also comprise at least about 80, 90 or 95% by weight of all macromolecular species present in the composition. The isolated species of interest may also be purified to substantially homogeneity (impurity species in the fraction not detectable by conventional detection methods), wherein the fraction consists essentially of derivatives of a single macromolecular species.

The term "isolated nucleic acid" may refer to a nucleic acid (such as DNA or RNA). Thus, the term includes fragments of cDNA or genomic DNA produced, for example, by polymerase chain reaction (PCR) or restriction endonuclease treatment, whether incorporated into a vector, integrated into its source organism A hybrid gene encoding a chimeric polypeptide may also be joined to other coding sequences in the genome of the same or a different species (including, for example, a virus), or be independent of any other DNA sequence. DNA can be double-stranded or single-stranded, sense or antisense.

Nucleic acids or polypeptides are said to be "recombinant" when they are artificial or genetically engineered, or are derived from artificial or engineered proteins or nucleic acids. The term "recombinant" when used in reference to e.g. cells, nucleotides, vectors or polypeptides generally means that said cells, nucleotides or vectors have been modified by introducing heterologous (or exogenous) nucleic acids or by altering native nucleic acids, Either means that the polypeptide has been modified by the introduction of heterologous amino acids, or that the cells are derived from cells so modified. Recombinant cells express nucleic acid sequences (eg, genes) that are not found in the native (non-recombinant) form of the cell, or express native nucleic acid sequences (eg, genes) that may be expressed abnormally, in small amounts, or not at all. The term "recombinant nucleic acid" (eg, DNA or RNA) molecule refers to, for example, a non-native nucleotide sequence, or through the participation of at least two sequence segments that generally do not co-occur, are not related to each other, or are otherwise separate from each other (e.g. artificially assembled) nucleotide sequences. A recombinant nucleic acid may comprise a nucleic acid molecule formed by joining or combining nucleic acid segments from different sources and/or artificially synthesized. The term "recombinantly produced" refers to an artificial combination, usually achieved by means of chemical synthesis, repeated sequence recombination of nucleic acid segments or other methods of generating nucleotide diversity (such as shuffling), or The isolated nucleic acid segments are manipulated, for example, by genetic engineering techniques known to those skilled in the art. "Recombinant expression" generally refers to the technique of producing recombinant nucleic acid in vitro, transferring the recombinant nucleic acid to cells in vivo, in vitro or from the body, and expressing or propagating the recombinant nucleic acid therein. A "recombinant polypeptide" or "recombinant protein" generally refers to a polypeptide or protein, respectively, produced from a cloned or recombinant gene or nucleic acid.

A "subsequence" or "fragment" is any portion of, up to and including the complete sequence.

When the position of any given polymer component (amino acid residue, incorporated nucleotide, etc.) The number for a given amino acid or nucleotide polymer "corresponds to" the number for a selected amino acid polymer or nucleic acid when specified by actual position.

A vector is a component that facilitates the transduction of a selected nucleic acid into a cell, or the expression of a nucleic acid in a cell. Vectors include, for example, plasmids, cosmids, viruses, YACs, bacteria, poly-lysine, and the like. An "expression vector" is a recombinantly or synthetically produced nucleic acid construct having a series of specific nucleic acid elements that permit transcription of a specific nucleic acid in a host cell. An expression vector can be part of a plasmid, virus or nucleic acid fragment. Expression vectors generally include a nucleic acid to be transcribed operably linked to a promoter.

"Essentially full-length polynucleotide or amino acid sequence" means at least about 50%, at least about 60%, usually at least about 70%, usually at least about 80%, or usually at least about 90% of the full length , 95%, 96%, 97%, 98% or 99% or longer amino acid sequence or nucleic acid sequence.

A "human alpha-interferon receptor" is a receptor that is naturally activated in human cells by alpha interferon.

A substance is "natural" in reference to the fact that the substance can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, including a virus, that can be isolated from its natural source and that has not been intentionally modified by man in a laboratory is natural. In one aspect, a "native" nucleic acid (eg, DNA or RNA) molecule is one that exists in the same state as it occurs in nature; ie, the nucleic acid molecule has not been isolated, recombinant or cloned.

As used herein, "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Well-known immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, and various 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 class as IgG, IgM, IgA, IgD, and IgE, respectively. Typical immunoglobulin (eg, antibody) structural units contain tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (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. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. Antibodies can exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody at the disulfide bonds in the hinge region, yielding the dimer F(ab)'2 of Fab, which itself is a light chain linked by a disulfide bond to VH-CH1. F(ab)'2 can be reduced under mild conditions to break the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab with part of the hinge region (for a detailed description of other antibody fragments see "Basic Immunology", edited by W.E. Paul, Raven Press, New York (1993)). Although antibody fragments are available by digestion of intact antibodies, those skilled in the art will appreciate that these Fab' fragments can be synthesized de novo either chemically or using recombinant DNA methodology. Accordingly, the term antibody as used herein also includes antibody fragments which have been produced by modification of intact antibodies or which have been synthesized de novo using recombinant DNA methodology. Antibodies include single-chain antibodies, including single-chain Fv (sFv) antibodies, in which a variable heavy chain and a variable light chain are linked together (directly or through a peptide linker) to form an extended polypeptide.

An "antigen-binding fragment" of an antibody is a peptide or polypeptide fragment of an antibody that binds to an antigen. Those amino acids in an antibody that facilitate, participate in, or affect binding to an antigen form the antigen-binding site. See Scott, T.A and Mercer, E.I., The Concise Encyclopedia: Biochemistry and Molecular Biology (deGruyter, 3rd ed., 1997) [hereinafter abbreviated as "Scott, The Concise Encyclopedia"] and Watson, J.D et al., Recombinant DNA (p. 2nd Edition, 1992) [hereinafter "Watson, Recombinant DNA"], each of which is incorporated herein by reference in its entirety.

"Immunogen" refers to a substance capable of eliciting an immune response. Examples of immunogens include, for example, antigens, autoantigens that function in inducing autoimmune diseases, and tumor-associated antigens expressed on cancer cells.

An "antigen" is a substance that elicits antibody formation in a host, or that produces a specific population of lymphocytes reactive with the substance. Antigens are generally large molecules (such as proteins and polysaccharides) that are foreign to the host.

The term "immunoassay" includes detection methods using antibodies or immunogens that bind or specifically bind to an antigen. Immunoassays are typically characterized by utilizing the specific binding properties of specific antibodies to isolate, target, and/or quantify antigens.

The term "homology" generally refers to the degree of similarity between two or more structures. The term "homologous sequences" refers to regions of a macromolecule that have similar monomer sequences. The term "homology" when applied to nucleic acid sequences refers to the degree of similarity between two or more nucleic acid sequences (eg, genes) or fragments thereof. Generally speaking, the degree of similarity between two or more nucleic acid sequences refers to: the composition of two or more nucleotide bases (or other genotype characteristics) in two or more nucleic acid sequences, Degree of similarity in order or arrangement. The term "homologous nucleic acid" generally refers to nucleic acids that contain nucleotide sequences that have a certain degree of similarity in nucleotide base composition, arrangement or order. The two or more nucleic acids may be of the same or different species or groups. The term "percent homology" when applied to nucleic acid sequences generally refers to the percent degree of similarity between the nucleotide sequences of two or more nucleic acids.

When applied to polypeptide (or protein) sequences, the term "homology" refers to the degree of similarity between two or more polypeptide (or protein) sequences (eg, genes) or fragments thereof. Generally speaking, the degree of similarity between two or more polypeptide (or protein) sequences refers to: the composition, sequence or arrangement of two or more amino acids in two or more polypeptides (or proteins) degree of similarity. The two or more polypeptides (or proteins) may be of the same or different species or groups. The term "percent homology" when applied to polypeptide (or protein) sequences generally refers to the percentage degree of similarity between the amino acid sequences of two or more polypeptide (or protein) sequences. The terms "homologous polypeptide" or "homologous protein" generally refer to polypeptides or proteins, respectively, having similar amino acid sequences and functions. Such homologous polypeptides or proteins are related in that they have similar amino acid sequence and function, but they can be derived or obtained from different or the same species using the techniques described herein.

The term "subject" as used herein includes, but is not limited to, living things; mammals, including, for example, humans, non-human primates (such as monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, Horses, monkeys, sheep or other non-human mammals; non-mammals including, for example, non-mammalian vertebrates such as birds (eg, chickens or ducks) or fish; and non-mammalian invertebrates.

The term "pharmaceutical composition" refers to a composition suitable for use as a medicine to a subject, including an animal or a human. Pharmaceutical compositions generally comprise an effective amount of an active agent and a pharmaceutically acceptable carrier.

The term "effective amount" refers to a dosage or amount sufficient to produce the desired effect. Desired effects include objective or subjective improvement in a subject at said dose or amount.

"Prophylactic treatment" is the administration of treatment to a subject who does not show signs or symptoms of disease, or only shows early signs or symptoms of disease, such that the administered treatment attenuates, prevents or reduces the risk of developing disease. The role of prophylactic therapy is to treat disease preventively. "Prophylactic activity" refers to the ability of agents such as nucleic acids, vectors, genes, polypeptides, proteins, substances, and compositions thereof to An activity that attenuates, prevents, or reduces a subject's risk of developing a disease. A "prophylactically useful" agent or compound (such as a nucleic acid or polypeptide) refers to an agent or compound that is useful for attenuating, preventing, treating or reducing the occurrence of a disease.

"Therapeutic treatment" is treatment administered to a subject exhibiting symptoms or signs of disease, where the purpose of administering the treatment to the subject is to attenuate or eliminate the signs or symptoms of disease. "Therapeutic activity" refers to the activity of an agent such as a nucleic acid, vector, gene, polypeptide, protein, substance, or a composition thereof that, after being administered to a subject showing signs or symptoms of a disease, can eliminate or weaken these signs or symptoms. A "therapeutically useful" agent or compound (such as a nucleic acid or polypeptide) refers to an agent or compound that is useful for attenuating, treating or eliminating signs or symptoms of a disease.

The term "gene" broadly refers to any segment of DNA associated with a biological function. A gene includes a coding sequence and/or the regulatory sequences required for its expression. Genes also include non-expressed DNA nucleic acid segments, such as those that form recognition sequences for other proteins.

In general, the nomenclature used hereinafter and the laboratory methods of cell culture, molecular genetics, molecular biology, nucleic acid chemistry and protein chemistry described below are those well known and commonly used by those skilled in the art. Such as Sambrook et al., Molecular Cloning - Laboratory Manual (Second Edition), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (hereinafter referred to as "Sambrook") and the latest molecular biology methods, F.M.Ausubel et al., Current Methods, Greene Publishing Associates, Inc and John Wiley & Sons, a joint venture (supplemented 1999) (hereinafter referred to as "Ausubel") can be used for recombinant nucleic acid methods, nucleic acid synthesis, cell culture methods and Transgene incorporation such as electroporation, injection and lipofection. In general, oligonucleotide synthesis and purification steps are performed according to the instructions. The techniques and methods are generally performed according to conventional methods in the art and various general references provided herein. The methods therein are believed to be well known to those skilled in the art and are provided for the convenience of the reader.

Various other terms are defined or characterized herein. Polynucleotide interferon-alpha homologue sequence of the present invention

The present invention provides isolated or recombinant interferon-alpha homologue polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides.

As described in detail below, according to the present invention, the polynucleotide sequence encoding the new interferon-alpha homologue polypeptide, the nucleotide sequence (such as subsequence) of the encoding interferon-alpha homologue polypeptide fragment and the encoding related The nucleotide sequences of fusion polypeptides or proteins or their functional equivalents are collectively referred to herein as "interferon-alpha homologues", "interferon homologue nucleic acids", "IFN-alpha homologues", "IFN homologs ", "IFN nucleic acid", "interferon homologue", "interferon nucleic acid", "recombinant interferon-α", "recombinant interferon-α nucleic acid", "nucleic acid of the present invention", "polynucleoside of the present invention acid" or "nucleotide of the invention". The polynucleotides, nucleotides and nucleic acids of the present invention also include and include the polynucleotides, nucleotides and nucleic acid fragments of each of the above terms. The term "nucleic acid" and the term "nucleotide" are used interchangeably.

Polynucleotides encoding polypeptides of the invention are found in libraries of shuffled interferon-alpha related sequences. Library members were screened for antiproliferative activity against human tumor cell lines and in some cases analyzed for antiviral activity against virus-infected human cells. A subset of the sequences provided herein were found in shuffled libraries screened for antiviral activity against virus-infected mouse cells. Coding sequences for interferon homologues were identified as described in the Examples.

Briefly, a library of shuffled mature interferon-alpha coding sequences was introduced into E. coli. As described in Example 1, colonies were screened in a high-throughput antiproliferative activity test against the human Daudi tumor cell line, colonies expressing active polypeptides were selected, re-screened, and expression levels determined. DNA from selected colonies was isolated, re-shuffled to generate a second generation library, which was introduced into E. coli and screened for antiproliferative activity in a cell proliferation assay based on the human Daudi cell line. DNA from colonies selected from the first and second generation library screens was transduced into Chinese hamster ovary (CHO) cells, resulting in stable cell lines. The protein expressed by CHO was purified, quantified and assayed for antiproliferative activity using the human Daudi cell line and optionally human WISH cells infected with encephalomyocarditis virus (EMCV) as described in Example 1. Examples of shuffled nucleic acids encoding interferon-alpha homologue polypeptides identified herein as SEQ ID NO: 1 to SEQ ID NO: 35 have anti-proliferative activity in assays based on the human Daudi cell line, so The nucleic acids encode mature interferon-alpha homologue polypeptides identified herein as SEQ ID NO: 36 to SEQ ID NO: 70, respectively. Additionally, a library of shuffled mature interferon-alpha coding sequences was screened in a high-throughput antiviral activity screening assay against EMCV-infected mouse cells. Examples of shuffled nucleic acids encoding polypeptides having antiviral activity in a murine cell/EMCV-based assay are identified herein as SEQ ID NO: 72 to SEQ ID NO: 78, the nucleic acid encoding is identified herein as SEQ ID NO: 72 Mature interferon homologue polypeptides of ID NO: 79 to SEQ ID NO: 85.

In another aspect, the present invention provides an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) SEQ ID NO: 1 to SEQ ID NO: 35, or a complementary polynucleotide sequence thereof; (b ) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO: 36 to SEQ ID NO: 71, or a complementary polynucleotide sequence thereof; (c) under at least stringent or at least highly stringent hybridization conditions (or super-highly stringent or ultra-ultra-highly stringent hybridization conditions), with substantially full-length polynucleotide sequence (a) or (b), or with 50, 120, 130 of polynucleotide sequence (a) or (b), A subsequence or fragment of 140, 145, 150, 155, 160 or 165 nucleotide bases hybridizes to a polynucleotide sequence; and (d) a polynucleotide comprising a fragment of (a), (b) or (c) acid sequences, said fragments encode complete or partial polypeptides having antiproliferative activity in assays based on the human Daudi cell line, or antiviral activity in assays known in the art for determining antiviral activity.

In another aspect, the present invention provides an isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from: (a) SEQ ID NO: 72 to SEQ ID NO: 78, or a complementary polynucleotide sequence thereof; (b ) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO: 79 to SEQ ID NO: 85, or a complementary polynucleotide sequence thereof; (c) under at least stringent or at least highly stringent hybridization conditions (or super-highly stringent or ultra-ultra-highly stringent hybridization conditions), with substantially full-length polynucleotide sequence (a) or (b), or with 50, 120, 130 of polynucleotide sequence (a) or (b), A subsequence or fragment of 140, 145, 150, 155, 160 or 165 nucleotide bases hybridizes to a polynucleotide sequence; and (d) a polynucleotide comprising a fragment of (a), (b) or (c) acid sequences, said fragments encode complete or partial polypeptides that have antiproliferative activity in assays based on the human Daudi cell line, or antiviral activity in assays based on the murine cell line/EMCV.

The invention also includes a mature interferon-alpha homologue polypeptide comprising the amino acid identified herein as SEQ ID NO: 71, the invention also includes a polynucleotide sequence encoding said polypeptide or a fragment of said polypeptide, The polypeptide fragments have antiproliferative activity in assays based on human Daudi cell line, and/or have antiviral activity in assays based on murine cells/EMCV.

The invention also includes isolated or recombinant nucleic acids comprising a polynucleotide sequence encoding a polypeptide comprising the following amino acid sequence: CDLPQTHSLG-X ₁₁ -X ₁₂ -RA-X ₁₅ -X ₁₆ -LL-X ₁₉ -QM- X ₂₂ -RX ₂₄ -SX ₂₆ -FSCLKDR-X ₃₄ -DFG-X ₃₈ -PX ₄₀ -EEFD-X ₄₅ -X ₄₆ -X ₄₇ -FQ-X ₅₀ -X ₅₁ -QAI-X ₅₅ -X ₅₆ -X ₅₇ -HE-X ₆₀ -X ₆₁ -QTFN-X ₆₇ -FSTK-X ₇₂ -SS-X ₇₅ -X ₇₆ -WX ₇₈ -X 79 -X ₈₀ -LL-X _{83 -KX 85 -X 86} -TX ₈₈ -LX ₉₀ -QQLN-X ₉₅ -LEACV-X ₁₀₁ -QX ₁₀₃ -VX ₁₀₅ -X ₁₀₆ -X ₁₀₇ -X ₁₀₈ -TPLMN-X ₁₁₄ -DX ₁₁₆ -ILAV-X ₁₂₁ -KY-X ₁₂₄ -QRITLYL-X ₁₃₂ -EX ₁₃₄ -KYSPC-X ₁₄₀ -WEVVRAEIMRSFSFSTNLQKRLRRKE, or conservatively substituted variants thereof, wherein X ₁₁ is N or D; X ₁₂ is R, S or K; X ₁₅ is L or M; X ₁₆ is I, M or V; X ₁₉ is A or G; X ₂₂ is G or R; X ₂₄ is I or T; X ₂₆ is P or H; X ₃₄ is H, Y or Q; X ₃₈ is F or L; X ₄₀ is X ₄₅ is G or S; X ₄₆ is N or H; X ₄₇ is Q or R; X ₅₀ is K or R; X ₅₁ is A or T; X ₅₅ is S or F; X ₅₆ is V or A; X ₅₇ is L or F; X ₆₀ is M or I; X ₆₁ is I or M; X ₆₇ is L or F; X ₇₂ is D or N; X ₇₅ is A or V; X ₇₆ is A or T; X ₇₈ is E or D; X ₇₉ is Q or E; X ₈₀ is S, R, T or N; X ₈₃ is E or D; X ₈₅ is F or L; X ₈₆ is S or Y; X ₈₈ is E or G; X ₉₀ is Y, H, N; X ₉₅ is D, E or N; X ₁₀₁ is I, M or V; X ₁₀₃ is E or G; X ₁₀₅ is G or W; X ₁₀₆ is V or M; X ₁₀₇ is E, G or K; X ₁₀₈ is E or G; X ₁₁₄ is V, E or G; X ₁₁₆ is S or P; X ₁₂₁ is K or R; X ₁₂₄ is F or L; X ₁₃₂ is T, I or M; X ₁₃₄ is K or R; and X ₁₄₀ is A or S. According to the general knowledge of those skilled in the art, each single letter in the amino acid sequence represents a specific amino acid residue. The polypeptide has antiproliferative activity (e.g., at least about 8.3×10 ⁶ units/mg) in an assay based on the human Daudi cell line, and/or has antiviral activity (at least About 2.1×10 ⁷ units/mg).

As detailed below, the polynucleotides of the invention are useful in a variety of ways, including but not limited to: as therapeutic and prophylactic agents in methods of treating a variety of diseases in a variety of subjects, both in vivo and ex vivo; For use in in vitro methods such as diagnostic methods to detect, diagnose and treat various diseases in multiple subjects; for use in, for example, gene therapy; for use as therapeutic and prophylactic agents in methods for, for example, therapeutically and prophylactically treating diseases ; use as an immunogen; use in diagnostic and screening assays; use as a diagnostic probe to detect the presence of complementary or partially complementary nucleic acids (including detection of nucleic acids encoding IFN-α). Preparation of polynucleotides of the invention

The polynucleotides and oligonucleotides of the invention can be prepared by standard solid phase methods according to known synthetic methods. Generally, fragments of about 20, 30, 40, 50, 60, 70, 80, 90 and/or 100 nucleotide bases in length are synthesized individually and then (by, for example, enzymatic or chemical ligation, or polymerase mediated recombination methods) to form virtually any desired contiguous sequence. On the other hand, more than 100 nucleotide bases (such as 150, 180, 200, 210, 240, 270, 300, 330, 360, 390, 400, 420, 450, 465, 474, 470, 475, 489, 490, 495, 496 bases), which are then joined (by, for example, enzymatic or chemical ligation, or polymerase-mediated recombination) to form virtually any desired continuous sequence. For example, the classical phosphoramidite method described in Beaucage et al. (1981) Tetrahedron Commun. 22: 1859-69, or the method described in Matthes et al. The methods are generally practiced in automated synthetic methods), and the polynucleotides and oligonucleotides of the invention, including fragments thereof (and those described herein), are prepared by chemical synthesis. According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, followed by purification, annealing, ligation, and cloning into appropriate vectors.

Additionally, virtually any nucleic acid can be ordered from any of a number of vendors, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc .(www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others. Similarly, peptides and antibodies can also be ordered from any of a number of vendors, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc. (http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc. and many others.

Specific polynucleotides of the invention can also be obtained by screening cDNA libraries (e.g., libraries generated by recombination of homologous nucleic acids as in typical diversity generation methods, such as shuffling methods) using oligonucleotide probes, wherein The probes are capable of hybridizing or PCR-amplifying polynucleotides encoding interferon homologue polypeptides and fragments thereof. Methods for screening and isolating cDNA clones are well known to those skilled in the art. This technique is described, for example, in: Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Edition), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (hereinafter referred to as "Sambrook") and Current Molecular Biology Ausubel et al., eds., Current Methods, A joint venture between Greene Publishing Associates, Inc and John Wiley & Sons (added 1999) (hereinafter referred to as "Ausubel").

As detailed herein, the polynucleotides of the invention include sequences encoding novel mature interferon-alpha homologues and sequences complementary to the coding sequences, as well as novel fragments of the coding sequences and complements thereof. A polynucleotide can be in the form of RNA, or in the form of DNA, including mRNA, cRNA, synthetic RNA and DNA, and cDNA. A polynucleotide can be double-stranded or single-stranded, and if single-stranded, coding or non-coding (antisense, complementary) strand. The polynucleotide optionally includes an isolated form of the coding sequence of an interferon-alpha homologue (i), (ii) in combination with other coding sequences to encode, for example, a fusion protein, a pre-protein, a pre-proprotein, etc., (iii) with Non-coding sequences, such as promoters, termination elements, or 5' and/or 3' untranslated regions associated with effective expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment, wherein interference A coding sequence for a prime-alpha homolog is a heterologous nucleic acid sequence or gene. Sequences can also be combined with typical nucleic acid combination preparations, including carriers, buffers, adjuvants, excipients, and the like.

The term DNA or RNA encoding the respective interferon-alpha homologue polypeptide includes any oligodeoxynucleotide or oligodeoxyribonucleotide sequence which, upon expression in a suitable host cell, results in the production of the interferon-alpha homologue of the present invention peptide. DNA or RNA can be produced in a suitable host cell, or in a cell-free (in vitro) system, or can be produced synthetically (by eg amplification techniques such as PCR) or chemically. Utilize the polynucleotide of the present invention

The polynucleotides of the present invention have multiple uses, such as: recombinant production (i.e., expression) of the interferon-alpha homologue polypeptides of the present invention; use as therapeutic and prophylactic agents in methods for, for example, therapeutically and prophylactically treating diseases used in gene therapy methods and related fields of application; used as an immunogen; used in diagnostic and screening assays; used as a diagnostic probe to detect the presence of complementary or partially complementary nucleic acids (including detection of nucleic acids encoding native IFN-α); Use as a substrate for other reactions, such as shuffling reactions or mutagenesis reactions to generate new and/or improved IFN-[alpha] homologues and the like. expression of polypeptide

According to the present invention, polynucleotide sequences encoding novel and/or mature interferon-alpha homologues, fragments of interferon-alpha proteins, related fusion proteins, or functional equivalents thereof are collectively referred to herein as "interferon-alpha -alpha homolog polypeptide", "interferon-alpha homolog protein", or "interferon-alpha homolog", "interferon homolog", "IFN-alpha homolog", "IFN homolog", "IFN Polypeptide", "IFN protein", "polypeptide of the invention" or "protein of the invention". Polypeptides or proteins of the invention are intended to include and comprise polypeptides or amino acid fragments of each of the above terms. The polynucleotide sequences of the present invention are used in those recombinant DNA (or RNA) molecules that mediate the expression of interferon-alpha homologue polypeptides in appropriate host cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences encoding substantially identical or functionally equivalent amino acid sequences may also be used to clone and express interferon homologs. modified coding sequence

Those skilled in the art will understand that it is advantageous to modify the coding sequence (including, for example, the nucleotide sequence encoding the interferon-α homologue or fragment thereof of the present invention) to enhance its expression in a specific host. The genetic code is redundant, with 64 possible codons, but most organisms prefer to use a subset of these codons. The codons that are most frequently used in a species are called optimal codons, and those that are less commonly used are classified as rare or uncommon codons (see, eg, Zhang S.P. et al. (1991) Gene 105:61-72). Codons can be substituted to reflect the preferred codon usage of the host, a process known as "codon optimization" or "control of species codon bias".

An optimized coding sequence can be prepared to contain codons preferred by a particular prokaryotic or eukaryotic host (Murray, E. et al. (1989) Nucleic Acids Res. 17:477-508), thereby allowing the coding sequence to be compared with the non-optimized sequence produced. The rate of translation can be increased, or recombinant RNA transcripts can be produced with desirable properties, such as longer half-lives, compared to other transcripts. Translation stop codons can also be modified to reflect host preference. For example, the preferred stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The preferred stop codon for monocots is UGA, while insects and E. coli prefer UAA as the stop codon (Dalphin M.E. et al. (1996) Nucleic Acids Res. 24:216-218).

The polynucleotide sequences of the present invention may be altered to alter interferon homolog coding sequences for a variety of reasons including, but not limited to: alterations in cloning, processing and/or expression of modified gene products. For example, changes can be introduced using techniques well known in the art, such as site-directed mutagenesis to insert new restriction sites, alter glycosylation patterns, alter codon bias, introduce splice sites, and the like. Vectors, Promoters and Expression Systems

The invention also includes recombinant constructs comprising one or more nucleic acid sequences broadly described herein (eg, nucleic acid sequences encoding interferon-alpha homologues or fragments thereof of the invention). Constructs include vectors such as plasmids, cosmids, phages, viruses (including retroviruses), bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), etc., into which the nucleic acid sequence of the present invention is inserted in forward or reverse direction . In a preferred aspect of this embodiment, the construct also contains regulatory sequences, including, for example, a promoter operably linked to the sequence. A large number of suitable vectors and promoters are known to those skilled in the art and are also commercially available.

Common textbooks describing the molecular biology techniques used herein, including vectors, the use of promoters, and many other related topics include: Juo, P-S., A Concise Dictionary of Biomedicine and Molecular Biology (CRC Press, 1996); Singleton et al., Microbiology Dictionary of Science and Molecular Biology (2nd Edition, 1994); Cambridge Dictionary of Science and Technology (Edited by Walker, 1988); Hale & Marham, HARPERCOLLINS Dictionary of Biology (1991); Scott and Mercer, The Concise Encyclopedia of Biochemistry and Molecular Biology Complete Book (3rd Edition, 1997); Berger and Kimmel, A Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Publishing Company, San Diego, CA (hereinafter referred to as "Berger"); Sambrook et al., Molecular Cloning-Experimental Laboratory Manual (2nd Edition), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Latest Methods in Molecular Biology, edited by F.M. Ausubel et al., Latest Methods, Greene Publishing Associates, Inc Joint Venture with John Wiley & Sons (added in 1999) ("Ausubel"). Sufficient to teach the skilled artisan to perform in vitro amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification and other RNA polymerase-mediated techniques (such as NASBA), to produce Techniques for homologous nucleic acids of the present invention can be found, for example, in Berger, Sambrook and Ausubel, and Mullis et al. (1987), U.S. Patent 4,683,202; Ed.) Academic Publishing Company, San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN36-47; NIH Research Journal (1991) 3, 81-94; KWoh et al., (1989) Proc .Natl Acad.Sci.USA86,1173; Guatelli et al., (1990) Proc.Natl Acad.Sci.USA87,1874; Lomell et al., (1989) Journal of Clinical Chemistry, 35, 1826; Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117 and Sooknanan and Malek (1995) Biotechnology 13: 563 -564.

PCR generally refers to the process of amplifying very small amounts of specific nucleic acid, RNA and/or DNA fragments by methods well known in the art (see, eg, US Patent 4,683,195 and other references mentioned above). Oligonucleotide primers are generally designed using sequence information at the end of the desired region or beyond. The sequences of the primers are identical or similar to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the opposite strand can coincide with the end of the amplified product. PCR can be used to amplify specific RNA or specific DNA sequences, recombinant DNA or RNA sequences, DNA and RNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, and the like. PCR is an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample using another (eg, known) nucleic acid as a primer. Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al., US Patent 5,426,039. An improved method for the amplification of large nucleic acids by PCR is briefly described in Cheng et al., (1994) Nature 369:684-685 and references therein, where PCR amplicons up to 40 kb in length were generated. Those skilled in the art will appreciate that virtually any RNA can be converted into double-stranded DNA suitable for restriction digestion, PCR extension and sequencing using reverse transcriptase and polymerase. See Ausubel, Sambrook and Berger, supra.

The present invention also relates to host cells transduced with the vectors of the present invention, and the production of polypeptides of the present invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (ie, transduced, transformed or transfected) with a vector of the invention, which may be, for example, a cloning vector or an expression vector. Vectors can be in the form of, for example, plasmids, virus particles, phage, and the like. The engineered host cells can be grown in conventional nutrient media, with media components modified as necessary to activate promoters, select for transformants, or amplify interferon homolog genes. Culture conditions such as temperature, pH, etc. are the same as those previously used to select host cells for expression, they will be obvious to those skilled in the art, see also the references mentioned herein, including for example Freshney (1994) animal Cell Culture, A Handbook of Essential Techniques, 3rd Edition, Wiley-Liss, New York and references mentioned therein.

The interferon homologue polypeptides and proteins of the present invention can also be produced in non-animal cells, such as plants, yeast, fungi, bacteria, and the like. In addition to Sambrook, Berger and Ausubel, details on cell culture can be found in Payne et al., (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995 ) Plant Cell, Tissue, and Organ Culture; Basic Methods, Springer Laboratory Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.), Handbook of Microbial Culture Media (1993), CRC Press, Boca Raton, FL.

The polynucleotides of the invention can be contained in any of a variety of expression vectors to express polypeptides. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences such as derivatives of SV40; bacterial plasmids; phage DNA; baculoviruses; yeast plasmids; derived from plasmids and phage DNA, viral DNA such as vaccinia virus, adenovirus, Combination vectors of fowl pox virus, pseudorabies virus, adeno-associated virus, retrovirus and many others. Any vector capable of transducing genetic material into a cell, replicating when required, and surviving in the relevant host can be used.

The nucleic acid sequence in the expression vector is operably linked to an appropriate transcriptional control sequence (promoter) to mediate mRNA synthesis. Examples of such promoters include: LTR or SV40 promoter, E. coli lac or trp promoter, bacteriophage lambda _PL promoter and other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. Expression vectors also contain a ribosome binding site for translation initiation and a transcription terminator. The vector optionally includes appropriate sequences for amplifying expression. In addition, the expression vector optionally contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, Or tetracycline or ampicillin resistance against E. coli.

A suitable host can be transformed with a vector containing the appropriate DNA sequence described herein, together with an appropriate promoter or control sequence, such that the host expresses the protein. Examples of suitable expression hosts include: bacterial cells such as Escherichia coli, Streptomyces and Salmonella typhimurium; fungal cells such as Saccharomyces cerevisiae, Pichia pastoris and Neurospora crassa; insect cells such as Drosophila and meadowfoam moth; mammalian cells, such as CHO, COS, BHK, HEK293 or Bowes melanoma; plant cells, etc. It will be appreciated that not all cells or cell lines need be capable of producing fully functional interferon homologs; for example, antigenic fragments of interferon homologs may be produced in bacteria or other expression systems. The present invention is not limited by the host cells used.

In bacterial systems, a variety of expression vectors can be selected according to the use of the interferon homologue. For example, when large quantities of interferon homologues or fragments thereof are required to induce antibodies, vectors that mediate high-level expression of fusion proteins that are readily purified are required. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), wherein the interferon homologue coding sequence is ligated into the vector, with an amino-terminal Met sequence followed by β-galactoside The 7 residues of the enzyme are located in the same reading frame to generate a hybrid protein; pIN vector (Van Heeke & Schuster (1989) Biochemical Journal 264: 5503-5509); pET vector (Novagen, Madison WI), etc.

Similarly, in the yeast Saccharomyces cerevisiae, a variety of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used to produce the interferon homologue proteins of the present invention. For reviews, see Ausubel et al., (supra) and Grant et al., (1987; Methods in Enzymology 153:516-544).

In mammalian host cells, a variety of expression systems, such as viral-based systems, are available. When an adenovirus is used as the expression vector, the coding sequence is optionally ligated into an adenoviral transcription/translation complex consisting of a late promoter and a tripartite leader sequence. Insertion into non-essential E1 or E3 regions of the viral genome results in the ability of the virus to express interferon homologues in infected host cells (Logan and Shenk (1984) Proc. Natl. Acad. Sci. 81: 3655-3659) . In addition, transcriptional enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to enhance expression in mammalian host cells. other expression elements

Specific initiation signals facilitate efficient translation of interferon homolog coding sequences. These signals include, for example, the ATG initiation codon and adjacent sequences. When inserting the interferon homolog coding sequence, its initiation codon and upstream sequences into an appropriate expression vector, no other translational control signals are required. However, when inserting only a coding sequence (such as that of a mature protein) or a portion thereof, exogenous transcriptional control signals must be provided, including the ATG initiation codon. Additionally, the initiation codon must be in the correct reading frame to ensure transcription of the complete insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. Expression potency can be enhanced by inclusion of an enhancer appropriate to the cell system used (Schaf, D. et al. (1994) Results Probl. Cell Differ. 20:125-62; Bittner et al. (1987) Methods in Enzymology, 153:516-544 ). secretory/localization sequence

A polynucleotide of the invention may also be fused, e.g., in-frame, to a nucleic acid encoding a secretion/localization sequence to target expression of the polypeptide to a desired cellular compartment, membrane or organelle, or to mediate secretion of the polypeptide into the periplasmic space or in cell culture medium. Such sequences are known to those skilled in the art and include secretory leader peptides, organelle targeting sequences (e.g. nuclear localization sequences, ER retention signals, mitochondrial translocation sequences, chloroplast translocation sequences), membrane localization/anchor sequences ( Such as termination transfer sequence, GPI anchor sequence) and so on. Polypeptides expressed from polynucleotides of the invention include amino acid sequences corresponding to secretion and/or localization sequences. expression host

In other embodiments, the present invention relates to host cells containing the constructs described above. The host cell can be a eukaryotic cell, such as a mammalian cell, a yeast cell or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The construct can be introduced by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, or other common techniques (Davis, L, Dibner, M and Battey, I. (1986) Methods in Fundamental Molecular Biology). host cell. A nucleic acid of the invention encoding a polypeptide may be contained in a cell, wherein the cell expresses the polypeptide (eg, an interferon-alpha homologue polypeptide having antiviral or antiproliferative activity as determined by the assays described herein). The invention also includes vectors comprising any of the nucleic acids of the invention, and cells transduced by said vectors. In addition, the present invention also includes cells and transgenic animals containing any of the polypeptides or nucleic acids described above or throughout the specification, such as cells and transgenic animals produced by transduction of the vectors of the present invention.

The host cell strain is optionally selected for its ability to modulate expression of the inserted sequence or to process the expressed protein in the desired manner. Protein modifications include, but are not limited to: acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing of the "pre" or "prepro" form of cleaved proteins is critical for proper insertion, folding and/or function. For this post-translational activity, different host cells such as CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms that can be selected among them to ensure correct modification and processing Imported foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression can be used. For example, a cell line capable of stably expressing a polypeptide of the invention may be transduced using an expression vector containing a viral origin of replication or endogenous expression elements and a selectable marker gene. Following vector introduction, cells are cultured in nourishing medium for 1 to 2 days before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the culture and recovery of cells that successfully express the introduced sequence. For example, resistant populations of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. Depending on the sequence and/or vector used, proteins or fragments thereof produced by recombinant cells can be secreted, membrane-associated or contained intracellularly. Those skilled in the art will understand that a signal sequence can be designed in the expression vector containing the polynucleotide encoding the mature interferon homologue of the present invention, and the signal sequence can mediate the secretion of the mature polypeptide through the prokaryotic or eukaryotic cell membrane. Other peptide sequences

A polynucleotide of the invention may also comprise a coding sequence fused in frame to a marker sequence (eg, to facilitate purification of a polypeptide-encoding sequence of the invention). Such purification-friendly domains include, but are not limited to: metal-chelating peptides that can be purified on immobilized metals, such as histidine-tryptophan modules, glutathione-binding sequences (such as GST), blood Hemagglutinin (HA) tag (corresponding to the epitope of influenza virus hemagglutinin protein; Wilson, I. et al. (1984) Cell 37:767), maltose binding protein sequence, FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA) used in the FLAG epitope and so on. The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and the interferon homolog sequence will facilitate purification. One expression vector expected to be useful in the compositions and methods described herein provides for the expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. open. The histidine residue facilitates purification on IMIAC (immobilized metal ion affinity chromatography, such as Porath et al. (1992) Expression and Purification of Proteins 3:263-281), while the enterokinase cleavage site allows interferon homologs Polypeptides are isolated from fusion proteins. The pGEX vector (Promega; Madison, WI) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Typically, such fusion proteins are soluble by adsorption to ligand-agarose beads (e.g., glutathione-agarose when fused to GST), followed by elution in the presence of free ligand, Fusion proteins can be readily purified from lysed cells. Peptide Production and Recovery

After transducing the appropriate host strain (line) and culturing the host strain (line) to an appropriate cell density, induce the selected promoter by appropriate means (such as temperature shift or chemical agent induction), and culture the cells for a period of time . Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells used to express proteins may be disrupted by any convenient method well known in the art, including repeated freeze-thaw cycles, sonication, mechanical disruption or use of cell lysing agents or other methods.

As noted above, numerous references are available for the culture and production of a wide variety of cells, including cells of bacterial, plant, animal (especially mammalian) and archaeal origin. See, for example, Sambrook, Ausubel and Berger (supra), and Freshney (1994) Animal Cell Culture, A Handbook of Basic Techniques, 3rd Edition, Wiley-Liss, New York and references cited therein, Doyle and Griffiths (1997) Mammalian Cell Culture: Basic Techniques, John Wiley and Sons, New York; Humason (1979) Animal Tissue Technology, 4th ed., W.H. Freeman and Company; and Ricciardelli et al. (1989) In Vitro Cell Developmental Biology, 25:1016-1024. For plant cell culture and regeneration, see Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc., New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue, and Organ Culture ; Basic Methods, Springer Laboratory Manual, Springer-Verlag (Berlin Heidelberg New York) and Plant Molecular Biology (1993) R.R.D. Croy eds., Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 1983706. Cell culture media can generally be found in Atlas and Parks (eds.) Handbook of Microbial Media (1993) CRC Press, Boca Raton, FL. Additional information on cell culture can be found in commercially available literature, such as Life Science Research, Cell Culture Catalog (1998) ("Sigma-LSRCCC") provided by Sigma-Aldrich Corporation (St. Louis, MO), and such as Sigma-Aldrich Plant Culture Catalog and Supplements (1997) ("Sigma-PCCS") provided by the Company (St. Louis, MO).

Polypeptides of the invention may also be recovered and purified from recombinant cell culture by any of a variety of methods well known in the art, including: ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography , phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (eg using any of the labeling systems described herein), hydroxyapatite chromatography and lectin chromatography. When necessary, protein refolding steps can be used to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used in the final purification step. In addition to the references mentioned above, various purification methods are well known in the art, including, for example, Sandana (1997) Bioseparation of Proteins, Academic Publishing Company; and Bollag et al. (1996) Protein Methods, 2nd Edition, Wiley- Liss, New York; Walker (1996) Handbook of Protein Methods, Humana Press, NJ, Hahis and Angal (1990) Protein Purification Applications: A Handbook, Oxford IRL Press, Oxford, UK; Harris and Angal, Protein Purification Methods: A Handbook , Oxford IRL Press, Oxford, UK; Scopes (1993) Protein Purification: Principles and Practice, 3rd ed., Springer Verlag, New York; Janson and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, pp. 2 editions, Wiley-VCH, New York; and Walker (1998) Protein Methods on CD-ROM, Humana Press, NJ. In vitro expression system

Cell-free transcription/translation systems can also be used to produce polypeptides using the DNA or RNA of the invention. Several such systems are commercially available. A general guide to in vitro transcription and translation methods can be found in Tymms (1995) In Vitro Transcription and Translation Methods: Methods in Molecular Biology, Vol. 37, Garland Publishing, New York. modified amino acid

A polypeptide of the invention may contain one or more modified amino acids. The presence of modified amino acids is advantageous, for example, to (a) extend the serum half-life of the polypeptide, (b) reduce the antigenicity of the polypeptide, (c) increase the storage stability of the polypeptide. For example, amino acids may be co-translationally or post-translationally modified during recombinant production (eg, N-linked glycosylation of the N-X-S/T motif when expressed in mammalian cells), or modified synthetically.

Non-limiting examples of modified amino acids include: glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids , PEGylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. There are many references that can guide those skilled in the art in detail to modify amino acids. For exemplary methods, see Walker (1998) CD-ROM version of Protein Methods, Human Press, Towata, NJ.

The polynucleotides and polypeptides of the invention have a number of uses, including, but not limited to, for example: recombinant production (i.e., expression) of recombinant interferon-alpha homologues of the invention; treatment of multiple subjects in vivo and ex vivo; For use as therapeutic and prophylactic agents in methods for various diseases; for use in in vitro methods such as diagnostic and screening methods to detect, diagnose and treat various diseases (e.g. cancer, virus-based diseases, angiogenesis-based diseases); as an immunogen; in gene therapy methods and DNA- or RNA-based delivery methods, for delivering or administering biologically active polypeptides of the invention in vivo, ex vivo or in vitro to a subject Tissues, populations or cells, organs, grafts, body systems (such as organ systems, lymphatic systems, blood systems, etc.) of a patient; as DNA vaccines, multi-component vaccines for therapeutic and prophylactic treatment of Various diseases (e.g. cancer, viral-based diseases, angiogenesis-based diseases) in subjects (e.g. mammals); use as adjuvants to enhance or amplify immune responses in subjects; use as multi-step booster immunization methods (e.g., including priming by delivery of DNA or RNA nucleotides (e.g., encoding a polypeptide of the present invention or encoding another polypeptide) followed by boosting immunization with a polypeptide (e.g., a polypeptide of the present invention or other polypeptides) again) Components; used as diagnostic probes to detect the presence of complementary or partially complementary nucleic acids (including detection of nucleic acids encoding natural interferon-alpha); used as a substrate for other reactions, such as shuffling reactions, mutation reactions, or other diversity-generating reactions to produce new and/or improved interferon-alpha homologues and new interferon-alpha nucleic acids encoding such homologues, for example to produce new therapeutic or prophylactic properties, etc.; for polymerase chain reaction ( PCR) or cloning methods, such as including digestion or ligation reactions, to identify new and/or improved natural or non-natural IFN-α nucleic acids and polypeptides encoded thereby. A polynucleotide encoding an interferon homologue of the present invention, or a complement of the polynucleotide, is optionally administered to a cell to perform a therapeutic or prophylactic method, or to express a therapeutic product in vivo, ex vivo or in vitro. Applications including in vivo or ex vivo, such as gene therapy, include a variety of techniques by which gene expression in cells can be altered. Such methods include, for example, introducing a gene expressing, for example, a therapeutically or prophylactically useful polypeptide, such as an interferon homologue of the invention. The method includes, for example, infection with a retrovirus comprising a polynucleotide and/or polypeptide of the invention. Optionally, the retrovirus also contains other exogenous, eg therapeutic or prophylactic gene construct sequences. In one aspect, as detailed below, the present invention provides a method for administering to a subject in need thereof, including an organism or a mammal, including, for example, a human, a primate, a mouse, a pig, a cow, a goat, a rabbit, a mammal Rats, guinea pigs, hamsters, horses, sheep; or non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, or one or more cells of invertebrates, from in vivo or in vitro administration of one or more A nucleic acid of the invention described herein, a gene therapy method for preventing or therapeutically treating a disease in said subject.

In another aspect, as detailed below, the present invention provides ex vivo or in vitro administration of one or more The polypeptide of the invention, the method of preventing or therapeutically treating the disease of the subject. Peptide expression

Polynucleotides encoding interferon homolog polypeptides of the present invention are particularly useful for therapeutic or prophylactic applications in vivo or ex vivo, using techniques well known to those skilled in the art. For example, ex vivo cultured cells are engineered with polynucleotides (DNA or RNA), and the engineered cells are then returned to the patient. Cells, either in vivo or ex vivo, can also be engineered to express polypeptides in vivo or ex vivo, respectively.

Various viral vectors are known suitable for transduction and expression of organisms in vivo or ex vivo. Such vectors include retroviral vectors (see Miller (1992) Current Topics in Microbiology and Immunology 158: 1-24; Salmons and Gunzburg (1993) Human Gene Therapy 4: 129-141; Miller et al. (1994) Methods in Enzymology 217:581-599) and adeno-associated virus vectors (see Carter (1992) Current Opinions in Biotechnology 3:533-539; Muzcyzka (1992) Current Topics in Microbiology and Immunology 158:97-129). Other viral vectors that may be used include adenoviral vectors, herpesviral vectors, and Sindbis viral vectors, generally described, for example, in JollY (1994) Cancer Gene Therapy 1:51-64; Latchman (1994) Molecular Biotechnology 2:179-195 and Johanning et al. (1995) Nucleic Acids Res. 23: 1495-1501.

Gene therapy offers a variety of approaches to combat chronic infectious diseases (such as HIV infection, viral hepatitis, herpes simplex virus (HSV), hepatitis B virus (HepB), dengue virus, etc.), as well as non-communicable diseases, including cancer and allergic diseases and some forms of birth defects, such as enzyme defects. Several methods have been used to introduce nucleic acids into the body, from cells in vivo and in vitro. These methods include: liposome-based gene delivery (Debs and Zhu (1993) WO93/24640 and US Patent 5,641,662; Mannino and Gould-Fogerite (1988) Biotechnology 6(7):682-691; Rose, US Patent 5,279,833 ; Brigham (1991) WO91/06309; and Felgner et al. (1987) Proc. Natl Acad. Sci. USA 84: 7413-7414); Brigham et al. : 1285-1288; Hazinski et al. (1991) Am.J.Resp.Cell Molec.Biol 4:206-209; and Wang and Huang (1987) Proc.NatlAcad.Sci. (USA) 84:7851-7855); Viral vector mediated gene delivery, e.g. for the treatment of cancer (see e.g. Chen et al. (1994) Proc. Natl Acad. Sci. USA 91:3054-3057; Tong et al. (1996) Gynecol. Oncol. 61:175-179; Clayman et al. (1995) Cancer Research 5:1-6; O'Malley et al. (1995) Cancer Research 55:1080-1085; Hwang et al. (1995) Am.J.Respir.Cell Mol.Biol.13:7-16; Haddada et al. (1995) Current Topics in Microbiology and Immunology 199 (Pt.3): 297-306; Addison et al. (1995) Proc. Natl Acad. Sci. USA 92: 8522-8526; Colak et al. (1995) Brain Research 691 : 76-82; Crystal (1995) Science 270: 404-410; Elshami et al (1996) Human Gene Therapy 7: 141-148; Vincent et al (1996) J Neurosurgery 85: 648-654), and many others. In addition, replication-deficient retroviral vectors have been used which carry a therapeutic polynucleotide sequence as part of the retroviral genome, of which the simple MuLV vector should be mentioned in particular, see e.g. Miller et al. (1990) Molecular Cell Biology 10:4239 (1990); Kolberg (1992) NIH Journal of Research 4:43, and Cornetta et al (1991) Human Gene Therapy 2:215). Additionally, nucleic acid transport in combination with ligand-specific, cation-based transport systems has also been used (Wu and Wu (1988) J. Biol. Chem. 263: 14621-14624). Naked DNA expression vectors have also been described (Nabel et al. (1990), supra; Wolff et al. (1990) Science 247: 1465-1468). In general, these methods can be adapted to the present invention by incorporating nucleic acids encoding interferon homologues described herein into appropriate vectors.

Common textbooks describing methods of gene therapy, adapted to the present invention by introducing nucleic acids of the invention into patients, include: Robbins (1996) Gene Therapy Methods, Humana Press, NJ, and Joyner (1993) Gene Targeting: A How-To, IRL Press, Oxford, UK. antisense technology

In addition to expressing the nucleic acids of the invention as gene replacement nucleic acids, the nucleic acids can also be used for sense and antisense inhibition of expression, eg downregulation of the expression of the nucleic acids of the invention, once expression of the nucleic acids is no longer required in the cell. Similarly, the nucleic acids of the invention, or subsequences or antisense sequences thereof, can also be used to block the expression of naturally occurring homologue nucleic acids. A variety of sense and antisense technologies are known in the art, see for example Nellen (1997) Antisense Technology: How-To, IRL Press, University of Oxford, Oxford, UK and Agrawal (1996) Antisense Therapeutics Humana Publishing Society, NJ and references mentioned therein. pharmaceutical composition

The polynucleotides and polypeptides of the present invention (including, for example, vectors, cells, antibodies, etc.) containing the polynucleotides or polypeptides of the present invention can be combined with appropriate pharmaceutical carriers for therapeutic and prophylactic use. The composition contains a therapeutically or prophylactically effective amount of the polynucleotide or polypeptide of the present invention, and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers include any standard pharmaceutical carriers, buffers and excipients. Said carrier or excipient includes but not limited to: saline, buffered saline (such as phosphate buffered saline solution), dextrose, water, glycerol, ethanol, emulsion (such as oil/water or water/oil emulsion), various types of emulsifiers Wetting agents and/or adjuvants, and combinations thereof. Suitable pharmaceutical carriers and agents are described in REMINGTON'S Pharmaceutical Sciences (Mack Publishing Company, Easton, 19th Edition, 1995). The formulation method should be compatible with the mode of administration of active agents (such as nucleotides, polypeptides, carriers, cells, etc.). Methods of administering nucleic acids, polypeptides, vectors, cells, antibodies and proteins are well known in the art and are discussed further below. used as a probe

It is also intended herein to include the use of polynucleotides (also referred to herein as oligonucleotides) generally having at least 12 bases, preferably at least 15 bases, more preferably at least 20, 30 or 50 bases, said The polynucleotide is capable of hybridizing under at least high stringency (or ultra-high stringency or ultra-ultra-high stringency conditions) polynucleotide sequences of the interferon homologues described above. Polynucleotides can be used as probes, primers, sense and antisense reagents, etc. according to the methods described in the above documents. sequence variation silent variation

Those skilled in the art will understand that: due to the degeneracy of the genetic code, many nucleic acid sequences encoding the interferon homologue polypeptide of the present invention can be produced, some of which have very low sequence homology with the nucleic acid sequences clearly disclosed herein .

Table 1 Codon table

amino acid codon

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic Acid Asp D GAC GAU

Glutamic Acid Glu E E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G G GGA GGC GGG GGU

Histidine His His H H CAC CAU

Isoleucine Ile I I AUA AUC AUU

Lysine Lys K K AAA AAG

Leucine Leu L L UUA UUG CUA CUC CUG CUU

Methionine Met M M AUG

Asparagine Asn N N AAC AAU

Proline Pro P P CCA CCC CCG CCU

Glutamine Gln Q Q CAA CAG

Arginine Arg R R AGA AGG CGA CGC CGG CGU

Serine Ser S S AGC AGU UCA UCC UCG UCU

Threonine Thr T T ACA ACC ACG ACU

Valine Val V V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

For example, it can be seen from the codon table (Table 1) that the codons AGA, AGG, CGA, CGC, CGG and CGU all encode the amino acid arginine. Thus, at each position designated by a codon as arginine in the nucleic acids of the invention, the codon can be changed to any of the corresponding codons described above without changing the encoded polypeptide. It is understood that U in RNA sequences is equivalent to T in DNA sequences.

For example, using the nucleic acid sequence TGT GATCTG CCT CAG corresponding to nucleotides 1-15 in SEQ ID NO: 1, silent variations of this sequence include TGC GAC TTA CCA CAA, these two sequences encoding the amino acid sequence CDLPQ are equivalent to SEQ ID NO: Amino acids 1-5 in ID NO:36.

Such "silent variations" are a type of "conservatively modified variations" discussed below. Those skilled in the art will appreciate that each codon in the nucleic acid (except AUG, which is usually the only codon for methionine) can be modified by standard techniques to encode a functionally equivalent polypeptide. Accordingly, every silent variation of a nucleic acid which encodes a polypeptide is implicit in any stated sequence. The invention provides each and every possible variation of a nucleic acid sequence encoding a polypeptide of the invention, which is combined according to possible codon usage. These combinations are prepared according to the standard triplet genetic code (shown in Table 1) used for nucleic acid sequences encoding interferon homolog polypeptides of the present invention. All of the aforementioned variants of each nucleic acid herein are specifically provided and described by consideration of the sequence as well as the genetic code. conservative variation

"Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical the sequence of. Those skilled in the art believe that: individual substitutions, deletions, alterations, additions or deletions of a single amino acid or a small percentage of amino acids (generally less than 5%, more preferably less than 4%, 3%, 2% or 1%) in the coding sequence Or additions are "conservatively modified variations" in which the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar amino acids are well known in the art. Table 2 lists six groups containing amino acids that can be "conservatively substituted" for another amino acid.

Table 2 Conservatively substituted groups 1 Alanine (A) Serine (S) Threonine (T) 2 Aspartic Acid (D) Glutamic Acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Thus, a "conservatively substituted variation" or simply "conservative substitution" of a polypeptide sequence set forth in the invention involves substituting a small percentage, generally less than 5%, more preferably less than at 4%, 3%, 2% or 1% amino acids.

For example, a conservative substitution variation of a polypeptide identified herein as SEQ ID NO: 36 would contain within about 8 or 9 residues (i.e., about 5% of the amino acids) of a 166 amino acid long polypeptide according to the above-defined 6 groups of "conservative substitutions".

In other examples, if the 4 conservative substitutions are located in a region corresponding to amino acid residues 141-166 of SEQ ID NO: 36, according to the conservative substitutions listed in Table 2, examples of conservative substitution variations in this region WEVVR AEIMRSFSFS TNLQK RLRRKE include : WEVVR S EIMRSFS Y S TNLQ R RLRRK D and WE L VR AEI V R SFSFS TNL N K RLR K KE etc. (In the above examples, the underlined ones are conservative substitutions). The protein sequences listed herein, in conjunction with the substitution tables above, provide a list of all proteins with conservative substitutions.

Finally, the addition of sequences that do not alter the coding activity of the nucleic acid molecule, eg, the addition of non-functional sequences, is also a conservative variation of the base nucleic acid.

Those of skill in the art will appreciate that many conservative variations of the disclosed nucleic acid constructs will result in functionally equivalent constructs. For example, as discussed above, due to the degeneracy of the genetic code, "silent substitutions" (ie, substitutions in a nucleic acid sequence that do not alter the encoded polypeptide) are an implicit feature of every nucleic acid sequence that encodes an amino acid. Similarly, "conservative amino acid substitutions" in which one or a few amino acids in an amino acid sequence are substituted with a different amino acid having highly similar properties can also be readily identified as being highly similar to the disclosed constructs. Such conservative variations of each of the disclosed sequences are also a feature of the invention. nucleic acid hybridization

"Hybridization" occurs when nucleic acids associate with each other, typically in solution. Nucleic acid hybridization is due to the existence of a variety of well-identified physical-chemical forces, such as hydrogen bonding, solvent repulsion, base stacking, etc. A comprehensive guide to nucleic acid hybridization can be found in Tijssen's (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization to Nucleic Acid Probes, Part I, Chapter 2, "A Review of Hybridization Principles and Nucleic Acid Probe Assay Strategies", and Ausubel , the literature is the same as above. Hames and Higgins (1995) Gene Probe 1, IRL Press of Oxford University Press, Oxford, UK (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probe 2, IRL Press of Oxford University Press, Oxford, UK (Hames and Higgins 2) provides details on the synthesis, labelling, detection and quantification of DNA and RNA, including oligonucleotides.

"Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments, such as Southern and Northern hybridization, are sequence-dependent and vary under different environmental parameters. Detailed guidance on nucleic acid hybridization can be found in Tijssen (1993), supra and Hames and Higgins 1 and Hames and Higgins2, supra.

For the purposes of the present invention, "highly stringent" hybridization and wash conditions are generally selected to be about 5°C or less lower than the thermal melting point ( _Tm ) for the specific sequence at a defined ionic strength and pH (see below). described, highly stringent conditions can also be described in equivalent terms). The _Tm is the temperature (under defined ionic strength and pH) at which 50% of the test sequences hybridize to an exact matching probe. Very stringent conditions are selected to be equal to the _Tm of a particular probe.

The _Tm is the temperature of the nucleic acid duplex at which the duplex is 50% denatured under the given conditions, which is a direct measure of the stability of the nucleic acid hybrid. Thus, _Tm corresponds to the temperature corresponding to the midpoint of the transition from helix to random coil; for long stretches of nucleotide sequences it depends on length, nucleotide composition and ionic strength.

Following hybridization, unhybridized nucleic acid material can be removed by a series of washes, the stringency of which can be adjusted according to the desired results. Wash conditions of low stringency (eg, using higher salt concentrations and lower temperatures) increase sensitivity but can produce nonspecific hybridization signals and strong background signals. Conditions of higher stringency (eg, using lower salt concentrations and higher temperatures closer to the hybridization temperature) reduce background signal, generally leaving only specific signal. See Rapley, R. and Walker, J.M., eds., Methods in Molecular Biology (Humana Publishing Co. 1998) (hereinafter "Rapley and Walker"), which is incorporated herein by reference in its entirety.

The _Tm of a DNA-DNA duplex can be estimated using the following equation:

_Tm (°C)=81.5°C+16.6(log ₁₀ M)+0.41(%G+C)-0.72(%f)-500/n, where M is the molar concentration of monovalent cations (usually Na+), ( %G+C) is the percentage of guanosine (G) and cytidine (C) nucleotides, %f is the percentage of formamide, and n is the number of nucleotide bases (i.e. length) of the hybrid, see Rapley and Walker, op. cit.

Estimate the _Tm of the RNA-DNA duplex as follows:

T _m (°C)=79.8°C+18.5(log ₁₀ M)+0.58(%G+C)-11.8(%G+C) ^2-0.56 (%f)-820/n, where M is a monovalent cation (usually is the molar concentration of Na+), (%G+C) is the percentage of guanosine (G) and cytidine (C) nucleotides, %f is the percentage of formamide, and n is the number of nucleotide bases in the hybrid Item (that is, length), the literature is the same as above.

Equations 1 and 2 are generally only accurate for hybrid duplexes longer than 100-200 nucleotides, supra.

The _Tm of nucleic acid sequences shorter than 50 nucleotides can be estimated as follows:

T _m (℃)=4(G+C)+2(A+T),

where A (adenine), C, T (thymine) and G are the numbers of the corresponding nucleotides.

An example of stringent hybridization conditions for hybridizing a complementary nucleic acid having more than 100 complementary residues on a Southern or Northern blot membrane is: overnight hybridization at 42°C in 50% formalin containing 1 mg heparin. An example of stringent washing conditions is: 0.2×SSC, 65° C. for 15 minutes (see Sambrook, supra for a description of SSC buffer). Often low stringency washes are substituted for high stringency washes to remove background probe signal. An example of a low stringency wash is 2X SSC, 15 minutes at 40°C.

In general, specific hybridization is detected if the signal-to-noise ratio is 2.5-fold to 5-fold higher (or higher) than the corresponding value observed with an unrelated probe in a particular hybridization assay. In the context of the present invention, detection of at least stringent hybridization between two sequences indicates a relatively high structural similarity or homology to a nucleic acid of the invention, eg as provided herein in the Sequence Listing.

As noted above, "highly stringent" conditions are selected to be about 5°C or less lower than the thermal melting point ( _Tm ) for the specific sequence at a defined ionic strength and pH. Target sequences closely related to or identical to a nucleotide sequence of interest (eg, a "probe") can be identified under high stringency conditions. Conditions of lower stringency are suitable for less complementary sequences, see eg Raoley and Walker, supra.

Nucleic acids of the invention can be identified using comparative hybridization and are a preferred method for distinguishing nucleic acids of the invention. In the context of the present invention, the detection of highly stringent hybridization between two sequences indicates a relatively high structural similarity/homology to the nucleic acids eg provided in the Sequence Listing herein. Highly stringent hybridization between two nucleotide sequences elucidates a higher level of similarity or homology in structure, nucleotide base composition, arrangement or sequence than is detectable under stringent hybridization conditions. In particular, in the context of the present invention, detection of highly stringent hybridization indicates high structural similarity or structural homology (e.g. nucleotide structure, base composition, arrangement or sequence). For example, it is desirable to identify test nucleic acids that hybridize under stringent conditions to the nucleic acids exemplified herein.

Thus, one measure of stringency is the ability to bind the listed nucleic acids under highly stringent conditions (or very stringent conditions, or ultra-highly stringent hybridization conditions, or ultra-ultra-highly stringent hybridization conditions) (for example nucleic acid sequence SEQ ID NO: 1 to SEQ ID NO: 35 and SEQ ID NO: 72 to SEQ ID NO: 78 and complementary polynucleotide sequences thereof) one of hybridization. Stringency conditions (including, for example, high stringency, ultra-high stringency or ultra-ultra-high stringency hybridization conditions) and wash conditions can be readily determined empirically for any test nucleic acid.

For example, when highly stringent hybridization and washing conditions are determined, the hybridization and washing conditions can be gradually increased (for example, by increasing the temperature during hybridization or washing, reducing the salt concentration, increasing the detergent concentration and/or increasing the organic solvent, such as concentration of formalin) until a selected set of criteria is reached. For example, the hybridization and washing conditions are gradually increased until the probe can bind to a perfectly matched complementary target (the target nucleic acid contains one or more selected from SEQ ID NO: 1 to SEQ ID NO: 35, SEQ ID NO: 72 to SEQ ID NO: 72 to SEQ ID NO: IDNO: 78 and the nucleic acid sequence of its complementary polynucleotide sequence), said probe also contains one or more selected from SEQ ID NO: 1 to SEQ ID NO: 35, SEQ ID NO: 72 to SEQ ID NO: 78 and its complementary polynucleotide sequence, when the signal-to-noise ratio is at least 2.5 times, optionally 5 times or higher than the corresponding value observed when the probe hybridizes to a mismatched target. In this case, a non-matching target is a nucleic acid corresponding to a known alpha interferon, eg, an alpha interferon nucleic acid as present in public databases such as GenBank ^™ at the time of filing this application. Examples of such mismatched target nucleic acids include, for example, nucleic acids having the following GenBank accession numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-A) -H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α- C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 ( α-Con1). Other such sequences can be identified in GenBank by those skilled in the art. The nomenclature of the human interferon gene and protein is discussed in Diaz et al. (1996) Journal of Interferon and Cytokine Research, 16:179-180 and Allen et al. (1996) Journal of Interferon and Cytokine Research, 16:181-184, both The entire text is incorporated herein by reference.

The test nucleic acid is said to be present when the test nucleic acid hybridizes to the probe nucleic acid to at least 1/2 the degree to which the probe hybridizes to an exactly matched complementary target, i.e. the signal-to-noise ratio is at least 1/2 that of the probe to the target. Nucleic acid specifically hybridizes to the probe nucleic acid, wherein under conditions of said hybridization, an exactly-matched probe binds to an exactly-matched complementary target with a signal-to-noise ratio of at least about 2.5-10 times, typically 5-10 times greater than Corresponding values observed for probes hybridized to any mismatched target nucleic acids having the following GenBank accession numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540( α-21), X02955 (α-4b), V00532 (α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 ( α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other similar interferon-α sequences provided in GenBank.

Ultra-highly stringent hybridization and wash conditions are conditions in which the stringency of the hybridization and wash conditions is increased until the signal-to-noise ratio of the probe binding to an exactly matched complementary target nucleic acid is at least 10 times greater than that observed for the probe. Corresponding values when the needle is hybridized to any mismatched target nucleic acid having the following GenBank accession numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6) , X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350(α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other similar IFN-α sequences provided in GenBank. If the target nucleic acid can hybridize to the probe under these conditions, and the signal-to-noise ratio is at least 1/2 that of an exactly matched complementary target nucleic acid, it can be said that the target nucleic acid and the probe can bind under ultra-high stringency conditions .

Similarly, even higher stringency levels can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, conditions can be identified wherein the stringency of the hybridization and wash conditions is increased until the signal-to-noise ratio of the probe binding to an exactly matched complementary target nucleic acid is at least 10-fold, 20-fold, 50-fold, 100-fold or 500-fold or higher The corresponding value when the probe is observed to hybridize to any mismatched target nucleic acid having the following GenBank accession numbers: J00210 (α-D), J00207 (α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540 (α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other similar interferon-α sequences provided in GenBank. If the target nucleic acid can hybridize to the probe under these conditions, and the signal-to-noise ratio is at least 1/2 that of an exactly matched complementary target nucleic acid, it can be said that the target nucleic acid and the probe can hybridize under ultra-ultra-high stringency conditions. Combined.

Target nucleic acids capable of hybridizing to nucleic acids shown in SEQ ID NO: 1 to SEQ ID NO: 35 and SEQ ID NO: 72 to SEQ ID NO: 78 under high, ultra-high and ultra-ultra-high stringency conditions are of the present invention feature. Examples of such nucleic acids include nucleic acids having one or several silent or conservative nucleic acid substitutions compared to a given nucleic acid sequence.

Nucleic acids that do not hybridize to each other under stringent conditions are considered to be substantially identical if the polypeptides encoded by the nucleic acids are substantially identical. For example, when a copy of the nucleic acid is produced using the maximum codon degeneracy permitted by the genetic code, or when targeting one of SEQ ID NO: 36 to SEQ ID NO: 70 and SEQ ID NO: 79 to SEQ ID NO: 85 This is the case when antisera are raised against one or more species that have been subtracted from polypeptides encoded by known interferon-alpha sequences, including, for example, the following interferon- α nucleic acid sequence, that is, the GenBank accession number is: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α-H), V00542 (α-14 ), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7) , X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1) nucleic acid sequence or GenBank Polypeptides encoded by other similar interferon-alpha sequences provided in . Details regarding the immunological characterization of the polypeptides of the invention can be found hereinafter. In addition, to distinguish duplexes with sequences less than about 100 nucleotides, the TMAC1 hybridization method known to those skilled in the art can be used, see for example Sorg, U et al., Nucleic Acids Res. (1991, 9, 11) 19(17) , the entirety of which is incorporated herein by reference.

In one aspect, the invention provides a nucleic acid comprising a unique subsequence in a nucleic acid selected from SEQ ID NO: 1 to SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78. This unique subsequence is unique compared to nucleic acids corresponding to any known interferon-alpha nucleic acid sequences, including, for example, known sequences with the following GenBank accession numbers: J00210(α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957 (α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or other similar interferon-α sequences provided in GenBank. This unique subsequence can be determined by aligning any one of SEQ ID NO: 1 to SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78 with the complete set of nucleic acids, the complete set The nucleic acid corresponds to a known interferon-alpha nucleic acid sequence having, for example, the following GenBank accession numbers: J00210 (alpha-D), J00207 (alpha-a), X02958 (alpha-6), X02956 (alpha-5), V00533 (alpha -H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α- C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 ( α-Con1), or other similar interferon-α sequences provided in GenBank. Sequence alignments can be performed using the BLAST algorithm set to default parameters. Any unique subsequence can be used as a probe to identify nucleic acids of the invention.

Similarly, the invention includes a polypeptide comprising a unique amino acid subsequence in a polypeptide selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85. This unique subsequence is unique compared to amino acid subsequences of known interferon-alpha polypeptide sequences comprising, for example, known interferon-alpha nucleic acids corresponding to any of the following GenBank accession numbers Or the amino acid subsequence of the polypeptide encoded by other similar interferon-α nucleic acids provided in GenBank: J00210(α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533 (α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532( α-C), X02960 (α-7), X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and Amino acid subsequences of I08313 (α-Con1), or other similar polypeptide sequences provided in GenBank. The polypeptide is sequence aligned with the complete set of known interferon-alpha polypeptide sequences, e.g., polypeptides encoded by nucleic acids corresponding to the following GenBank accession numbers or other similar interferon-alpha nucleic acids provided in GenBank: J00210 (α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125( α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), (referred to as "control polypeptide") (note that when the sequence corresponds to a non- - Translated sequences, such as pseudogenes, the corresponding polypeptides can be produced simply by translating the nucleic acid sequence into an amino acid sequence in a computer, where the reading frame is selected to correspond to that of the homologous interferon-alpha nucleic acid) or GenBank Other similar polypeptide sequences provided in .

In addition, the present invention provides target nucleic acids that hybridize under at least stringent or highly stringent conditions (or conditions of higher stringency) to a unique encoding oligonucleotide selected from the group consisting of SEQ ID NO: A unique subsequence in a polypeptide from 36 to SEQ ID NO: 70 or from SEQ ID NO: 79 to SEQ ID NO: 85, which corresponds to an amino acid subsequence of a known interferon-alpha polypeptide sequence shown in GenBank or corresponds to any control This unique subsequence is unique compared to polypeptides. Unique sequences were determined as described above.

In one embodiment, stringent conditions are selected such that an oligonucleotide that is exactly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide and has a signal-to-noise ratio that is greater than that of an exactly complementary oligonucleotide to the coding oligonucleotide. The signal-to-noise ratio of the control nucleic acid hybridization for any control polypeptide is at least about 5 to 10 times higher. Conditions can be selected to observe a higher signal-to-noise ratio in the particular assay used, for example about 15-fold, 20-fold, 30-fold, 50-fold or higher. In this embodiment, a target nucleic acid hybridizes to a unique encoding oligonucleotide with a signal-to-noise ratio that is at least about 2-fold higher than the signal-to-noise ratio of a control nucleic acid hybridizing to an encoding oligonucleotide. Additionally, a higher signal-to-noise ratio may be selected, for example about 2.5 times, about 5 times, about 10 times, about 20 times, about 30 times, about 50 times or higher. The specific signal will depend on the label used for the assay in question, e.g. fluorescent, colorimetric, radioactive, etc.

In another aspect, the present invention provides a polypeptide comprising a unique subsequence in a polypeptide selected from SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85, wherein with This unique subsequence is unique compared to polypeptide sequences corresponding to known interferon-alpha polypeptides, such as interferon-alpha polypeptide sequences provided in GenBank. Substrates and forms of sequence recombination

The polynucleotides of the invention are useful in a variety of recombination and regressive recombination (e.g., DNA shuffling) reactions, as well as in other diversity-generating techniques including, for example, mutagenesis techniques and standard cloning methods described by Ausubel, Berger and Sambrook, supra. substrates for the production of other interferon-alpha homologues with desirable properties. Depending on the screening or selection method used, recombinant, e.g., shuffled interferon-alpha homologue polypeptides can be produced and isolated that are endowed with various desired properties, e.g., enhanced antiviral activity, enhanced antiproliferative activity, enhanced growth inhibition against specific target cells, cytostatic and/or cytotoxic activity, reduced immunogenicity, etc.

Various methods of diversity generation can be used, including nucleic acid shuffling methods, which are well described in the art. These methods can be used alone and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, and variants of the encoded protein. These methods, used alone and in combination, can provide powerful, broadly applicable approaches to generate diverse nucleic acids and nucleic acid combinations (sets) (including, for example, nucleic acid libraries) for, e.g., engineering or rapid evolution with new and/or improved Characterization of nucleic acids, proteins, pathways, cells and/or organisms.

Although methods are distinguished and categorized in the course of the illustrative discussion that follows, it should be understood that techniques are often mutually inclusive. Indeed, multiple methods can be used alone or in combination, in parallel or sequentially, to generate a variety of sequence variants.

The result of any of the diversity generating methods described herein can be the production of one or more nucleic acids from which nucleic acids can be selected or screened for proteins that encode or confer desired properties. After generating diversity by one or more of the methods described herein, or other methods available to those skilled in the art, any nucleic acid produced may be selected for a desired activity or property, such as enhanced antiviral activity, enhanced Antiproliferative activity, enhanced anti-angiogenic activity, enhanced growth inhibition against specific target cells, cytostatic and/or cytotoxic activity, reduced immunogenicity, etc. Methods for determining nucleic acids with enhanced antiviral, antiproliferative, growth inhibitory, cytostatic and/or cytotoxic activity or reduced immunogenicity include those described herein. The method may comprise identifying any detectable activity by any assay in the art, for example in an automated or automatable manner. Multiple related (or even unrelated) properties can be evaluated sequentially or in parallel, at the operator's discretion.

The following publications describe a variety of methods for generating diversity, including regressive recombination, and/or methods for generating modified nucleic acid sequences that can be used in the methods of the present invention, including the following publications and those mentioned therein References: Soong, N.W et al (2000) "Molecular propagation of viruses", Nature Genetics 25: 436-439; Stemmer, W et al (1999) "Molecular propagation of viruses for targeting and other clinical properties", Tumor targeting 4 : 1-4; Ness et al. (1999) "DNA shuffling of subgenomic sequences of subtilases", Nature Biotechnology 17: 893-896; Chang et al. (1999) "Evolution of cytokines using DNA family shuffling", Nature Biotechnology 17: 793-797; Minshull and Stemmer (1999) "Protein evolution through molecular propagation", Current Opinion in Chemical Biology 3:284-290; Christians et al. (1999) "Directed evolution of thymidine kinases for AZT phosphorylation using DNA family shuffling", Nature Biotechnology 17: 259-264; Crameri et al. (1998) "DNA shuffling of gene families from multiple species promotes directed evolution", Nature 391:288-291; Crameri et al. (1997) "Arsenate detoxification pathways by DNA shuffling Molecular evolution", Nature Biotechnology 15: 436-438; Zhang et al. (1997) "Directed evolution of galactosidases into effective fucosidases by DNA shuffling and screening", Proc. Natl Acad Sci. USA 94: 4504-4509 ; Patten et al. (1997) "DNA shuffling in medicine and vaccines", Current Opinion in Biotechnology 8:724-733; Crameri et al. (1996) "Construction and evolution of antibody-phage libraries by DNA shuffling", Nature Medicines 2:100 -103; Crameri et al. (1996) "Improvement of green fluorescent protein by molecular evolution using DNA shuffling", Nature Biotechnology 14:315-319; Gates et al. Affinity-Selective Separation of Ligands from Peptide Libraries", Journal of Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and Assembly PCR", Encyclopedia of Molecular Biology, VCH Publishers, New York, p447-457 ; Crameri and Stemmer (1995) "Combined multiplex cassette mutagenesis yields transformations of all mutant and wild-type cassettes", Biotechnology 18: 194-195; Stemmer et al. (1995) "Single-step assembly of genes and entire plasmids to form large oligodeoxy Ribonucleotides", Genes 164: 49-53; Stemmer (1995) "Evolution of Molecular Algorithms", Science 270: 1510; Stemmer (1995) "Searching for sequence gaps", Biotechnology 13: 549-553; Stemmer (1994 ) "Rapid protein evolution in vitro by DNA shuffling", Nature 370:389-391; and Stemmer (1994) "DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution", Proc. Natl Acad. Sci . USA91: 10747-10751.

Additional details on DNA shuffling and other methods of generating diversity can be found in the following U.S. patents, PCT applications and EP applications: USPN 5,605,793 (February 25, 1997) to Stemmer, "In Vitro Recombination Methods"; USPN 5,811,238 to Stemer et al. (September 22, 1998), "Method for Producing Polynucleotides with Desired Characteristics by Repeated Selection and Recombination"; USPN 5,830,721 (November 3, 1998) to Stemmer et al. DNA mutagenesis by assembly"; Stemmer's USPN 5,834,252 (November 10, 1998), "Polymerase Reaction for End Complementation"; Minshull's USPN 5,837,458 (November 17, 1998), "Cell and Metabolic Engineering Methods and compositions"; WO95/22625, Stemmer and Crameri, "Mutagenesis by random fragmentation and reassembly"; WO96/33207, Stemmer and Lipschutz, "Polymerase chain reaction of terminal complementarity"; WO97/20078, Stemmer and Crameri, "Methods for producing polynucleotides with desired characteristics by repeated selection and recombination"; WO97/35966, Minshull and Stemmer, "Methods and compositions for cellular and metabolic engineering"; WO99/41402, Punnonen et al., " Targeting of Gene Vaccine Vectors”; WO99/41383, Punnonen et al., “Immunization with Antigen Libraries”; WO99/41369, Punnonen et al., “Engineering Genetic Vaccine Vectors”; WO99/41368, Punnonen et al. optimization”; EP752008, Stemmer and Craneri, “DNA mutagenesis by random fragmentation and reassembly”; EP0932670, Stemmer, “Changing cellular DNA uptake by regressive sequence recombination”; WO99/23107, Stemmer et al. Shuffling modifies viral tropism and host range"; WO99/21979, Apt et al., "Human papillomavirus vectors"; WO98/31837, Del Cardayre et al., "Changing whole cells and organisms by recurrent sequence recombination"; WO98/27230, Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering"; EP0946755, Patten and Stemmer, "Methods and Compositions for Polypeptide Engineering"; Methods"; WO00/00632, "Methods for Generating Libraries of High Diversity"; WO00/09679, "Methods for Obtaining Libraries of Polynucleotide Sequences Recombined in Vitro and Obtaining Sequences"; WO98/42832, Arnold et al., "Using random or Defined primers for recombinant polynucleotide sequences"; WO99/29902, Arnold et al., "Methods for generating polynucleotide and polypeptide sequences"; WO98/41653, Vind, "In vitro methods for constructing DNA libraries"; WO98/41622, Borchert et al. , "Methods for constructing libraries using DNA shuffling"; and WO98/42727, Pati and Zarling, "Altering sequences using homologous recombination".

Certain U.S. applications provide additional details on DNA shuffling and related techniques and other methods of generating diversity, these applications include: Patten et al. September 29, 1998 (USSN 60/102,362), January 29, 1999 (USSN 60/117,729 ) and "Genes with Altered Shuffled Codons" filed September 28, 1999; Del Cardayre et al. "Changing Whole Cells and Organisms by Recurrent Sequence Recombination"; Crameri et al. February 5, 1999 (USSN60/118,813), June 24, 1999 (USSN60/141,049) and September 28, 1999 (USSN09/408,392) "Oligonucleotide-mediated Nucleic Acid Recombination," submitted; Welch et al., "Synthetic Shuffling Using Codon-Based Oligonucleotide Synthesis," filed Sept. 28, 1999 (USSN 09/408,393); Selifonov and Stemmer at "Methods for Preparing Characteristic Strands, Polynucleotides, and Polypeptides Having Desired Properties," filed Feb. 5, 1999 (USSN 60/118854) and Oct. 12, 1999 (USSN 09/416,375); Patten et al. 1999 Filed March 5 (USSN 60/122,943), July 2, 1999 (USSN 60/142,299), November 10, 1999 (USSN 60/164,618) and November 10, 1999 (USSN 60/164,617) "Modified Nucleic Acids Inserted by Recombination"; and USSN 60/186,482, "Single-Stranded Nucleic Acid Template-Mediated Recombination and Isolation of Nucleic Acid Fragments," filed March 2, 2000 by Affholter.

As disclosed in the aforementioned publications, patents, published foreign applications and reviews of U.S. patent applications, diversity generation methods can be implemented by a variety of established methods, such as shuffling (or "recursive recombination") nucleic acids to provide Novel nucleic acids with desired properties. Any of these methods are suitable for use in the present invention and can be used to form alpha interferon as discussed herein, resulting in new alpha interferon homologues with new or improved properties. Two methods of preparing the above-mentioned interferons (eg IFN homologues) produced by these methods are also a feature of the invention. Briefly, several different broad classes of sequence modification methods, such as recombination, can be used in the present invention, and these methods are described, for example, in the references mentioned above. First, nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the above references, including, for example, DNase digestion of the nucleic acids to be recombined, followed by ligation and/or reassembly of the nucleic acids by PCR. Second, the recombination of nucleic acids is performed in vivo or ex vivo, eg, by allowing recombination between nucleic acids in cells. Third, whole-genome recombination approaches can be used, in which the entire genome of a cell or other organism is recombined, optionally including spiking a genomic recombination mixture with desired library components (eg, genes corresponding to pathways of the invention). Fourth, synthetic recombination methods can be used, in which oligonucleotides corresponding to the desired target are synthesized and reassembled in a PCR or ligation reaction, resulting in a new recombinant nucleic acid comprising Nucleic acid oligonucleotides. Oligonucleotides can be prepared by standard nucleotide addition methods, or by, for example, tri-nucleotide synthesis. Fifth, in silico recombination methods can be implemented, in which genetic algorithms are used in silico to recombine sequence strands corresponding to homologous (or even non-homologous) nucleic acids. The resulting strand of the recombinant sequence is optionally converted to nucleic acid by synthesizing nucleic acid corresponding to the recombinant sequence, for example using a combination of oligonucleotide synthesis/gene reassembly techniques. Any of the general recombination approaches described above can be repeated to produce a more diverse set of recombinant nucleic acids. Sixth, methods of increasing natural diversity can be used, such as by hybridizing a wide variety of nucleic acids or nucleic acid fragments to single-stranded templates, followed by polymerization and/or ligation to regenerate full-length sequences, optionally, followed by degradation template and recover the resulting modified nucleic acid. The above references provide these and other basic recombinant formats as well as various modified methods based on these formats. Regardless of the form of recombination used, the nucleic acids of the invention can be recombined (with each other, or with related (or even unrelated) nucleic acids) to produce a diverse set of recombinant nucleic acids, including, for example, homologous nucleic acids. In general, the sequence recombination techniques described herein provide particular benefits because they allow the sequence of SEQ ID NO: 1 to SEQ ID NO: 35 and SEQ ID NO: 72 to SEQ ID NO: 78, or fragments or variants thereof The sequences can be recombined in any available manner, providing a very rapid way to study the way in which different combinations of sequences affect the desired outcome.

Following recombination, any nucleic acids produced can be screened or selected for the desired activity. In the context of the present invention, any activity that can be detected by any assay known in the art, eg, detected and identified in an automated manner, is included. Additionally, useful properties such as low immunogenicity, increased half-life, improved solubility, orally available properties, etc. may also be selected. Various alpha-interferon-related (or even unrelated) properties can be detected using any available assay.

DNA mutagenesis and shuffling provide powerful, broadly applicable methods to generate diversity that can be used to engineer proteins, pathways, cells and organisms with improved properties. In addition to the basic methods described above, it is sometimes necessary to combine shuffling methodologies with other techniques for generating diversity. A variety of diversity generation methods can be used in conjunction with (or separately from) shuffling methods, and the results (ie, diverse nucleic acid populations) can be screened in the systems of the invention. Additional diversity can be introduced by methods that result in changes in individual nucleotides or groups of contiguous or noncontiguous nucleotides, ie, mutagenesis. Various mutagenesis methods are described in the above references; additional details regarding mutagenesis methods can be found in the following references.

Mutagenesis methods for generating diversity include, for example: recombination (PCT/US98/05223; publication number WO98/42727); site-directed mutagenesis (Ling et al. (1997) "Approaches to DNA mutagenesis: an overview," Anal.Biochem.254 (2 ): 157-178; Dale et al. (1996) "Oligonucleotide-directed random mutagenesis using the phosphorothioate method," Methods Mol.Biol.57: 369-374; Smith (1985) "In vitro mutagenesis," Ann.Rev.Genet.19 : 423-462; Botstein & Shortle (1985) "Strategies and applications of in vitro mutagenesis," Science 229: 1193-1201; Carter (1986) "Site directed mutagenesis," Biochem.J.237: 1-7; and Kunkel ( 1987) "The efficiency of oligonucleotide directed mutagenesis," Nucleic Acids & Molecular Biology (Eckstein, F and Lilley, D.M.J eds., Springer Verlag, Berlin)); Mutagenesis using uracil-containing templates (Kunkel (1985) "Rapid and efficient site -specific mutagenesis without phenotypic selection, "Proc. Nat'l Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) "Rapid and efficient site-specific mutagenesis without phenotypic selection, "Results Probl. 382; and Bass et al. (1988) "Mutant Trprepressors with new DNA-binding specificities," Science 242: 240-245); Oligonucleotide-mediated mutagenesis (Results Probl. Cell Differ. 100: 468-500 (1983 ); Results Probl.Cell Differ.154: 329-350(1987); Zoller & Smith(1982) "Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any" DNA f Nucleic Acids Res.10: 6487-6500; Zoller & Smith (1983) "Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, "Results Probl. Cell Differ. 100: 468-500; simple method using two oligonucleotide primers and a singlestranded DNA template, "Results Probl.Cell Differ.154:329-350); DNA mutagenesis modified by phosphorothioate (Taylor et al. (1985) "The use of phosphorothioate-modified DNAin restriction enzyme reactions to prepare nickeled DNA, "Nucl.Acids Res.13:8749-8764; Taylor et al. (1985) "The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, "Nucl.Acids-Res.1 8787(1985)；Nakamaye & Eckstein(1986)″Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis，″Nucl Acids Res.14：9679-9698；Sayers等(1988)″Y-T Exonucleases in phosphorothioate -based ligonucleotide-directed mutagenesis, "Nucl. Acids Res. 16: 791802; and Sayers et al. (1988) "Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, "03.1-Acids: 8 814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) "The gapped duplex DNA approach to oligonucleotide-directed mutation construction," Nucl. Acids Res. 12:9441-9456; Kramer & Fritz (1987) "Oligonucleotide directed construction of mutations via gapped duplex DNA, "Results Probl.CellDiffer.154:350-367; Kramer et al. (1988) "Improved enzymatic in vitroreactions in the gapped duplex DNA approach to oligonucleotide-directed constructionids of Nmutations.1" : 7207; and Fritz et al. (1988) "Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro," Nucl. Acids Res. 16: 6987-6999).

Other suitable methods include point mismatch repair (Kramer et al. (1984) "Point Mismatch Repair," Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13 vectors, "Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved oligonucleotide-directed mutagenesis using M13 vectors, "Results Probl. Cell Differ. 154: 382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) "Use of oligonucleotides to generate largedeletions," Nucl.Acids Res. 14:5115), restriction-selection and restriction-selection and restriction-purification (Wells et al. (1986) "Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, "Phil.Trans.R.Soc.Lond.A317:415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning of a genecoding for the ribonuclease S protein, "Science223:1299-1301; Sakamar and Khorana (1988) "Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducing), "Nucl.Acids Res.14:6361 -6372; Wells et al. (1985) "Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites," Gene34: 315323; and Grundstrom et al. (1985) "Oligonucleotide-directed mutagenesis by microscale' shot-gun's" Nucl.Acids Res.13:3305-3316), double-strand break repair (Mandecki (1986) "Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis," Proc. Nat'l Acad. Sci. USA, 83:7177-7181). Additional details on many of the above methods can be found in: Methods in Enzymology, Vol. 154, which also describes useful controls for vexing problems in various mutagenesis methods.

Random or semi-random mutagenesis using added or degenerate oligonucleotides can also be utilized (Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis," Biotechnology 10:297300; Reidhaar -Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes," Methods Enzvmol.208: 564-86; Lim and Sauer (1991) "The role of internal packing interactions in determining the structure and stability of a. protein," Mol.Biol.219:359-76; Breyer and Sauer (1989) "Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambdarepressor," J.Biol.Chem.264:13355-60); " (Crea, R; US Patents 5,830,650 and 5,798,208, and EP Patent 0527809B1) generate diversity.

In one aspect of the invention, error-prone PCR can be used to generate nucleic acid variants. Using this technique, PCR is performed under conditions of low copy fidelity of the DNA polymerase, such that a high frequency of point mutations are available along the full-length PCR product. Examples of this technique can be found in the above references, eg Leung et al. (1989) Technique 1:11-15 and Caldwell et al. (1992) PCR Methods Applic. 2:28-33. Similarly, assembly PCR can be used in methods involving the assembly of PCR products from a mixture of small DNA fragments. Multiple different PCR reactions can be performed in parallel in the same vial, using the product of one reaction to prime the product of another reaction. Sexual PCR mutagenesis can be used in which DNA molecules are randomly fragmented based on sequence homology, followed by exchange immobilization by primer extension in a PCR reaction, whereas in vitro on different, but sequence-related DNA Homologous recombination occurs between molecules. This method is described in the above references, see eg Stemmer (1994) Proc. Nat'l Acad. Sci. USA 91:10747-10751. Regression ensemble mutagenesis can be used, in which a protein mutagenesis algorithm is used to generate a diverse population of phenotypically related mutants, members of which differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. An example of this method is found in Arkin & Youvan (1992) Proc. Nat'l Acad. Sci. USA 89:7811-7815.

As noted above, oligonucleotide-mediated mutagenesis can be used in a method that allows site-specific mutations to be produced in any nucleic acid sequence of interest. Examples of this technique can be found in the above references, eg Reidhaar-Olson et al. (1988) Science, 241:53-57. Similarly, cassette mutagenesis can be used in methods for replacing small regions of double-stranded DNA molecules with synthetic oligonucleotide cassettes that differ from the native sequence. Oligonucleotides may contain, for example, complete and/or partially randomized native sequences.

In vivo (or ex vivo) mutagenesis can be used as a method of generating random mutations in desired DNA of any clone comprising, for example, propagation of DNA in E. coli strains whose DNA repair pathway or pathways carry There are mutations. These "mutator strains" have a higher rate of random mutations relative to their wild-type parents. Propagating the DNA in one of these strains will eventually produce random mutations within the DNA.

Exponential ensemble mutagenesis can be used to generate combinatorial libraries with a high percentage of unique, functional mutants, where a small set of residues is randomized in parallel to identify at each altered position the sequence that results in a functional protein. amino acid. An example of this approach can be found in Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552. Similarly, random and site-directed mutagenesis can be used. An example of this approach can be found in Arnold (1993) Current Opinion in Biotechnology 4:450-455.

Kits for mutagenesis, library construction and other diversity generating methods are also commercially available. For example, kits are commercially available from, e.g., Stratagene (e.g., QuickChange ^™ Site-Directed Mutagenesis Kit; and Chameleon ^™ Double-Stranded, Site-Directed Mutagenesis Kit), Bio/Can Scientific, Bio-Rad (e.g. using the Kunkel method described above), Boehringer Mannheim Corp., Clonetech Laboratories, DNATechnologies, Epicentre Technologies (e.g. 5prime 3prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), New England Biolabs, PharmaciaBiotech, Promega Corp., Quantum Biotechnologies, Amersham International plc (e.g. using Eckstein method above) and Anglian Biotechnology Ltd (for example using the Carter/Winter method above).

Any of the shuffling or mutagenesis techniques described may be used in conjunction with methods for introducing additional diversity into genomes, such as bacterial, fungal, animal or plant genomes. For example, in addition to the methods described above, techniques have been proposed that produce chimeric nucleic acid polymers suitable for transformation into multiple species (see, eg, Schellenberger US Patent 5,756,316 and the above references). Nucleic acid can be provided when such chimeric multimers are composed of genes that diverge from each other (e.g., generated from natural diversity or using site-directed mutagenesis, error-prone PCR, mutagenic bacterial strain pathways, etc.), and transformed into an appropriate host Diversity sources to diversify DNA.

Chimeric multimers transformed into host species are suitable as substrates for in vivo (or ex vivo) shuffling methods. Alternatively, multiple polynucleotides sharing regions of partial sequence similarity or homology can be transformed into a host species and recombined in vivo (or ex vivo) by the host cells. Subsequent rounds of cell division can be used to generate libraries whose members contain a single, uniform population of monomeric or focused nucleic acids. Alternatively, monomeric nucleic acids can be recovered by standard techniques and recursively recombined in any of the described shuffling methods.

Chain termination methods that generate diversity have also been suggested (see eg US Patent 5965408 and references cited above). In this method, double-stranded DNA corresponding to one or more genes that share regions of sequence similarity or homology is mixed and denatured in the presence or absence of gene-specific primers. The single-stranded polynucleotides are then annealed, and exposed to polymerase and chain-terminating reagents (such as ultraviolet, gamma, or X-ray irradiation; ethidium bromide or other intercalators; DNA-binding proteins, such as single-strand binding proteins, transcription activators or moieties). proteins; polycyclic aromatic hydrocarbons; trivalent chromium or trivalent chromium salts; or shortening polymerization mediated by rapid thermal cycling, etc.), resulting in partial duplex molecules. Partial duplex molecules, such as those containing partially extended strands, are then denatured and re-annealed in subsequent rounds of replication or partial replication, resulting in polynucleotides that share varying degrees of sequence similarity or homology, and are chimeric polynucleotides with respect to the original population of DNA molecules. Optionally, the product or a partial collection of the product may be amplified at one or more stages of the method. Polynucleotides generated by, for example, chain termination methods described above are suitable substrates for diversity generation methods (eg RSR, DNA shuffling) according to any of the described forms.

Diversity can be further increased by using non-homology-based methods and DNA shuffling (which can be homology-based or non-homology-based, depending on the exact format, as described in the aforementioned publications and applications). For example, the initial recombinant library can be generated using incremental truncation (ITCHY) for generating hybrid enzymes described in Ostermeier et al. (1999) "A combinatorial approach to hybridenzymes independent of DNA homology" Nature Biotech. 17:1205, which Libraries can be used as substrates for one or more rounds of in vitro, ex vivo or in vivo diversity generation methods such as RSR or shuffling methods.

Methods for generating multi-species expression libraries have been described (eg US Patents 5,783,431; 5,824,485 and the above references) and it has been suggested that these methods can be used to identify desired protein activities (US Patent 5,958,672 and the above references). In general, multi-species expression libraries contain cDNA or genomic sequences from multiple species or strains operably linked in expression cassettes to appropriate regulatory sequences. Optionally random concatenation of cDNA and/or genomic sequences is performed to further increase diversity. The vector may be a shuttle vector suitable for transformation and expression in more than one host organism, such as bacterial species, eukaryotic cells. In some cases, the library is biased by pre-selecting sequences encoding the desired protein, or hybridizing to the desired nucleic acid. Any such library can be a substrate for any of the methods described herein.

In some applications, preselection or prescreening of libraries (e.g., amplified libraries, genomic libraries, cDNA libraries, normalized libraries, etc.) or other substrate nucleic acids prior to shuffling is required, or substrates are biased towards encoding functional products of nucleic acids (the shuffling method itself also has these effects). For example, in engineering antibodies, recombination events in vivo (or ex vivo or in vitro) can be used to bias the shuffling approach towards those with functional antigen binding sites prior to generating diversity by any of the methods described (e.g., DNA shuffling). Antibody. For example, recombinant CDRs from a B cell cDNA library can be amplified and assembled into framework regions prior to generating diversity according to any of the methods described herein (e.g., DNA shuffling) (e.g., Jirholt et al. (1998) "Exploiting sequence space : shuffling in vivo formed complementarity determining regions into a master framework, "Gene 215:471).

Libraries can be biased towards nucleic acids encoding proteins with desired activities (eg, binding affinity, enzymatic activity, antiviral activity, ability to induce an immune response, antiproliferative activity, adjuvant properties, etc.). For example, after identifying clones from a library with specific activities, the clones can be mutagenized using any known method for introducing DNA changes, including but not limited to DNA shuffling or other forms of recurrent sequence recombination or diversity generation. . Libraries containing mutagenized homologues are then screened for the desired activity, which may or may not be the same as the originally specified activity. An example of this approach can be found in US Patent 5,939,250. Desired activities can be identified by any method known in the art. For example, WO99/10539 mentions that gene libraries can be screened by mixing extracts of the gene library with fractions obtained from metabolically active cells and identifying combinations exhibiting the desired activity. It has also been proposed (for example WO98/58085): by inserting a substrate with biological activity into a library sample, and using a fluorescence analyzer, such as a flow cytometer, CCD, fluorometer or spectrophotometer, to detect the substrate corresponding to the desired activity. The bioactive fluorescence of the product allows the identification of clones with the desired activity.

Libraries can also be biased towards nucleic acids with particular characteristics, such as nucleic acids that hybridize to selected nucleic acid probes. For example, application WO99/10539 proposes that the coding desired activity (e.g. enzymatic activity such as lipase, esterase, protease, glycosidase, glycosyltransferase, phosphatase, kinase) can be identified from the genomic DNA sequence as follows: , oxygenase, peroxidase, hydrolase, hydratase, nitrilase, transaminase, amidase or acyltransferase) polynucleotide. Single-stranded DNA molecules in a population of genomic DNA are hybridized to ligand-conjugated probes. Genomic DNA can be obtained from cultured or uncultured microorganisms, or from environmental samples. Alternatively, genomic DNA can be obtained from a multicellular organism or tissue thereof.

Second strand synthesis can be performed directly from the hybridization probes used in capture, which may or may not have been previously released from the capture medium, or by a variety of other strategies known in the art. chain. Alternatively, isolated single-stranded genomic DNA populations can be fragmented without further cloning and used directly in shuffling-based gene reassembly methods. In one such method, a population of fragments from a genomic library is annealed to portions, or often about full length, of ssDNA or RNA corresponding to opposite strands. Assembly of complex chimeric genes from this population is mediated by ribozyme-based removal of unhybridized fragment ends, polymerization to fill gaps between these fragments, and subsequent single-strand ligation. The parental strand is removed by digestion (if RNA or containing uracil), magnetic separation under denaturing conditions (if labeled in a manner to facilitate such separation), or other isolation/purification methods that may be used. Alternatively, the parental strand is optionally purified together with the chimeric strand, and the parental strand is removed in subsequent screening and processing steps. As stated in "Single-stranded nucleic acid template-mediated recombination and nucleic acid fragment isolation" by Affholter (USSN60/186,482, filed March 2, 2000), and WO98/27230, "Methods and Compositions for Polypeptide Engineering" by Patten and Stemmer As described above, shuffling using a single-stranded template and a desired nucleic acid that binds to a portion of the template can also be performed.

In one method, single-stranded molecules are converted to double-stranded DNA (dsDNA), and the dsDNA molecules are bound to a solid support by ligand-mediated binding. Following isolation of unbound DNA, selected DNA molecules are released from the support and introduced into appropriate host cells to generate library-enriched sequences that hybridize to the probes. Libraries generated according to this method provide desirable substrates for any of the shuffling reactions described herein.

"Non-stochastic" methods of generating nucleic acids and polypeptides are described in Short, J. "Non-Stochastic Generation of Genetic Vaccines and Enzymes" WO 00/46344. Such methods including proposed non-random polynucleotide reassembly and gene site saturation mutagenesis, as well as the synthetically linked polynucleotide reassembly methods described herein are also useful in the present invention.

It will be readily appreciated that any of the techniques described above that are suitable for enriching libraries prior to diversification can also be used to screen products or product libraries generated by diversity generating methods.

Recombinant nucleic acids produced by regressive recombination of one or more polynucleotides of the invention with one or more other nucleic acids also form part of the invention. One or more other nucleic acids may comprise another polynucleotide of the invention; optionally, alternatively, or in addition, one or more other nucleic acids may comprise, for example, a sequence encoding native interferon-alpha or a subsequence thereof, or Nucleic acid of any homologous interferon-alpha sequence or subsequence thereof, or interferon-beta sequence or subsequence thereof (such as interferon-alpha or interferon-beta sequences described in GenBank or other available literature), Or for example any other nucleic acid, homologous or non-homologous (certain forms of recombination mentioned above, especially recombination carried out by synthetic or in silico methods, do not require homology).

As described in detail in the above references, the recombination step can be performed in vivo, ex vivo, in vitro or in silico. The invention also includes cells containing any of the resulting recombinant nucleic acids, nucleic acid libraries produced by multiplicity, recombination or regressive recombination of the nucleic acids described herein, cell populations containing said libraries or containing any of the recombinant nucleic acids, vectors, viruses, plasmids etc., wherein the recombinant nucleic acid is produced by diversification or recombination (or regressive recombination) of a nucleic acid described herein with another such nucleic acid or other nucleic acid. The corresponding sequence chain in a database stored in a computer system or computer readable medium is also a feature of the invention. Other polynucleotide compositions

The invention also includes compositions comprising two or more polynucleotides of the invention (eg, for use as substrates for recombination). The composition may contain a library of recombinant nucleic acids, wherein the library contains at least 2, 3, 5, 10, 20 or 50 or more nucleic acids. The nucleic acid is optionally cloned into an expression vector, providing an expression library.

The invention also includes compositions produced by digesting one or more polynucleotides of the invention (in some of the above recombinant formats) with restriction endonucleases, RNases or DNases; and by mechanically Compositions produced by fragmenting or shearing one or more polynucleotides of the invention by means (eg, sonication, vortexing, etc.) can also be used to provide recombinant substrates in the methods described above. Similarly, compositions containing sets of oligonucleotides corresponding to more than one nucleic acid of the invention are also useful as recombination substrates and are also a feature of the invention. For convenience, these fragmented, sheared or oligonucleotide-synthesized mixtures are referred to as fragmented nucleic acid sets.

The invention also includes compositions produced by incubating one or more fragmented nucleic acid sets in the presence of ribonucleotide- or deoxyribonucleotide triphosphates and a nucleic acid polymerase. The resulting composition forms a reconstitution mixture that can be used in the various forms of recombination described above. The nucleic acid polymerase can be RNA polymerase, DNA polymerase or RNA-mediated DNA polymerase (eg "reverse transcriptase"); the polymerase can be eg a thermostable DNA polymerase (eg VENT, TAQ, etc.). interferon homologue polypeptide

The invention provides isolated or recombinant interferon-alpha homolog polypeptides, also referred to herein as "interferon-alpha homologs" or "interferon homologs" or "IFN-alpha homologs" or "IFN homologs" . The isolated or recombinant interferon homologue polypeptides of the present invention include: polypeptides containing sequences selected from SEQ ID NO: 36 to SEQ ID NO: 70 and SEQ ID NO: 79 to SEQ ID NO: 85, and conservative modifications thereof variants, and fragments thereof, which have anti-proliferative activity in, for example, an assay based on the human Daudi cell line (or other similar assays), and/or in an assay based on, for example, the murine cell line/EMCV (or other similar assays) ) has antiviral activity. Figure 1 provides a sequence alignment of exemplary interferon homologue polypeptide sequences of the present invention. The polypeptide sequences of the invention can be readily aligned to each other, or to known native interferon-alpha sequences, by those skilled in the art using public databases and sequence alignment programs.

The present invention also provides a polypeptide comprising at least about 100, 120, 130, 140, 150, 155, 160, 163, 165 or 166 of any one of SEQ ID NO: 36-70 or SEQ ID NO: 71 consecutive amino acids. In one aspect, the amino acid sequence contains amino acids Lys160 and Glu166, wherein the amino acid numbers in the sequence correspond to those in SEQ ID NO: 36 one-to-one.

Comparing the exemplified sequences of the invention (Figure 1) with known natural interferon-α and other type I interferons (including β, δ, ω, τ-interferons) sequences derived from humans and non-humans, Several conclusions can be drawn. These sequences are readily available from a variety of sources such as GenBank and the Pfam (Protein Families) database at http://www.sanger.ac.uk/Software/Pfam/index.Shtml.

It should be noted that the following amino acid residues (represented as "Group I" residues) exist in some interferon homologue polypeptide sequences of the present invention, and these residues do not appear in known natural human or non-human I At the equivalent position of the type interferon sequence.

Group I: Asp11; Pro14; Arg50; Phe55; Asp75; Asn80; Pro111; Leu124; Glu134; Ser140 and Ala143; residue numbering corresponds to the mature interferon homologue sequence identified in SEQ ID NO:36.

It should be noted that the following amino acid residues (represented as "group II" residues) exist in some interferon homologue polypeptide sequences of the present invention, and these residues do not appear in known natural human interferon-alpha At the equivalent position of the subtype sequence.

Group II: Pro9; (Lys, Ser) 12; (Thr, Val) 24; Gln34; Arg40; Ser45; Arg47; Leu56; 104; Val112; Gly114; Pro116; Lys133 and His136.

In other embodiments, the interferon homologue polypeptide comprises at least 20, 50, 100, 150, 155 or 160 or more contiguous amino acids of any one of SEQ ID NOS: 36-70, and/or one or more of the following amino acids Ala19, (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein the numbering of amino acids corresponds to the numbering in SEQ ID NO: 36, or one or more of the following amino acids Pro9, (Lys or Ser) 12, (Thr or Val) 24, Gln34, Arg40, Ser45, Arg47, Leu56, Ile60, Phe67, Ala79, Gly88, His90, Arg91, Glu95, Val101, (Gly, Ala) 104 , Val112, Gly114, Pro116, Lys133 and His136, wherein the numbering of amino acids in the polypeptide sequence corresponds to the numbering of each amino acid in the amino acid sequence of SEQ ID NO:36. Thus, for example, in this embodiment, the interferon polypeptide comprises an amino acid sequence comprising a proline residue at amino acid position 9 of the sequence, a lysine or serine residue at position 12, and a residue at amino acid position 24. A threonine or valine residue is contained at the 34th position, a glutamine residue at the 34th position, an arginine residue at the 40th position, etc. The polypeptide exhibits antiproliferative activity (e.g., at least about 8.3 x ¹⁰⁶ units/mg) in a proliferation assay based on the human Daudi cell line, and/or exhibits antiviral activity in an assay based on human WISH cells/EMCV Activity (at least about 2.1×10 ⁷ units/mg). Some of these polypeptides bind human alpha interferon receptors. Some such polypeptides are 166 amino acids in length. In another aspect, such a polypeptide may comprise a sequence selected from any one of SEQ ID NO:36 to SEQ ID NO:54.

The antiproliferative activity of any polypeptide of the invention is generally related to the ability of the polypeptide to cause growth of cells or parts thereof or to rapidly, often repeatedly generate new cell growth.

The present invention also includes polypeptides (such as any of SEQ ID NOs: 36-71 or SEQ ID NOs: 79-85) or nucleic acids encoding polypeptides (such as SEQ ID NOs: 1-35 or SEQ ID NOs: 72-78) any of ), wherein the polypeptide has anti-angiogenic activity as determined by an anti-angiogenic assay well known to those skilled in the art.

The present invention also includes:

(a) Any interferon-alpha polypeptide comprising one or more of the Group I amino acid residues described above.

(b) In human-like interferon sequence (that is, a sequence showing a high level of similarity or homology to human interferon), or highly similar or homologous to any sequence or fragment thereof listed in the attached sequence listing (i.e. having a sequence homology or percent sequence identity of at least about 80%, 90%, 95%, 96%, 97%, 98% or more) in the context of sequences containing one or more of the above Group II Any interferon-alpha polypeptide of amino acid residues.

(c) any interferon-alpha polypeptide containing the following combination of residues located at or near a region of the interferon-alpha molecule known or believed to interact with a type I interferon receptor, wherein the sequence combination (motif) does not occur in any known equivalent position of natural human or non-human type I interferon:

(i) (Tyr or Gln)34; plus one or more of Ile132 or Arg134; or

(ii) Asp78, Glu79 or (Asp or Thr)80; plus one or more of Ile132 or Arg134.

In another embodiment, the present invention provides a sequence comprising SEQ ID NO: 71: CDLPQTHSLG-X ₁₁ -X ₁₂ -RA-X ₁₅ -X ₁₆ -LL-X ₁₉ -QM-X ₂₂ -RX ₂₄ - SX ₂₆ -FSCLKDR-X ₃₄ -DFG-X ₃₈ -PX ₄₀ -EEFD-X ₄₅ -X ₄₆ -X ₄₇ -FQ-X ₅₀ -X ₅₁ -QAI-X ₅₅ -X ₅₆ -X ₅₇ -HE-X ₆₀ -X ₆₁ -QTFN-X ₆₇ -FSTK-X ₇₂ -SS-X ₇₅ -X ₇₆ -WX ₇₈ -X ₇₉ -X ₈₀ -LL-X ₈₃ -KX ₈₅ -X ₈₆ -TX ₈₈ -LX ₉₀ -QQLN- X ₉₅ -LEACV-X ₁₀₁ -QX ₁₀₃ -VX ₁₀₅ -X ₁₀₆ -X ₁₀₇ -X ₁₀₈ -TPLMN-X ₁₁₄ -DX ₁₁₆ -ILAV-X ₁₂₁ -KY-X ₁₂₄ -QRITLYL-X ₁₃₂ -EX ₁₃₄ -KYSPC -X ₁₄₀ -WEVVRAEIMRSFSFSTNLQKRLRRKE, or conservatively substituted variants thereof, wherein X ₁₁ is N or D; X ₁₂ is R, S or K; X ₁₅ is L or M; X ₁₆ is I, M or V; X ₁₉ is A or G; X ₂₂ is G or R; X ₂₄ is I or T; X ₂₆ is P or H; X ₃₄ is H, Y or Q; X ₃₈ is F or L; X ₄₀ is Q or R; X ₄₅ X ₄₆ is N or H; X ₄₇ is Q or R; X ₅₀ is K or R; X ₅₁ is A or T; X ₅₅ is S or F; X ₅₆ is V or A; X ₅₇ is L or F; X ₆₀ is M or I; X ₆₁ is I or M; X ₆₇ is L or F; X ₇₂ is D or N; X ₇₅ is A or V; X ₇₆ is A or T; X ₇₈ is E or D; X ₇₉ is Q or E; X ₈₀ is S, R, T or N; X ₈₃ is E or D; X ₈₅ is F or L; X ₈₆ is S or Y; X ₈₈ is E or G; X ₉₀ is Y, H, N; X ₉₅ is D, E or N; X ₁₀₁ is I, M or V; X ₁₀₃ is E or G; X ₁₀₅ is G or W; X ₁₀₆ is V or M; X ₁₀₇ is E, G or K; X ₁₀₈ is E or G; X ₁₁₄ is V, E or G; X ₁₁₆ is S or P; X ₁₂₁ is K or R; X ₁₂₄ is F or L; X ₁₃₂ is T, I or M; X ₁₃₄ is K or R; and X ₁₄₀ is A or S; or an interferon alpha homolog of a fragment of said SEQ ID NO:71. In another aspect, the interferon homologue polypeptide of SEQ ID NO: 71 or a fragment thereof exhibits anti-proliferative activity (at least about 8.3×10 ⁶ units/mg) in a proliferation assay based on human Daudi cell line, and/or Shows antiviral activity (at least about 2.1 x ¹⁰⁷ units/mg) in a human WISH cell/EMCV-based assay. Both tests are discussed in detail below. Such a polypeptide may comprise an amino acid sequence selected from SEQ ID NO:36 to SEQ ID NO:54, or may be encoded by a nucleotide sequence selected from SEQ ID NO:1 to SEQ ID NO:19.

Fragments of the interferon homolog polypeptides described herein are also a feature of the invention. Interferon alpha homologue fragments of the present invention generally comprise interferon homologue polypeptides containing at least about 20, 25 or 30, generally at least about 40, of any of SEQ ID NOs: 36-71 or SEQ ID NOs: 79-85, 50, 60, 70, 80, 90 or 100 consecutive amino acids. In other embodiments, the fragment generally contains at least about 100, 110, 120, 125, 130, 140, 150, 155, 158, 160, 162 of any of SEQ ID NO: 36-71 or SEQ ID NO: 79-85 , 163, 164 or 165 consecutive amino acids. Such polypeptide fragments have antiproliferative activity in assays based on the human Daudi cell line, and/or antiviral activity in assays based on human or murine cell lines/EMCV.

In other embodiments, the invention provides polypeptides that are 166 amino acids in length, and in some such embodiments, the polypeptides have antiproliferative activity in human Daudi cell line based assays (or other similar assays), including For example at least about 8.3 x ¹⁰⁶ units/mg, and/or have antiviral activity in a human WISH cell/EMCV based assay (or other similar assay), including for example at least about 2.1 x ¹⁰⁷ units/mg .

In other embodiments, the invention provides polypeptides comprising at least 100, 150, 155 or 160 contiguous amino acids of a protein encoded by an encoding polynucleotide sequence comprising any of : (a) SEQ ID NO: 1 to SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78; (b) coded from SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to The polynucleotide sequence encoding the first polypeptide of any one of SEQ ID NO: 85; and (c) under hybridization conditions of at least high stringency (or ultra-high stringency or ultra-ultra-high stringency conditions), with basic A complementary polynucleotide sequence hybridized to the full-length polynucleotide sequence of (a) or (b). Such polypeptides have antiproliferative activity in an assay based on the human Daudi cell line (or other similar assays), and/or have antiviral activity in an assay based on human WISH cells/EMCV (or other similar assays). Some such polypeptides of the invention specifically bind human alpha interferon receptors. The polypeptides and nucleic acids of the present invention need not be associated with target molecules or related molecules, including polypeptides or fragments thereof of any one of SEQ ID NO: 36-71 (including those having antiviral or antiproliferative activity in the assays described herein), Or the corresponding sequences of any one of the nucleic acids of SEQ ID NO: 1-35 or fragments thereof (including those having antiviral or antiproliferative activity in the assays described herein) are identical, but may be substantially identical thereto. Various changes can be made to the polypeptide, such as conservative or non-conservative insertions, deletions and substitutions, where such changes confer advantages in its use. Polypeptides of the invention can be modified in a variety of ways so long as they contain sequences that are substantially identical (as defined below) to, or have a certain percentage identity to, the sequence of a native or known interferon polypeptide molecule.

Aligning and comparing relatively short amino acid sequences (less than about 30 residues) is generally facile. Comparing longer sequences requires more sophisticated methods to obtain an optimal alignment of two sequences. By the local homology algorithm of Smith and Waterman (1981) Adv.Appl.Math.2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol.Biol 48:443, by Pearson and Lipman ( 1988) Proc.Nat'l Acad.Sci.(USA) 85:2444 similarity retrieval method, by running these algorithms (GAP, BESTFIT, FASTA and TFASTA, Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI), or by examining the best sequence alignment for which an alignment comparison window can be made, selecting the best alignment (ie, resulting in the highest percent sequence similarity over the comparison window) produced by multiple methods.

The term sequence identity means that two polynucleotide sequences are identical (ie, on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions in the two sequences at which the same residue occurs to yield the number of matching positions, using the matching position The number is divided by the total number of positions in the comparison window (ie, the window size) and the result is multiplied by 100 to give the percent sequence identity. In one aspect, the invention provides an interferon homologue nucleic acid having at least about 80 %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher percent sequence identity.

As applied to polypeptides, the term substantially identical means that two peptide sequences share at least about 80% when optimally aligned, for example by the programs GAP or BESTFIT using default gap weighting values (described in more detail below). % sequence identity, preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity or higher (eg, 97, 98 or 99% sequence identity). Preferably, different residue positions differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; a The group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is Lysine, arginine, and histidine; a group of amino acids with sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine- glutamine. In one aspect, the invention provides an interferon homologue polypeptide having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher percent sequence identity.

A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm described in Pearson, W.R. & Lipman, D.J., 1988, Proc. Nat'l Acad. Sci. USA 85:2444. See also W.R. Pearson, 1996, Methods Enzymol. 266:227-258. The preferred parameters used in the FASTA alignment of DNA sequences for calculating percent identity are optimized, BL50 matrix 15:-5, k-tuple=2; join penalty=40, optimization=28; gap penalty-12, Gap Length Penalty = -2; and Width = 16.

Another preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST2.0 algorithms, respectively described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST and BLAST2.0 with the parameters described herein are used to determine percent sequence identities for nucleic acids and proteins of the invention. Software for performing BLAST analyzes is publicly available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, can match Or coincide with a partially positive threshold T. T is referred to as the adjacent byte score threshold (Altschul et al., supra). These raw adjacent byte hits can be used as clues to initiate searches to find longer HSPs containing them. Hotspot bytes are extended in both directions along each sequence until the cumulative alignment score is increased. 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 is used to calculate the cumulative score. Stop extending hotspot bytes in each direction when: the cumulative alignment score drops by X compared to its achievable maximum; Accumulated, the cumulative score is zero or below; or the end of the sequence has been reached. The BLAST algorithm parameters W, T and X measure the sensitivity and speed of the sequence alignment. The BLASTN program (for nucleotide sequences) uses the following defaults: wordlength (W) of 11, expectation (E) of 10, M=5, N=-4, and compares both strands. For amino acid sequences, the BLASTP program uses the following defaults: wordlength 3, expectation (E) 10, BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Nat'l Acad. Sci. U.S.A. 89: 10915 ) sequence alignment (B) is 50, expectation (E) is 10, M=5, N=-4, and both strands are compared.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, eg, Karlin & Altschul (1993) Proc. Nat'l Acad. Sci. U.S.A. 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the likelihood by which a match between two nucleotide or amino acid sequences occurred by chance. For example, a nucleic acid may be considered similar to a reference sequence if the smallest sum probability found when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP generates multiple sequence alignments from a set of related sequences using progressive pairwise sequence alignments to show relationships and percent sequence identities. The algorithm can also draw dendrograms showing the clustering relationships used to generate sequence alignments. PILEUP uses a simplified approach to the incremental sequence alignment method described by Feng & Doolittle (1987) J. Mol. Evol. 35:351-360. The method used was similar to that described by Higgins & Sharp (1989) CABIOS 5: 151-153. The program can align up to 300 sequences, each of a maximum length of 5000 nucleotides or amino acids. Multiple alignment methods begin with the pairwise alignment of the two most similar sequences, which produces a cluster of two aligned sequences. This cluster is then sequence aligned to the next most related sequence or cluster of aligned sequences. Sequence alignment of two clusters of sequences is performed by simply extending the pairwise alignment of two independent sequences. The final sequence alignment is obtained through a series of progressive pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison, and by designating program parameters. Using PILEUP, a reference sequence is compared to other subject sequences to determine percent sequence identity relationships using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP is available from the GCG sequence analysis software package, eg, version 7.0 (Devereaux et al. (1984) Nuc. Acids Res. 12:387-395)).

Another preferred example of an algorithm suitable for alignment of multiple DNA and amino acid sequences is the CLUSTALW program (Thompson, JD et al. (1994) Nucl. Acids. Res. 22:4673-4680). ClustalW performs multiple pairwise comparisons between sets of sequences and assembles them into multiple sequence alignments based on homology. Gap opening and gap extension penalties are 10 and 0.05, respectively. For amino acid sequence alignments, the BLOSUM algorithm can be used as a protein weighting matrix (Henikoff and Henikoff (1992) Proc. Nat'l Acad. Sci. USA89:1091510919). Preparation of polypeptides of the invention

Recombinant methods for producing and isolating interferon homolog polypeptides of the invention are described above. In addition to recombinant production, polypeptides can be produced by direct peptide synthesis using solid-phase techniques (see Stewart et al. (1969) Solid-Phase Peptide Synthesis, W.H. Freeman Co, San Francisco; Merrifield, J. (1963) J. Am. Chem. Soc. 85:2149-2154). Peptide synthesis can be performed using manual techniques or by automation. For example, automatic synthesis can be performed using the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) according to the instructions provided by the manufacturer. For example, the subsequences can be chemically synthesized separately and combined using chemical means to provide a full-length interferon homologue. Fragments of the interferon homologue polypeptides of the invention discussed in detail above are also a feature of the invention and may be synthesized by using the methods described above.

The polypeptide is produced by introducing into a population of cells a nucleic acid of the invention operably linked to regulatory sequences effective to produce the encoded polypeptide, culturing the cells in culture medium, and optionally isolating the polypeptide from the cells or culture medium, i.e. Polypeptides of the invention can be produced.

On the other hand, by introducing a recombinant expression vector containing at least one nucleic acid of the present invention (wherein at least one nucleic acid is operably linked to a regulatory sequence capable of effectively producing the encoded polypeptide) in the cell population, under appropriate conditions in the culture medium The polypeptides of the invention are produced by culturing cells to produce the polypeptide encoded by the expression vector, and optionally isolating the polypeptide from the cells or culture medium. Peptide Antibody

In another aspect of the invention, the interferon homolog polypeptides of the invention are used to produce antibodies that have, for example, diagnostic, prophylactic and therapeutic uses related to, for example, the activity, distribution and expression of the interferon homolog.

Antibodies against interferon homologues of the invention can be raised by methods well known in the art. Such antibodies include, but are not limited to: polyclonal, monoclonal, chimeric, humanized, single chain antibodies, Fab fragments and fragments produced by Fab expression libraries. Antibodies that block receptor binding are particularly preferred for therapeutic or prophylactic use.

Interferon homolog polypeptides used to induce antibodies need not be biologically active; however, the polypeptide or oligopeptide must be antigenic. The peptide used to induce specific antibodies may have an amino acid sequence consisting of at least 10 amino acids, preferably at least 15 or 20 amino acids. A short stretch of interferon homolog polypeptide can be fused to another protein, such as keyhole limpet hemocyanin, and antibodies raised against the chimeric molecule.

Methods for producing polyclonal and monoclonal antibodies are known to those skilled in the art, and many antibodies are available. See, eg, Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications , Los Altos, CA, and references cited therein; Goding (1986) Monoclonal Antibodies: Principles and Practice (2ded.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256:495-497. Other suitable antibody production techniques include selection of recombinant antibody libraries in phage or similar vectors, see Huse et al (1989) Science 246:1275-1281; and Ward et al (1989) Nature 341:544-546. Certain monoclonal and polyclonal antibodies and antisera generally bind with a _KD of at least about 0.1 μM, preferably at least about 0.01 μM or better, most commonly and preferably 0.001 μM or better.

Detailed methods for making chimeric (humanized) antibodies can be found in US Patent No. 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed.) (1995) Antibody Engineering, ^2nd Edition, Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A Practical Approach , IRL at Oxford Press, Oxford, England (McCafferty), and Paul (1995) Antibody Engineering Protocols, Humana Press, Towata, NJ (Paul).

In a useful embodiment, the invention provides fully humanized antibodies directed against interferon homologs of the invention. Humanized antibodies are particularly desirable in applications where antibodies are used as prophylactic and therapeutic agents in vivo and ex vivo in human patients. Human antibodies consist of sequences characteristic of human immunoglobulins. Human antibodies of the invention can be produced using a variety of methods (see, eg, Larrick et al., US Patent 5,001,065 and Borrebaeck McCafferty and Paul, supra, for reviews). In one embodiment, the human antibodies of the invention are initially produced in trioma cells. The gene encoding the antibody is then cloned into and expressed in other cells, such as non-human mammalian cells. General methods for the production of human antibodies by trioma technology are described in Ostberg et al. (1983), Hybridoma 2:361367, Ostberg, US Patent 4,634,664 and Engelman et al., US Patent 4,634,666. The antibody-producing cell lines obtained by this method are called triomas because they are the descendants of three cells; two human cells and one mouse cell. Trioma has been found to produce antibodies more stably than normal hybridomas prepared from human cells. Adjuvant

In one aspect, the interferon homologue polypeptides or fragments thereof of the invention are useful as adjuvants to stimulate, enhance, potentiate or amplify the immune response associated with the antigen when administered with the antigen or after or prior to delivery of the antigen. In another aspect, the invention provides methods of administering to a subject one or more polypeptides of the invention described herein. Therapeutics and Preventives

As described in detail below, the interferon homologue polypeptides or fragments thereof of the present invention can be used for the prophylaxis and/or therapeutic treatment of various diseases.

For example, the invention provides interferon-alpha homolog polypeptides (and interferon-alpha homolog nucleic acids encoding such polypeptides) that have antiviral and antiproliferative activity in the assays described herein. In one aspect, the invention provides interferon-alpha homolog polypeptides (and interferon-alpha homolog nucleic acids encoding such polypeptides) wherein the ratio of antiviral activity to antiproliferative activity is greater than that of other known interfering agents mentioned herein Prime-alpha, the corresponding ratios as those listed in GenBank. The polypeptides (and nucleic acids encoding them) are useful in the therapeutic and/or prophylactic treatment of various diseases, for example in the treatment regimens for hepatitis B, hepatitis C, HIV and HSV. Some of these polypeptides (and nucleic acids encoding them), such as the interferon-alpha homologue 2BA8, have significant advantages over known interferon-alpha compounds in this treatment regimen because, relative to known interfering As far as interferon-alpha compounds are concerned, such as interferon-alpha 2a, they may exhibit lower side effects after administration, higher potency, and thus require lower doses, resulting in less immunogenic consequences. Sequence variation Conservatively modified variation

The interferon homolog polypeptides of the present invention include one or more conservatively modified variations ( or "conservative variation" or "conservative substitution"). Such conservatively modified variations include substitutions, additions or deletions which are altered, added or deleted in any of SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85 A single amino acid or a small percentage of amino acids (generally less than about 5%, more preferably less than about 4%, 2% or 1%) are omitted.

For example, conservatively modified variations (e.g. deletions) of the 166 amino acid long polypeptide identified herein as SEQ ID NO: 36 are at least about 157 or 158 amino acids in length, preferably at least about 159 or 160 amino acids in length, more preferably at least about 159 or 160 amino acids in length. Preferably at least about 162 or 163 amino acids, still more preferably at least about 164 or 165 amino acids, which correspond to a deletion of less than about 5%, 4%, 2% or 1% of the polypeptide sequence, respectively.

Another example of a conservatively modified variation (e.g., a "conservatively substituted variation") identified herein as SEQ ID NO: 36 is in about 8 residues (i.e., less than about 5% of the 166 amino acid long polypeptide). ) contains "conservative substitutions" according to the six substitution groups shown in Table 2 (see above).

The interferon homolog polypeptide sequences of the invention, including conservatively substituted sequences, may exist as part of a larger polypeptide sequence, for example, by adding one or more domains to facilitate protein purification (e.g., polyhistidine acid segment, FLAG epitope segment, etc.), for example when other functional domains have little or no effect on the activity of the interferon-alpha portion of the protein, or by post-synthetic processing steps, for example by using Protease treatment can remove other domains.

In another embodiment, an interferon homolog polypeptide of the invention comprises the following sequence identified herein as SEQ ID NO: 71: CDLPQTHSLG-X ₁₁ -X ₁₂ -RA-X ₁₅ -X ₁₆ -LL-X ₁₉ -QM-X ₂₂ -RX ₂₄ -SX ₂₆ -FSCLKDR-X ₃₄ -DFG-X ₃₈ -PX ₄₀ -EEFD-X 45 -X ₄₆ -X _{47 -FQ} -X ₅₀ -X ₅₁ -QAI-X ₅₅ - X ₅₆ -X ₅₇ -HE-X ₆₀ -X ₆₁ -QTFN-X ₆₇ -FSTK-X ₇₂ -SS-X ₇₅ -X 76 -WX ₇₈ -X ₇₉ -X _{80 -LL} -X ₈₃ -KX ₈₅ -X ₈₆ -TX ₈₈ -LX ₉₀ -QQLN-X ₉₅ -LEACV-X ₁₀₁ -QX ₁₀₃ -VX ₁₀₅ -X ₁₀₆ -X ₁₀₇ -X ₁₀₈ -TPLMN-X ₁₁₄ -DX ₁₁₆ -ILAV-X ₁₂₁ -KY-X ₁₂₄ -QRITLYL-X ₁₃₂ -EX ₁₃₄ -KYSPC-X ₁₄₀ -WEVVRAEIMRSFS FSTNLQKRLRRKE, or conservatively substituted variants thereof, wherein X ₁₁ is N or D; X ₁₂ is R, S or K; X ₁₅ is L or M; X ₁₆ is I, M or V; X ₁₉ is A or G; X ₂₂ is G or R; X ₂₄ is I or T; X ₂₆ is P or H; X ₃₄ is H, Y or Q; X ₃₈ is F or L; X ₄₀ is Q or R; X ₄₅ is G or S; X ₄₆ is N or H; X ₄₇ is Q or R; X ₅₀ is K or R; X ₅₁ is A or T; X ₅₅ is S or F ; X ₅₆ is V or A; X ₅₇ is L or F; X ₆₀ is M or I; X ₆₁ is I or M; X ₆₇ is L or F; X ₇₂ is D or N; X ₇₅ is A or V; X ₇₆ is A or T; X ₇₈ is E or D; X ₇₉ is Q or E; X ₈₀ is S, R, T or N; X ₈₃ is E or D; X ₈₅ is F or L; X ₈₆ is S or Y; X ₈₈ is E or G; X ₉₀ is Y, H, N; X ₉₅ is D, E or N; X ₁₀₁ is I, M or V; X ₁₀₃ is E or G; X ₁₀₅ is G or W ; X ₁₀₆ is V or M; X ₁₀₇ is E, G or K; X ₁₀₈ is E or G; X ₁₁₄ is V, E or G; X ₁₁₆ is S or P; X ₁₂₁ is K or R; X ₁₂₄ is F or L; X ₁₃₂ is T, I or M; X ₁₃₄ is K or R; and X ₁₄₀ is A or S; or a fragment of said SEQ ID NO:71. As defined above, conservatively modified variations of the sequence of SEQ ID NO: 71 may include deletions, insertions or conservative substitutions of a total of about 8 amino acids in the 166 amino acid polypeptide, which is referred to as X in SEQ ID NO: 71 The positions do not vary, they correspond to precisely defined amino acids.

For example, if the 4 conservative substitutions are located in a subsequence corresponding to amino acids 141-166 of SEQ ID NO: 71, examples of conservative substitution variations of the subsequence WEVVR AEIMR SFSFS TNLQK RLRRKE include: WEVVR S EIMR SFS Y S TNLQ R RLRRK D and WE L VRAEI V R SFSFS TNL N K RLR K KE etc., where underlined are conservative substitutions.

A feature of the invention is an interferon homologue polypeptide comprising at least about 20, usually at least about 25, usually at least about 30, 40, 50 of any of SEQ ID NO: 36-71 or SEQ ID NO: 79-85 , 60, 70, 80, 90 or 100 consecutive amino acids. In other embodiments, the polypeptide generally comprises at least about 100, 110, 120, 125, 130, 140, 150, 155, 158, 160, 163, 164 or 165 consecutive amino acids.

In other embodiments, the interferon homologue polypeptides of the present invention comprise an amino acid sequence containing one or more of the following amino acid residues (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134 , Phe152, Lys160 and Glu166, wherein the amino acid numbering corresponds to the amino acid numbering in the amino acid sequence SEQ ID NO:36. In a preferred embodiment, the interferon homologue polypeptide comprises an amino acid sequence comprising at least 150, 155 or 166 contiguous amino acid residues of any one of SEQ ID NO: 36-70, and further comprising Lys160 and Glu166, Wherein the amino acid number corresponds to the amino acid number in the amino acid sequence SEQ ID NO:36. Some of these polypeptides exhibit an antiproliferative activity of at least about 8.3 x ¹⁰⁶ units/mg in a human Daudi cell line based assay, or at least about 2.1 x 106 units/mg in a human WISH cell/EMCV based assay. Antiviral activity of ⁷ units/mg. Defining Peptides by Immunoreactivity

Since the polypeptides of the invention provide various novel polypeptide sequences compared to other alpha interferon homologues, the polypeptides also provide novel structural features which can be identified, for example, in immunological assays. The generation of antisera that specifically bind a polypeptide of the invention, and the polypeptides bound by such antisera, are also features of the invention.

The present invention includes interferon-alpha homologue polypeptides, which can specifically bind to or have specific immune reactions with antibodies or antiserum produced against an immunogen, and the immunogen contains a protein selected from the group consisting of SEQ ID NO: 36 to the amino acid sequence of one or more of SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:79 to SEQ ID NO:85. To eliminate cross-reactivity with other interferon-alpha polypeptides, such as known interferon-alpha polypeptides, available known interferon-alpha subtracted antibodies or antisera, including, for example, those produced by Polypeptides encoded by nucleic acids of the following GenBank accession numbers: J00210(α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14 ), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7) , X02961 (α-10 pseudogene), R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1), or any other Known interferon-alpha polypeptides (commonly referred to as "control interferon-alpha polypeptides"). When the accession number corresponds to the nucleic acid, the polypeptide encoded by the nucleic acid is produced and used for antibody/antiserum subtraction. When the nucleic acid corresponds to a non-coding sequence, such as a pseudogene, the amino acids corresponding to the reading frame of the nucleic acid are produced (eg, synthetically) or are minimally modified to include an initiation codon for recombinant production.

In a typical approach, the immunoassay uses polyclonal antisera raised against one or more polypeptides comprising one or more amino acid sequences corresponding to SEQ ID NO: 36 through SEQ ID NO: 70, one or more of SEQ ID NO: 71 and SEQ ID NO: 79 to SEQ ID NO: 85, or a substantial subsequence thereof (i.e. at least about 30% of the full-length sequence provided, 40 %, 50%, 60%, 70%, 80%, 90%, 95% or 98% or longer). The repertoire of potential polypeptide immunogens derived from one or more of SEQ ID NO: 36 to SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 79 to SEQ ID NO: 85 is hereinafter collectively referred to as "immunogens Sexual peptides". Optionally, antisera with low cross-reactivity to control alpha interferon polypeptides and/or other known interferon polypeptides are selected from the resulting antisera and removed by immunoadsorption with one or more control alpha interferon polypeptides Any cross-reactivity, then, was detected using polyclonal antisera in the immunoassay.

To generate antisera for use in immunoassays, one or more immunogenic polypeptides are produced and purified as described herein. For example, recombinant proteins can be produced in mammalian cell lines. Using immunogenic polypeptides mixed with standard adjuvants, such as Freund's adjuvant, and standard mouse immunization methods (see Harlow and Lane (1998) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, where standard descriptions production of antibodies, the format of the immunoassay and the conditions used to measure specific immunoreactivity) immunized with inbred strains of mice (inbred strains were used in this assay because the substantial genetic identity of the mice makes The test results are more reproducible). Alternatively, one or more synthetic or recombinant polypeptides derived from the sequences described herein are conjugated to a carrier protein and used as an immunogen.

Polyclonal sera are collected and titrated for immunogenic polypeptides in an immunoassay, eg, a solid phase immunoassay in which one or more immunogenic polypeptides are immobilized on a solid support. Polyclonal antisera with a titer of ¹⁰⁶ or higher are selected, pooled, and subtracted with a control interferon alpha polypeptide to generate subtracted, pooled, and titrated polyclonal antisera.

Subtracted, pooled and titrated polyclonal antisera were tested for cross-reactivity to control alpha interferon polypeptides. Preferably at least two immunogenic alpha interferon polypeptides are used in this assay, preferably in combination with at least two control alpha interferon polypeptides, to identify antibodies that specifically bind the immunogenic polypeptides.

In this comparative assay, the differential binding conditions of the subtracted, titrated polyclonal antiserum that result in the signal-to-noise ratio of the titrated polyclonal antiserum binding to immunogenic alpha interferon relative to the control The signal-to-noise ratio for alpha interferon binding is at least about 5-10 times higher. That is, the stringency of the binding reaction can be adjusted by adding non-specific competitors, such as albumin and skimmed milk powder, or by adjusting salt conditions, temperature, etc. These binding conditions are used in subsequent assays to determine whether the test polypeptide binds specifically to the pooled subtracted polyclonal antisera. In particular, the signal-to-noise ratio is at least 2-5 times higher than that of the control polypeptide under the discriminative binding conditions, and the signal-to-noise ratio of the test polypeptide is at least about 1/2 of the former compared with the immunogenic polypeptide. Compared with interferon-alpha, it shares higher structural similarity or homology with immunogenic polypeptides and is therefore a polypeptide of the present invention.

In another example, a test polypeptide is detected using an immunoassay in a competitive binding format. For example, cross-reacting antibodies are removed from the pooled antiserum mixture by immunoadsorption with a control interferon alpha polypeptide, as described above. Immunogenic polypeptides are then immobilized on the solid support exposed to the subtracted, pooled antiserum. The test protein is added to the assay to compete for binding to the pooled subtracted antisera. The ability of the test protein to compete for binding to the pooled subtracted antiserum compared to the immobilized protein was compared to the ability of the immunogenic polypeptide added to the assay (the immunogenic polypeptide effectively binds to the immobilized The immunogenic polypeptides compete for binding to pooled antisera). The percent cross-reactivity of the tested proteins was calculated using standard algorithms.

In a parallel assay, the ability of the control protein to compete for binding to the pooled subtracted antisera was determined in comparison to the ability of the immunogenic polypeptide to compete for binding. The percent control polypeptide cross-reactivity was calculated using standard algorithms. The test polypeptide is said to specifically bind to the pooled subtracted antisera when the percent cross-reactivity of the test polypeptide is at least 5-10 fold higher.

In general, immunoabsorbed and collected antisera can be used in the competitive binding immunoassays described herein to compare any test polypeptide to the immunogenic polypeptide. To make this comparison, a wide range of concentrations of each of the two polypeptides can be assayed and the amount of each polypeptide required to inhibit 50% of the binding of the subtracted antiserum to the immobilized protein determined using standard techniques. If the amount of the desired test polypeptide is two-fold lower than the amount of the desired immunogenic polypeptide, it is said that the test polypeptide is capable of specifically binding antibodies raised against the immunogenic polypeptide, provided that the amount is at least two times lower than the amount of the control polypeptide. About 5-10 times higher.

The final measure is specificity, optionally by fully immunoabsorbing the collected antisera with the immunogenic polypeptide (instead of the control polypeptide) until only the resulting immunogenic polypeptide-subtracted antisera pool and immunosorbent are detectable. Weak or undetectable binding of the immunogenic polypeptides used in The fully immunosorbed antisera are then tested for reactivity with the test polypeptide. If only weak or no reactivity is observed (i.e., the signal-to-noise ratio does not exceed 2-fold compared to the observed binding of fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide Antisera raised against immunogenic proteins can specifically bind. Antiproliferative properties of interferon homologs

As described in Example 1, the effect of interferon homologs on cell growth was examined in an assay based on the human Daudi cell line. Figure 2 shows the antiproliferative activity of exemplified interferon homologues of the present invention containing SEQ ID NO: 36 compared to control interferon, human IFN-α2a and shared human IFN-α (Con1) to the amino acid sequence of SEQ ID NO:54. The figure shows the number of active units (Y-axis) per milligram (mg) of interferon test sample for a set of exemplary interferon alpha homologues compared to human IFN-alpha 2a and shared human IFN-alpha, Each of the congener samples has a name (clone name) on the x-axis. These results indicate that compositions containing interferon-alpha homologues of the present invention can be used in methods for inhibiting or reducing the proliferation of tumor cells, including but not limited to: human cancer cells, hematopoietic cancer cells, human leukemia cells, human lymphoid cells tumor cells and human melanoma cells. Inhibition can be performed in vitro (for eg in various proliferation assays), ex vivo or in vivo (for eg as a therapeutic or prophylactic agent).

The interferon-alpha homologues of the invention exhibit a variety of modes of action against various cancer cell lines (see eg Example 2). Use in vitro cell line screens (described in, e.g., Monks, A. et al. (1991) J. Nat'l Cancer Inst. 83:757-766) to detect selective growth inhibition and/or Cell killing effect on specific cancer cell lines. Human cancer cell lines that were screened (see eg Example 2, Table 3) included leukemia, melanoma and lung cancer, colon cancer, brain cancer, central nervous system cancer, ovarian cancer, breast cancer, prostate cancer and kidney cancer.

Three activity parameters were determined in cancer cell line screening: 1) GI50 ("50% growth inhibition"), a measure of growth inhibitory activity, GI50 is the time when cell growth is inhibited by 50% of the interferon test sample (IFNα homolog or control IFNα) by the net increase in protein/polypeptide in the interferon test sample compared to that observed in the control cells (no test sample) at the end of the incubation period 2) TGI ("Total Growth Inhibition"), a measurement of cell quiescence activity, TGI is the concentration of the interferon test sample when the cell growth of a specific cell line is completely inhibited, wherein The amount of cellular protein at the end of the incubation period is equal to the amount of cellular protein at the beginning of the incubation period; and 3) LC50, a measure of cytotoxic activity, LC50 is the amount of cellular protein observed at the beginning of the incubation period compared to the amount of cellular protein observed at the beginning of the incubation period. At the end, the concentration of the interferon test sample at which the amount of cellular protein was reduced by 50% was determined, which shows the net loss of cells after addition of the interferon test sample. Additional details of the assay and data analysis methodology are provided in Example 2.

Activity parameters of an exemplary interferon-alpha homologue 3DA11 (SEQ ID NO: 40) against various cancer cell lines are shown in Figures 3A, 3B and 3C compared to interferon-alpha Con1 and human interferon-alpha 2a controls.

Regarding growth inhibitory activity, the homologue 3DA11 and the control Interferon-αCon1 in particular showed activity against most of the cell lines tested, with Interferon-αCon1 generally showing higher activity and Interferon-α2a generally showing lower overall activity, and only in a subset of cell lines (Fig. 3A).

In particular contrast, significant differences were observed in the cytotoxic and cytostatic activity of the homolog 3DA11 compared to the interferon-Con1 and human interferon-α2a controls. Within the concentration range tested, the homolog 3DA11 showed significant cytostatic activity against a cell population containing 11 cell lines, while Interferon-Con1 only showed activity against a cell population containing 1 cell line, while the homolog The compound 3DA11 was also active against this population of cells (Fig. 3B). IFN-[alpha]2a, on the other hand, was not active in any of the cell lines tested in this assay. Thus, the homologue 3DA11 has a broader profile of cytostatic activity than the shared human interferon-α (Con1) and human interferon-α2a.

The homolog 3DA11 also showed significant cytotoxic activity compared to the interferon-Con1 and human interferon-α2a controls (Fig. 3C). Surprisingly, the homolog 3DA11 showed cytotoxic activity against cell populations containing 8 cell lines, whereas the interferon-Con1 and interferon-α2a controls were effective against cells containing any cell line within the concentration range used in the assay None of the populations showed measurable activity. Thus, the homologue 3DA11 also has a broader profile of cytotoxic activity compared to interferon-Con1 and human interferon-α2a.

Figures 4A-4D illustrate the cytostatic activity (reflected by TGI values) of exemplary interferon-alpha homologues of the invention. In each figure, the relative cytostatic activity (expressed as -log TGI) diagram.

Of the exemplified congeners tested, the congeners 1D3 (SEQ ID NO: 54) and 3DA11 (SEQ ID NO: 40), but not either of the control interferons, were shown to target leukemia cell lines over the range of concentrations tested. Significant cytostatic activity of the RPMI-8226 cell population (Fig. 4A). In this example, the 1D3 and 3DA11 homologues exhibit at least about 100% higher activity against cell populations of leukemia cell lines relative to either control (Interferon-Con1 or Interferon-α2a) against cell populations of leukemia cell lines. A 25-fold corresponding activity (corresponding to a TGI difference of at least about 1.4 log units).

Homologs 1D3, 2G5 (SEQ ID NO: 45), 6CG3 (SEQ ID NO: 52) and 3DA11, but not any of the control interferons, showed significant cytostatic activity against the lung cancer cell line NCI-H23 (Fig. 4B). In this example, 1D3, 2G5, 6CG3 and 3DA11 homologues exhibit at least about A 12-fold higher corresponding activity (corresponding to a TGI difference of at least about 1.1 log units).

The homologues 1D3, 2G5 and 3DA11, but not any of the control interferons showed significant cytostatic activity against the renal carcinoma cell line ACHN cell population (Fig. 4C). In this example, the 1D3, 2G5, and 3DA11 homologues showed activity against the cell population of renal cancer cell lines relative to the cytostatic activity of interferon-Con1 or interferon-α2a against the cell population of the renal cancer cell line A corresponding activity that is at least about 35-fold higher (corresponding to a TGI difference of at least about 1.55 log units).

Homologs 1D3, 2G5, 3DA11, 2CA5 (SEQ ID NO: 42) and 2DB11 (SEQ ID NO: 41), as well as the interferon-Con1 control, but not the interferon-α2a control, were shown to target ovarian cancer cell line OVCAR-3 cells Significant cytostatic activity of the population (Fig. 4D). In this example, the homologue 1D3 exhibited at least about 2-fold higher relative activity (compared to at least about 0.3 log units) relative to interferon-Con1 in terms of cytostatic activity against each population of ovarian cancer cell line cells. TGI difference corresponding), whereas 1D3, 2G5, 3DA11, 2CA5 and 2DB11 homologues showed corresponding activities at least about 40-fold higher than interferon-α2a (corresponding to TGI difference of at least about 1.6 log units).

It is evident from the exemplary data presented herein that the interferon-alpha homologues of the present invention exhibit a diverse profile of cytostatic activity that is significantly different from that of interferon-alpha Con1 and interferon-alpha-2a .

The invention includes interferon-alpha homologues having increased cytostatic activity relative to human interferon-alpha 2a or the consensus human interferon-alpha Con1. In various embodiments, the cytostatic activity of the interferon-alpha homolog on the cancer cell line cell population is at least about 2 times higher than the corresponding activity of human interferon-alpha 2a (i.e., the TGI value is at least about 2 times lower), or interferes with The cytostatic activity of a homologue of K-alpha on a cell population selected from one or more of the following cancer cell lines is at least about 2-fold higher than the corresponding activity of Interferon-Con1: leukemia cell lines; melanoma cell lines; lung cancer cell lines a colon cancer cell line; a central nervous system (CNS) cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line and a kidney cancer cell line.

In other embodiments, the cytostatic activity of the interferon-alpha homologue on the cancer cell line cell population is up to about 5-fold greater (i.e., the TGI value is at least about 5-fold lower) than the corresponding activity of human interferon-alpha 2a, or the interferon-alpha - the cytostatic activity of the alpha homologue on a cell population selected from one or more of the following cancer cell lines is at least about 5-fold greater than the corresponding activity of interferon-Con1: a leukemia cell line; a melanoma cell line; a lung cancer cell line; Colon cancer cell lines; central nervous system (CNS) cancer cell lines; ovarian cancer cell lines; breast cancer cell lines; prostate cancer cell lines and kidney cancer cell lines. In other embodiments, the cell quiescence activity of the cancer cell line cell population is at least about 10-fold higher (i.e., has a TGI value at least about 10-fold lower) than the corresponding activity of human interferon-α 2a when the interferon-alpha homolog is at least about 10-fold lower, or the interferon - the alpha homologue has a cytostatic activity at least about 10 times greater than the corresponding activity of interferon-Con1 on a cell population selected from one or more of the following cancer cell lines: a leukemia cell line; a melanoma cell line; a lung cancer cell line; Colon cancer cell lines; central nervous system (CNS) cancer cell lines; ovarian cancer cell lines; breast cancer cell lines; prostate cancer cell lines and kidney cancer cell lines.

The invention includes interferon-alpha homologues having increased cytotoxic activity relative to human interferon-alpha 2a or interferon-Con1. In various embodiments, the cytotoxic activity of the interferon-alpha homolog to a cell population selected from one or more of the following cancer cell lines is at least about 2-fold higher than the corresponding activity of human interferon-alpha 2a (i.e., the LC50 value is at least about 2-fold lower), at least 5-fold higher, or at least 10-fold higher: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; CNS cancer cell lines; ovarian cancer cell lines; breast cancer cell lines ; Prostate and kidney cancer cell lines. In other embodiments, the cytotoxic activity of the interferon-α homologue on a cell population selected from one or more of the following cancer cell lines is at least about 2-fold higher than the corresponding activity of interferon-Con1 (i.e., the LC50 value is at least about 2-fold lower) 2-fold), at least about 5-fold higher, or at least about 10-fold higher: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; CNS cancer cell lines; ovarian cancer cell lines; breast cancer cell lines ; Prostate and kidney cancer cell lines.

The present invention includes interferon-alpha homologues having increased growth inhibitory activity relative to human interferon-alpha 2a or interferon-Con1. In various embodiments, the growth inhibitory activity of the interferon-alpha homologue on a cell population selected from one or more of the following cancer cell lines is at least about 2 times higher than the corresponding activity of human interferon-alpha 2a (i.e. the GI50 value is at least about 2-fold lower), at least about 5-fold higher, or at least about 10-fold higher: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; CNS cancer cell lines; ovarian cancer cell lines; breast cancer cell lines Cell lines; prostate cancer cell line and kidney cancer cell line. In other embodiments, the growth inhibitory activity of the interferon-α homologue on a cell population selected from one or more of the following cancer cell lines is at least about 2-fold higher than the corresponding activity of interferon-Con1 (i.e., the GI50 value is at least about 2 times lower) 2-fold), at least about 5-fold higher, or at least about 10-fold higher: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; CNS cancer cell lines; ovarian cancer cell lines; breast cancer cell lines ; Prostate and kidney cancer cell lines.

The discovery described herein that interferons (such as the interferon-alpha homologues described herein) can be evolved, modified or recombined to exhibit multiple activity profiles provides a basis for the evolution and generation of custom, specific Interferon homologues offer the opportunity to be useful in the treatment of a variety of specific diseases including, for example, various cancers or related diseases. For example, an interferon homologue of the invention that is optimized for enhanced potency against a particular target cancer cell type may also be optimized so that it (advantageously) has reduced toxicity against non-target cells and thus, is less effective against Subjects (eg, patients) administered the homologue experience lower side effects.

The present invention further provides the opportunity to optimize interferon homologues for tumor cells taken from subpopulations of subjects such as mammalian or human patients, or even from individual subjects such as mammalian or human patients, In order to provide customized treatment or preventive treatment plan for each subject. The optimized interferon homologues of the present invention can provide therapeutic or prophylactic effects against cancer or related diseases, or other diseases that can be treated with interferon, or diseases that are unresponsive to currently used interferon or other treatment regimens. Antiviral properties of interferon homologues

The antiviral activity of the interferon homologues of the invention was evaluated in the human WISH cell/EMCV assay as described in Example 1. Figure 2 shows the antiviral activity of the exemplary interferon homologues of the present invention comprising the amino acid sequence of SEQ ID NO: 36 to SEQ ID NO: 54.

The improved in vitro antiviral activity of the exemplified IFN-[alpha] homologues of the present invention has been demonstrated to be maintained in vivo in a murine model system. It was previously confirmed that the two IFN-α homologues CH2.2 and CH2.3 (SEQ ID NOs: 84 and 85, respectively) of the present invention, compared to human IFN-α 2a, respectively, in mouse cell-based assays Has about 206,000-fold and 138,000-fold improved antiviral activity, and in the same assay, compared with natural mouse interferon, has significantly higher activity (Chang et al. (1999) Nature Biotechnol.17:793- 797). As described in Example 3 below, Balb/c mice challenged with a lethal dose of vesicular stomatitis virus (VSV) were administered different doses of the IFN-α homologues CH2.2 and CH2.3, the natural murine interferon Mu - IFNα4 and human IFN-α2a. The high in vitro activity correlates well with the observed in vivo activity (Figure 5). CH2.2 and CH2.3 homologues were completely effective in protecting mice from lethal virus challenge, while natural mouse interferon was partially effective and human IFN-α2a was completely ineffective at the same dose. These results indicate that compositions containing interferon homologues of the present invention are useful in methods of inhibiting viral replication in a subject infected with viruses including, but not limited to, human immunodeficiency virus (HIV), hepatitis C Virus (HCV), Herpes Simplex Virus (HSV) and Hepatitis B Virus (HBV). Can be used in vitro (for example, in various antiviral assays), ex vivo (for use, for example, as a therapeutic or prophylactic agent in the ex vivo methods discussed herein), or in vivo (for use in, for example, a therapeutic or prophylactic agent in the in vivo methods discussed herein) agent) for inhibition. Use of interferon homologues in the treatment of autoimmune and other immune-related diseases

The compositions of the present invention are useful in the therapeutic or prophylactic treatment to alleviate a variety of immune system-related diseases characterized by hyper or hypoactive immune system function or other characteristics. Such diseases include hyperallergenic and autoimmune diseases such as multiple sclerosis, type I (insulin-dependent) diabetes, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis , asthma, allergies, psoriasis, etc. Therapeutic and prophylactic compositions

According to methods well known in the art, in appropriate in vitro, from in vivo and in vivo disease animal models, test the therapeutic or prophylactic compositions containing one or more interferon homologue polypeptides or nucleic acids of the present invention, to confirm efficacy, tissue Metabolism and estimated dose. In particular, dosage can be determined by comparing the activity of alpha interferon homologues with existing alpha interferon therapeutic or prophylactic agents in relevant assays. In one aspect, the invention provides a method comprising administering to a mammal or a non-mammalian vertebrate, such as a bird (such as a chicken or a duck), one or more of the above-described inventive Interferon homologue nucleotides or polypeptides (or fragments thereof), said mammals include, for example, humans, primates, mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, sheep . The composition generally contains one or more interferon homologue nucleotides or polypeptides (or fragments thereof) of the invention and an excipient, such as a pharmaceutically acceptable excipient.

In one aspect, a composition of the invention is produced by digesting one or more nucleic acids of the invention (or fragments thereof) with a restriction endonuclease, RNase or DNase.

In another aspect of the invention there is provided a nucleic acid produced by incubating one or more of the nucleic acids described above in the presence of deoxyribonucleotide triphosphates and a nucleic acid polymerase, such as a thermostable polymerase. combination.

The invention also includes compositions comprising two or more of the nucleic acids described above. The composition may contain a library of nucleic acids, wherein the library contains at least about 5, 10, 20, 50, 100, 150 or 200 or more nucleic acids.

Administration is by any route commonly used to introduce molecules into contact with blood or tissue cells. The interferon-alpha homologues of the invention may be administered in any suitable manner, preferably together with a pharmaceutically acceptable carrier. Appropriate methods of administering interferon homologues in the context of the present invention to a patient are available, and although more than one route may be used to administer a particular composition, a particular route will often provide a more immediate and more direct response than another. effective response.

The pharmaceutically acceptable carrier will be determined in part by the particular composition being administered, as well as the particular method by which the composition is administered. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention.

Polypeptide compositions may be administered for use in any of the prophylactic, therapeutic and diagnostic methods described herein by a variety of routes including, but not limited to: oral, intravenous, intraperitoneal, intramuscular, via Skin, subcutaneous, topical, sublingual, vaginal or rectal routes, or by inhalation. Interferon homolog polypeptide compositions can also be administered via liposomes. Such routes of administration and appropriate formulations are generally known in the art.

Interferon homologue polypeptides or nucleic acids, alone or in combination with other suitable components, may also be formulated for administration via inhalation as aerosols (ie, they may be "nebulized"). Aerosols can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, for example, by intra-articular, intravenous, intramuscular, transdermal, intraperitoneal and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions containing antioxidants , buffers, bacteriostatic agents and solutes that make the formulation isotonic with the blood of the recipient to whom it is administered, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives agent. Packaged nucleic acid preparations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

Parenteral and intravenous administration are preferred methods of administration. In particular, routes of administration already used by existing interferon-alpha therapeutics or preventives, and formulations currently in use are preferred routes of administration, as well as preferred formulations of interferon-alpha homologue polypeptides and nucleic acids of the present invention.

In the context of ex vivo or in vivo therapy, cells transduced with interferon homologues as described above may also be administered intravenously or parenterally as described above. It will be appreciated that delivery of cells to a subject (eg, a human patient) is a routine technique, eg, via intravenous or intraperitoneal administration to the blood.

In the context of the present invention, the dose of interferon homologue polypeptide or nucleic acid of the present invention administered to a subject (eg patient) is sufficient to produce a favorable therapeutic or prophylactic response in the subject (eg patient) for a certain period of time , or sufficient to inhibit pathogenic infection, depending on the specific application. The dosage will be determined by the potency of the particular carrier or formulation, and the activity of the interferon homologue employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose will be determined by the existence, nature and extent of any adverse side effects accompanying the administration of a particular vector, formulation, transduced cell type, etc. in a particular patient.

In the therapeutic and prophylactic treatment methods of the invention described herein, the general range of the effective amount (such as nucleic acid dose) of the interferon-alpha nucleic acid (such as DNA or mRNA) of the present invention is, for example: about 0.05 microgram/kilogram (kg) to about 50mg/kg, usually about 0.005-5mg/kg. However, it should be understood that the effective amount of the nucleic acid (e.g., nucleic acid dose) and/or polypeptide (e.g., polypeptide dose) may vary in a manner well known to those skilled in the art, including: the activity or potency of the polypeptide, the The activity or efficacy of any nucleic acid construct (eg, vector, promoter, expression system) administered, the disease to be treated (eg, a particular cancer) and the subject to whom the nucleic acid is delivered.

For the delivery of some polypeptides, eg, by delivery of nucleic acid encoding the polypeptide, sufficient levels of translation and/or expression can be achieved with nucleic acid dosages, eg, from about 0.005 mg/kg to about 5 mg/kg. Dosages for other polypeptides (and nucleic acids encoding them) with known biological activity can be readily determined by those skilled in the art based on the factors mentioned above. The doses of other known interferon-alphas used for specific diseases provide a guide for determining the dosage and treatment regimen of the nucleic acid or polypeptide of the invention. An effective amount of an interferon-alpha homologue polypeptide ranges from about 1 microgram to about 1 milligram, more preferably from about 1 microgram to about 100 micrograms.

Compositions for use in the therapeutic and prophylactic treatment methods of the invention described herein may contain, for example, an interferon-alpha homologue nucleic acid (e.g., DNA or mRNA) of the invention at a concentration of about 0.1 microgram/milliliter (ml) to about 20 mg/ml. ), and a pharmaceutically acceptable carrier (such as an aqueous carrier).

Compositions for use in the therapeutic and prophylactic treatment methods of the invention described herein may contain, for example, an interferon-alpha homologue polypeptide of the invention in concentrations as described above and herein, and a pharmaceutically acceptable carrier (eg, an aqueous carrier).

In determining the effective amount of vector, cell type, or administered agent in the treatment or prophylaxis of cancer or viral disease, physicians need to evaluate circulating plasma levels, toxicity of vector/cell/agent/interferon homologue, disease progression and anti-vector/interferon homologue antibody production.

To administer a dose range equal to the dose of interferon-alpha therapeutic or prophylactic protein currently used to a patient of eg 70 kg, the dose of vector or cells producing the interferon homolog sequence is calculated to produce the same amount of interferon homolog nucleic acid or expressed protein. The vectors of the present invention can complement the treatment of cancer and virus-mediated diseases by any known conventional therapy, including cytotoxic agents, nucleotide analogs (such as when used to treat HIV infection), biological response modifiers, etc. .

For administration purposes, when applied to the general and overall health of a patient, the interferon homologue of the invention and the transduced cells should be administered at a ratio determined by the LD50 of the interferon homologue polypeptide or nucleic acid , vector, or transduced cell type, and side effects of various concentrations of interferon homologue polypeptide or nucleic acid, vector, or cell type are determined. Administration can be via a single dose or in divided doses.

To introduce cells that have been transduced with recombinant alpha-interferon nucleic acid into a subject (eg, a patient), a blood sample is obtained prior to perfusion, and the blood sample is stored for analysis. 1 x ¹⁰⁶ to 1 x ¹⁰¹² transduced cells were perfused intravenously over 60-200 min. Monitor vital signs closely and monitor oxygen saturation by pulse oximeter. Blood samples were obtained 5 minutes and 1 hour after perfusion and saved for later analysis. Leukopheresis, transduction and reperfusion are optionally repeated every 2 to 3 months for a total of 4 to 6 treatments over 1 year. After the first treatment, perfusion is performed primarily on an outpatient basis, at the discretion of the clinician. If reperfusion is performed on an outpatient basis, monitor the participant for at least 4 hours, preferably 8 hours, after treatment. Transduced cells for reperfusion were prepared according to established methods. See Abrahamsen et al. (1991) J. Clin. Apheresis 6: 48-53; Carter et al. (1988) J. Clin. Arpheresis 4: 113-117; Aebersold et al. (1988), J. Immunol. Methods 112: 1-7; Muul et al. (1987) J. Immunol. Methods 101:171181 and Carter et al. (1987) Transfusion 27:362-365. After about 2 to 4 weeks of culture, the number of cells should reach 1×10 ⁶ to 1×10 ¹² . In this regard, the growth characteristics of the cells vary from patient to patient and from cell type to cell type. Approximately 72 hours prior to reperfusion of transduced cells, aliquots were removed for analysis of phenotype, and the percentage of cells expressing therapeutic or preventive agents.

If a subject (e.g., a patient) undergoing infusion of a vector or transduced cell or protein preparation develops fever, chills, or muscle pain, appropriate doses of aspirin, ibuprofen, acetaminophen, or other antipyretic sedatives may be administered. pain medication. Subjects (eg, patients) who experience infusion reactions, such as fever, muscle aches, and chills, can be pre-medicated with aspirin, acetaminophen, or, for example, diphenhydramine 30 minutes prior to infusion. Demerol may be used for more severe chills and muscle pains that do not respond quickly to antipyretics and antihistamines. Depending on the severity of the reaction, cell perfusion may be slowed or canceled. Methods of treatment and preventive treatment

The present invention also includes methods for treating or preventing diseases, said methods comprising administering to a subject in vivo or ex vivo one or more nucleic acids or polypeptides of the invention described above (or containing pharmaceutically acceptable excipients). agent and one or more compositions of said nucleic acid or polypeptide), said subjects include, for example, mammals, including, for example, humans, primates, mice, pigs, cows, goats, rabbits, rats, Guinea pigs, hamsters, horses, sheep; or non-mammalian vertebrates such as birds (such as chickens or ducks) or fish or invertebrates.

In one aspect of the invention, in an ex vivo method, one or more cells or cell populations of interest (e.g. tumor cells, tumor tissue samples, organ cells, blood cells) are obtained or removed from a subject. , skin, lung, heart, muscle, brain, mucous membrane, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, oral cavity, tongue, etc.), and with a certain amount of Polypeptide contacts of the invention. The contacted cells are then returned or delivered to the site in the subject from which the cells were obtained, or to another site of interest in the subject to be treated (eg including those defined above). If desired, the contacted cells can be transplanted to a tissue, organ or systemic site of interest (including all sites described above) in a subject using standard and well-known transplantation techniques, or for example It is delivered to the blood or lymphatic system using standard delivery or infusion techniques.

The invention also provides in vivo methods wherein one or more cells or cell populations of interest in a subject are contacted, directly or indirectly, with an amount of a polypeptide of the invention effective to prevent or treat a disease. In direct contact/administration forms, typically by any of a variety of methods, including topical administration, injection (e.g., by use of a needle or syringe), or vaccine or gene gun delivery, propelled into a tissue, organ, or skin site point, the polypeptide is directly administered or transferred to the cells to be treated or tissue sites of interest (e.g. tumor cells, tumor tissue samples, organ cells, blood cells, skin, lung, heart, muscle, brain, mucosa, liver, cells of the intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, oral cavity, tongue, etc.). Polypeptides may be delivered, for example, intramuscularly, intradermally, subcutaneously, orally, intraperitoneally, intrathecally, intravenously, or placed in a body cavity (including, for example, during a surgical procedure), or administered by inhalation or vaginal or rectal Delivery of peptides.

In indirect contact/administration forms in vivo, the polypeptide of the invention is administered or transferred indirectly to the cells or cells to be treated, typically by directly contacting or administering the polypeptide to one or more cells or cell populations that facilitate treatment. Tissue sites of interest include those described above (eg, skin cells, organ systems, lymphatic or blood cell systems, etc.). For example, by contacting cells of the blood or lymphatic system, skin or organ with a sufficient amount of the polypeptide so that the polypeptide is delivered to a site of interest (e.g., a tissue, organ or cell of interest in the body or the blood or lymphatic system), and result in an effective prophylactic or therapeutic effect, ie, treatment of tumor cells in the subject. Such contacting, administration or transfer will generally be accomplished using one or more of the routes or modes of administration described above.

In another aspect, the invention provides an ex vivo method wherein one or more cells or cell populations (e.g. tumor cells, tumor tissue samples, organ cells, blood cells) of interest are obtained or removed from a subject. , skin, lung, heart, muscle, brain, mucous membrane, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, oral cavity, tongue, etc.), and by making one or more of the cells or cell populations are transformed by contacting these cells with a polynucleotide construct containing a target nucleic acid sequence of the present invention that encodes a desired biologically active polypeptide (e.g., a polypeptide of the present invention) that is effective in preventing or treating a disease . One or more cells or populations of cells are contacted with a polynucleotide construct and a promoter controlling expression of said nucleic acid sequence in sufficient quantities such that the cells take up the polynucleotide construct (and promoter) and adequately express the present The target nucleic acid sequence of the invention produces a certain amount of biologically active polypeptides that can effectively prevent or treat diseases. The polynucleotide construct may include a promoter sequence (such as a CMV promoter sequence) that controls the expression of a nucleic acid sequence of the present invention and/or, if necessary, one or more other nucleotide sequences that encode At least one or more another polypeptide, cytokine, adjuvant or co-stimulatory molecule of the invention, or other desired polypeptide.

After transfection, the transformed cells are returned, delivered or transferred to the tissue site or system in the subject from which the cells were obtained, or to another site in the subject to be treated (e.g., tumor cells, tumor tissue samples, Organ cells, blood cells, skin, lung, heart, muscle, brain, mucous membrane, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, oral cavity, tongue, etc.). If necessary, the cells may be transplanted into a tissue, skin, organ or body system of interest in a subject using standard and well-known transplantation techniques, or delivered, for example, into the blood or blood using standard delivery or infusion techniques. lymphatic system. Transformed cells are typically delivered, administered or transferred using one or more of the routes or modes of administration described above. Expression of the target nucleic acid can occur naturally, or can be induced (as described in detail below), and the amount of the encoded polypeptide expressed should be sufficient and effective to treat the disease at the site or tissue system.

In another aspect, the present invention provides in vivo methods wherein one or more cells or cell populations of interest (e.g. including those cells and cell systems and subjects described above) in a subject are transformed , transformed by contacting the cell or cell population with (or administering to or transferring to the cell or cell population using one or more of the routes or modes of administration described above) a polynucleotide construct, said The construct comprises a nucleic acid sequence of the invention encoding a desired biologically active polypeptide (eg, a polypeptide of the invention) that is effective in preventing or treating a disease.

A polynucleotide construct can be administered directly to or transferred to a diseased cell (eg, by direct contact using one or more of the routes or modes of administration described above). Alternatively, non-diseased cells or other diseased cells are first brought into direct contact with a sufficient amount of a polynucleotide construct (the construct containing the The nucleic acid sequence of the active polypeptide and the promoter that controls the expression of the nucleic acid sequence), so that the cells can take in the polynucleotide construct (and the promoter), fully express the target nucleic acid sequence of the present invention, and produce a certain amount of polynucleotide that can effectively prevent or treat diseases. a biologically active polypeptide such that the polynucleotide construct or the resulting expressed polypeptide is capable of transferring naturally or automatically from the original delivery site, system, tissue or organ in the subject to the diseased site in the subject, A tissue, organ or system (eg, via the blood or lymphatic system), while the polynucleotide construct is administered or transferred indirectly to diseased cells. Expression of the target nucleic acid can occur naturally, or can be induced (as described in more detail below) so that the encoded polypeptide is expressed in an amount sufficient and effective to treat the disease at the site or tissue system. The polynucleotide construct may include a promoter sequence (such as a CMV promoter sequence) to control the expression of a nucleic acid sequence and/or, if necessary, one or more other nucleotide sequences encoding at least one or more of another polypeptide, cytokine, adjuvant or co-stimulatory molecule of the invention, or other desired polypeptide.

In each of the in vivo and ex vivo methods of treatment described above, a composition comprising an excipient and a polypeptide or nucleic acid of the invention may be administered or delivered. In one aspect, a composition comprising a pharmaceutically acceptable excipient and a polypeptide or nucleic acid of the invention is administered or delivered to a subject in an amount effective to treat a disease, as described above.

In another aspect, in each of the in vivo and ex vivo methods of treatment described above, the amount of polynucleotide administered to the cell or subject may be sufficient to cause one or more cells of the subject to Ingesting the polynucleotide and fully expressing the nucleic acid sequence produces a certain amount of biologically active polypeptide that can effectively enhance the immune response of the subject, including the immune response induced by the immunogen (such as an antigen). On the other hand, for each method, the amount of polypeptide administered to a cell or subject can be an amount sufficient to enhance an immune response in the subject, including an immune response induced by an immunogen (eg, an antigen).

On the other hand, in in vivo or in vivo therapeutic methods using polynucleotide constructs (or compositions containing polynucleotide constructs) to deliver physiologically active polypeptides to subjects, the inducible open- and close- Gene expression systems to induce expression of polynucleotide constructs. Examples of such on- and off-gene expression systems include Tet-On(TM) Gene Expression System and Tet-Off ^(TM) Gene Expression System, respectively (see eg Clontech Catalog 2000, pg. 110-111 for a detailed description of each system). Other controllable or inducible on- and off-gene expression systems are known to those skilled in the art. Using this system, the expression of a target nucleic acid in a polynucleotide construct can be precisely, reversibly and quantitatively regulated. For example, gene expression of a target nucleic acid can be induced after delivery or transfer or contact of stably transfected cells containing a polynucleotide construct to a tissue site, organ or system of interest. The construct contains a target nucleic acid. Such a system is particularly advantageous for methods and modalities of therapy, where it can advantageously delay or precisely control the expression of a target nucleic acid (e.g., to allow time for completion of surgery and/or post-surgery recovery; to allow polynucleotide constructs containing the target nucleic acid to time to reach the site, cell, system or tissue to be treated; time for the graft containing cells transformed by the construct to incorporate into the tissue or organ to which it is grafted or attached, etc.). integrated system

The invention provides computers, computer readable media and integrated systems containing signature strands corresponding to the sequence information for the polypeptides and nucleic acids described herein, including, for example, those sequences listed herein and their various silent and conservative substitutions.

Various methods and genetic algorithms (GO) known in the art can be used to detect homology or similarity between different characteristic strands, or to perform other desired functions, such as controlling output files, providing descriptions including sequence etc. information basis. Such as BLAST discussed above.

Thus, different types of homology and similarity of various stringencies and lengths can be detected and identified in the integrated system herein. For example, a variety of homology assays have been devised for comparative analysis of sequences in biopolymers, spell checking in word processing, and retrieval of data from various databases. Having understood the interaction of double-helix paired complements in the four main nucleobases of natural polynucleotides, models that simulate the annealing of polynucleotide strands of complementary homology can also be used as the basis for sequence alignments or other manipulations , the operations are generally performed on the feature chains corresponding to the sequences herein (for example, word-processing operations, constructing graphs containing sequence or subsequence feature chains, output tables, etc.). An example of a software package containing GO for calculating sequence similarity or homology is BLAST, which can be applied to the present invention by inputting a characteristic chain corresponding to the sequence herein.

Similarly, standard desktop computer application software such as word processing software (such as Microsoft Word ^™ or Corel WordPerfect ^™ ) and Database software (eg spreadsheets such as Microsoft Excel ^™ , Corel Quattro Pro ^™ or database programs such as Microsoft Access ^™ or Parados ^™ ) are suitable for use with the present invention. For example, an integrated system may include the aforementioned software with appropriate feature chain information, eg, for interfacing with a user interface (eg, a graphical user interface in a standard operating system such as Windows, Macintosh or LINUX systems) to operate the feature chain. As mentioned above, programs dedicated to sequence alignment such as BLAST can also be incorporated into the system of the present invention to align nucleic acids or proteins (or corresponding characteristic strands).

An integrated system used in the analysis of the present invention will generally include a digital computer with GO software for aligning sequences, and a data set capable of accessing a software system containing any of the sequences described herein. The computer can be, for example, a personal computer (Intel x86 or Pentium chip-compatible DOS ^™ , OS2 ^™ WINDOWS ^™ WINDOWS NT ^™ , WINDOWS95 ^™ , WINDOWS98 ^™ , a LINUX-based machine, MACINTOSH ^™ , Power PC, or a UNIX-based (e.g., SUN ^™ workstation ) machine) or other commercially available common computers known to those skilled in the art. Software for aligning or manipulating sequences is available or can be readily created by one skilled in the art using standard programming languages such as Visualbasic, Fortran, Basic, Java, and the like.

Any controller or computer optionally includes a monitor, which is often a cathode ray tube ("CRT") display, a flat panel display (eg, active matrix liquid crystal display, liquid crystal display), or other display. Computer circuits are often housed in a box that includes multiple integrated circuit chips, such as microprocessors, memory, interface circuits, and the like. The box may also optionally include hard drives, floppy drives, high capacity removable drives such as writable CD-ROMs and other common peripherals. An input device such as a keyboard or mouse is optionally provided for user input and selection of sequences to be compared or manipulated in the associated computer system.

Computers generally include suitable software for accepting user instructions, which may be in the form of user input into a set of parameter fields, such as in a GUI, or in the form of pre-programmed instructions, such as pre-programmed for a number of different specific operations . The software then translates these instructions into the appropriate language, instructing the errant direction of operation and the transfer controller to perform the desired operation.

The software may also include output elements that control nucleic acid synthesis (eg, based on the sequences or an alignment of the sequences herein), or other operations that occur downstream of the sequence alignment, or other operations using signature strands corresponding to the sequences herein.

In one embodiment, the invention provides an integrated system comprising a computer or computer readable medium comprising a database having one or more sequence records therein. Each sequence record contains one or more characteristic strands corresponding to nucleic acid or polypeptide or protein sequences selected from SEQ ID NO: 1 to SEQ ID NO: 85. The integrated system also includes a user input interface that allows the user to selectively view one or more sequence records. In one such integrated system, a computer or computer readable medium contains a set of alignment instructions for aligning a signature with one or more other signatures corresponding to nucleic acid or polypeptide or protein sequences.

One such integrated system includes a set of instructions comprising at least one of: a local homology comparison assay, a homology sequence alignment assay, a similarity search assay, and a BLAST assay. In some embodiments, the system also contains a readable output element that can display the alignment results generated by the sequence alignment instruction set. In another embodiment, the computer or computer readable medium further contains a set of instructions capable of translating at least one nucleic acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 35 or The sequence of SEQ ID NO:72 to SEQ ID NO:78. The set of instructions can select nucleic acids by applying a set of codon usage instructions or a set of instructions for determining sequence identity to a test nucleic acid sequence.

The present invention also provides a method of presenting information inherent to at least one of a plurality of sequence records stored in a database using a computer system. Each sequence record contains at least one characteristic chain corresponding to SEQ ID NO:1 to SEQ ID NO:85. The method comprises determining at least one signature chain corresponding to one or more of SEQ ID NO: 1 to SEQ ID NO: 85 or a subsequence thereof; determining which of the at least one signature chain listed is selected by the user ; and display each selected feature chain, or compare each selected feature chain with other feature chains. The method also includes displaying a sequence alignment and/or display list for each selected signature and other signatures. Reagent test kit

In other aspects, the invention provides kits embodying the methods, compositions, systems and apparatus described herein. Kits of the invention optionally include one or more of the following components: (1) an apparatus, system, system component or instrument component described herein; (2) performing a method described herein, and/or operating a method described herein Instruments or instrument components, and/or instructions for using the compositions described herein; (3) one or more alpha interferon homologue compositions (such as containing at least one interferon alpha homologue nucleic acid or polypeptide of the present invention or Compositions of its fragments, cells, carriers, etc.) or components (interferon alpha homologue nucleic acid or polypeptide of the present invention or fragments thereof, cells, vectors, etc.); (4) placing one or more articles of the present invention, A container comprising said component or composition; and (5) packaging material.

In another aspect, the present invention provides the use of any apparatus, apparatus assembly, composition or kit described herein for carrying out any of the methods or assays described herein, and/or any apparatus or kit for carrying out any of the methods or assays described herein. use of any test or method described.

Example Example 1: Preparation and Screening of Shuffled Interferon-alpha Libraries

Prepare about 20 human interferon-alpha subclass gene fragments (25-60 base pairs (bp) in length) by PCR amplification and DNase treatment, basically according to Crameri A et al. (1998; Nature 15: 288-291 ) to generate a shuffled mature coding sequence for interferon-alpha. An expression library was prepared by subcloning the mature shuffled interferon-α coding sequence into an E. coli secretion vector. The shuffled interferon polypeptides were expressed as mature proteins fused at the C-terminus to an E-tag (Amersham-Pharmacia) to facilitate quantification and purification from the periplasmic space. E. coli transformants were picked into microtiter plates using a robotic colony picker (Q-Bot, Genetix Pharmaceuticals) and periplasmic extracts were prepared.

The periplasmic extracts were tested for antiproliferative activity on the human Daudi cell line as described by Scarozza, A.M. et al. (1992) J. Interferon Res. 12:35-42.

Clones exhibiting antiproliferative activity in the Daudi assay were rescreened and expression levels were determined by Western blotting using an anti-E tag antibody (Amersham-Pharmacia). The most active clone normalized to expression level was selected for sequencing and used as substrate for additional rounds of shuffling and screening as described above.

Clones obtained from the first and second rounds of shuffling and having relatively high antiproliferative activity in the Daudi assay were subcloned into a CHO expression vector (pDEI-1011) in which E-tag/6-His tag (Amersham- Pharmacia) fused to the C-terminus of the shuffled interferon. Clones were transfected into CHO cells and stable cell lines were selected with 1 mg/ml G418. CHO-expressed mature interferon was purified on an anti-E-labeled Sepharose column (Amersham-Pharmacia) and quantified by the Bradford assay (Biorad). Antiproliferative activity of CHO-purified shuffled interferon was analyzed by Daudi assay and antiviral activity was analyzed using human WISH cell/EMCV assay as described below. Human WISH cell/EMCV antiviral test

In 100 μl of RPMI medium (Gibco-BRL) supplemented with 10% fetal bovine serum, penicillin (100 μg/ml) and streptomycin (100 μg/ml) in a 96-well culture plate, 6×10 ⁴ cells/ WISH cells were seeded at the density of the wells and incubated at 37°C for 24 hours. The medium containing the interferon-α polypeptide sample (total volume 100 μl) was added to the well, and incubated at 37° C. for 3 hours in an atmosphere of 5% CO ₂ . A volume of 50 μl of EMCV (encephalomyocarditis virus) dilution was added to the wells and incubated for 24 hours as described above. The medium was carefully removed and the wells were rinsed twice with warm phosphate-buffered saline (PBS). Neutral red (diluted 1:50 with culture medium, 100 μl/well) was added to the wells, and incubated for 2 hours as above. Glutaraldehyde (0.5% in PBS, 50 μl/well) was added and incubated for 30 minutes as above. The wells were washed twice with PBS, and 100 μl/well of 50% methanol, 1% acetic acid solution was added. Absorbance at 540 nanometers (nm) was measured using a microtiter plate reader.

Figure 2 shows the antiproliferative and antiviral activities of exemplified interferon homologues of the present invention compared to interferon-α2a and interferon-αCon1. The figure shows the number of units of activity per mg of homologue (Y-axis) for an exemplary set of interferon alpha homologues (Y-axis), where each homologue sample has a name on the X-axis. Example 2: Screening of cancer cell lines in vitro

Screening using in vitro cancer cell lines (described, e.g., in Monks, A. et al. (1991) J. Nat'l Cancer Inst. 83:757-766 (hereinafter referred to as "Monks") and http://dtp.nci.gov. /branches/btb/ivclsp.html, both of which are incorporated herein by reference in their entirety) to analyze the selective growth inhibitory and/or cell killing effects of the interferon-alpha homologues of the present invention on specific cancer cell lines. The 60 cancer cell lines used (Table 3) included leukemia, melanoma, and lung, colon, brain, ovarian, breast, prostate, central nervous system, renal system, and kidney cancers. Human tumor cell lines were cultured according to the method described in "Monks" and http://dtp.nci.gov./branches/btb/ivclsp.html.

Screened Human Cancer Cell Lines cancer type cell line leukemia CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR colon cancer COLO205, HCC-2998, HCT-15, HCT-116, HT29, KM12, SW-620 CNS cancer SF-268, SF-295, SF-539, SNB-19, SNB-75, U251 lung cancer A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522 breast cancer MCF-7, NCI/ADR HS578T, MDA-MB-231/ATCC, MDA-MB-435, MDA-N, BT-549, T-47D melanoma LOX IMVI, M14, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL28, UACC-62, UACC-257 ovarian cancer IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3 prostate cancer DU-145, PC-3 kidney cancer 786-0, A498, ACHN, CAKI-1, RXF393, SN12C, TK-10, UO-31

Briefly, cells are seeded in 96-well microtiter plates at a density of about 5,000 to about 40,000 cells/well, depending on the growth characteristics of the particular cell line. After inoculation, the microtiter plate is incubated at 37°C for 24 hours before adding the test sample (eg, an interferon homologue of the invention or a control interferon). After 24 hours, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA) to determine the cell population of each cell line at the time of addition of the test samples (T ₀ ). In the remaining culture plates, five 10-fold serial dilutions of interferon samples (affinity-purified from CHO cell supernatants) at a concentration of 10 ^−0.8 to 10 ^−4.8 μg/ml were added.

After adding the samples, the plates were incubated for an additional 6 days. The experiment was terminated by the addition of TCA.

Cell populations were determined by measuring cellular proteins in a quantitative protein dye binding assay. A 1% acetic acid solution (100 µl) containing 0.4% (w/v) sulforhodamine B was added to each well, followed by incubation at room temperature for 10 minutes. Unbound dye was removed by washing 5 times with 1% acetic acid and the plates were allowed to air dry. The protein-bound dye was dissolved in 10 millimolar (mM) Tris and the absorbance at 515 nanometers (nm) was read on an automated plate reader.

Seven absorbance measurements were taken for each dose-response experiment, and they corresponded to: the amount of protein in the cells before the addition of the sample (time 0; T ₀ ), the amount of protein in the cells at the end of the incubation period in the absence of the test sample The amount of protein (control growth, C), and 5 parts correspond to when there is the interferon test sample of 5 kinds of concentrations, at the end of the incubation period, the assay results of the protein amount of cells (detection of 5 concentration levels of interferon Growth of test sample, T _i ). Using these assay results, the following 3 parameters were calculated for each sample tested:

GI50, or "50% Growth Inhibition" is the concentration of interferon test sample required to inhibit cell growth by 50%, this value is determined by the net protein/peptide concentration in the interferon test sample at the end of the incubation period The increase in is determined by a 50% reduction relative to that in control cells (without test sample). GI50 was calculated as the concentration of the test sample, where [(T _i - T _o )/(CT _o )] x 100 = 50. See Figure 3A.

TGI, or "Total Growth Inhibition," is the concentration of the test sample of interferon required to cause total inhibition of cell growth in which the amount of cellular protein at the end of the incubation period is equal to the amount of cellular protein at the beginning of the incubation period. The concentration of the interferon test sample that produced total growth inhibition (TGI) was calculated as the concentration of the test sample, where T _i =T _o .

LC50 is compared with the cell protein amount observed at the beginning of the incubation period, the concentration of the interferon test sample when the measured cell protein amount is reduced by 50% at the end of the incubation period. Net loss of cells after sample. LC50 was calculated as the concentration of the test sample, where [(T _i -T _o )/T _o ] x 100 = -50.

If, for a particular sample tested, the effect is not achieved or the effect is very good within the concentration range tested, the value of said parameter is expressed as greater or less than the maximum or minimum concentration tested. Example 3: In vitro activity of IFN-alpha homologues correlates with in vivo potency

Human interferon-alpha gene fragments were shuffled and screened for activity in a murine cell-based antiviral assay as described by Chang et al. (1999) Nature Biotechnol. 17:793-797. An interferon-α homologue with ¹⁰⁵ -fold higher antiviral activity than human interferon-α2a against mouse cells was isolated. The antiviral activity of various interferon-α homologues even significantly exceeds that of native mouse interferon, including Mu-IFN-α4 (Chang et al., supra). Recurrent sequence recombination (eg, DNA shuffling) of human interferon-α gene segments to generate new interferon-α homologues, followed by screening of such homologs against the mouse interferon receptor, results in the identification and isolation of Closely related murine interferon-alpha homologue.

Dose-response studies were performed in mice to determine whether the high antiviral activity observed in vitro was also maintained in vivo. Two mouse-optimized interferon-alpha homologues, referred to herein as CH2.2 and CH2.3 (SEQ ID NO: 84 and 85, respectively), were used in this study. CH2.2 and CH2.3 have approximately 138,000-fold and 206,000-fold higher activity than human interferon-α2a in the in vitro mouse cell antiviral assay, compared to natural mouse interferon-α4 , with about 2.5-fold higher and about 1.6-fold higher activity (Chang et al., supra).

Groups of Balb/c mice received subcutaneous doses of 2, 10 or 50 μg per day (in a total volume of 50 μl) of phosphate-buffered saline (PBS), interferon-α homologue CH2.2, interferon-α Homolog CH2.3, murine IFN-α4 or human interferon-α2a. On day 2, mice were exposed to a lethal intranasal dose (10 times the LC50) of vesicular stomatitis virus (VSV). Data are expressed as the number of mice surviving to day 21.

Figure 5 shows that mouse-optimized interferon-α homologues CH2.2 and CH2.3 are as effective or more effective than native mouse interferon Mu-IFN-α4 in protecting mice from VSV infection efficient. Human IFN-α2a at the concentrations tested was almost completely ineffective at protecting mice from viral infection. Therefore, the in vivo potency of the interferon-alpha homologues of the present invention correlates significantly with the antiviral activity observed in in vitro assays.

Although the foregoing invention has been described in some detail for purposes of illustration and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail could be made without departing from the true scope of the invention. For example, all of the techniques, methods, compositions, apparatus and systems described above can be used in various combinations. All publications, patents, patent applications or other documents mentioned in this application are hereby incorporated by reference in their entirety for all purposes as if each individual publication, patent, patent application or other document were incorporated by reference for all purposes. Individual mentions are incorporated herein by reference.

sequence serial number clone number sequence SEQ ID NO: 1 2DH12 TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATGCTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGACCCTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTAGGGGTGAAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 2 2CA3 TGTGATCTGCCTCAGACCCACAGCCTTGGTGACAGGAGGGCCATGATACTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGAACTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGGAGAGACTCCCCTGATGAATGGGGACTCCATCCTGGCTGTGAAGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 3 4AB9 TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCGGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 4 2DA4 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATGCTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATAGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGC AAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 5 3DA11 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGGTA CTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 6 2DB11 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATGCTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 7 2CA5 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCGGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 8 2G6 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACCCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 9 3AH7 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGCGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATAGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTCACCAGCAACTGAATGAACTG GAAGCATGTGTAGTACAGGAGGTTGGGGTGGAAGAGACTCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACCTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAGTATAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 10 2G5 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATGCTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 11 2BA8 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCCTGATACTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTAATGAATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 12 1F3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGGACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAACCTCTTCAGCACAAAGGACTCATCTGTTGCTTGGGATGAGAGGCTTCTAGACAAACTCTATACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATGCAGGAGGTGTGGGTGGGAGGGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAGAAAATACTTCCAAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 13 4BE10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAGATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATAATGCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTGGAAGCATGTGTGATACAGGGGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCTTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 14 2DD9 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATGCTCCTGGCACAAATGGGAAGAATCTCCCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGGACTCTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 15 3CA1 TGTGATCTGCCTCAGACCCACAGCCTTGGCAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTACCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTGGAAGCATGTGTGATACAGGAGGTTGGGATGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAGTATAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 16 2F8 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGAACTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 17 6CG3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAAGAGGGCCATGATGCTCCTGGCACAAATGGGAAGAACCTCTCCTTTCTCCTGTCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAGGGCTCAAGCCATCTTTGTCCTCCATGAGATGATCCAGCAGACCTTCAATTTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAAGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 18 3CG7 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGTAGGGCCTTGATGCTCCTGGCACAAATGGGAAGAATCTCCCCTTTCTCCTGCCTGAAGGACAGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACCTTCAATCTTCAGCACAAAGGATTCATCTGAGCTGATTG CTCCTAGAAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTGGAAGCATGTGTGATACAGGAGGTTGGGATGGAAGAGACTCCCCCTGATGAATGTGGACTCCATCCCTGCTGTGTGAGGAAGTACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTAGACTTTCAAGAAG SEQ ID NO: 19 1D3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGACAGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCCACCAGTTCCAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 20 2G4 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCATGATGCTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCCTGTCTGATGGACAGACATGACTTTGAATTTCCCCAGGAGGAATTTGATGATAAACAGTTCCAGAAGGCTCCAGCCATCTCTGTCCTCCATGAGGTGATTCAGCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGACCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTGGAAGCATGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTTTATCTGACAAAGAAGAAGTATAGCCCTTGTTCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 21 1A1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTCCAGAAGGCCCAAGCCATCTCTGCCTTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTCACCAGCAACTGAATGACCTGGAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAGGAAATACTTTCAAAGAATCACTCTTTATCTAATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 22 1D10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGACAGACATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCCACCAGTTCCAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAA AGATTAAGGAGGAAGGAA SEQ ID NO: 23 1F6 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATGATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGCTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTAACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 24 2A10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCATTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAGGAAATACTTTCAAAGAATCACTCTTTATCTGATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 25 2C3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTCCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGATACTTGGGATGCGACCCTTTTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 26 2D1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACAAGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCGGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 27 2D10 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAGTCTCTCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTC CAGAAGGCTCAAGCCATCTCTGCCTTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 28 2D7 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGCGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGAAGGACAGACATGACTTCAGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATAACCTGGAAGCTTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCTATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 29 2D9 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACTTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGGTGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 30 2DA2 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGCCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACAGGACTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTCCACCAGCAACTGAATGAACTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCATGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 31 2DH9 TGTGATCTGCCTCAGACCCACAGCCCTGGTAACAGGAGGGCCTTGATGCTCCTGGCACAAATGGGACGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGGGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTCTACCGGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGAGGTTGGGGTGGAAGAGACCCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAGGAAGTACTTCCAAAGAATCACT CTTTATCTGACAGAGAAGAAGCATAGCCCTTGTTCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 32 2G11 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGACTTCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGATACTTGGGAACAGAGCCTCCTAGAAAAATTCTACATTGAACTTTTCCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAGAAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGGAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 33 2G12 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATGCTCATGGCACAAATGAGGAGAATCTCTCCTTTCCCCCGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGTGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCTATCTTCCTTTTCCATGAGATGATGCAGCAGACCTTCAATCTCTTCAGCACAAAGAACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGGGAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGCTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 34 2H9 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTAGAAGCCTGTGTGACACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCTATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 35 6BC11 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGATATGATTTCGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGCTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGATTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGAGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 36 2DH12 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQTLLEKFSTELYQQLNDLEACVIQEVGVKETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 37 2CA3 CDLPQTHSLGDRRAMILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNELEACVIQEVGVGETPLMNGDSILAVKKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 38 4AB9 CDLPQTHSLGNRRALILIAQMGRISPFSCLKDRHDFGFPREEFDGNQFQKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNELEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 39 2DA4 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDSNQFQKAQAISVLHEMMQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 40 3DA11 CDLPQTHSLGNRRALVLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 41 2DB11 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDETLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 42 2CA5 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRQDFGFPQEEFDGNRFQKAQAISVLHEMIQQTFNLFSTKNSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 43 2G6 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDPILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 44 3AH7 CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDSNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELHQQLNELEACVVQEVGVEETPLMNEDSILAVKKYLQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 45 2G5 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRQDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 46 2BA8 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRRE SEQ ID NO: 47 1F3 CDLPQTHSLGNRRALILLGQMGRISHFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSVAWDERLLDKLYTELYQQLNDLEACVMQEVWVGGTPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 48 4BE10 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEIMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNELEACVIQGVGVEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCSWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 49 2DD9 CDLPQTHSLGNRRALMLLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTGLYQQLNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 50 3CA1 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 51 2F8 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQFQKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFSTELYQQLNELEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCSWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 52 6CG3 CDLPQTHSLGNKRAMMLLAQMGRTSPFSCLKDRHDFGFPQEEFDGNQFQRAQAIFVLHEMIQQTFNFFSTKDSSAAWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 53 3CG7 CDLPQTHSLGNSRALMLLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISAFHEMIQQTFNLFSTKDSSAAWEQNLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNVDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 54 1D3 CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPQEEFDGHQFQKTQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 55 2G4 CDLPQTHSLGNRRAMMLLAQMSRISPSSCLMDRHDFEFPQEEFDDKQFQKAPAISVLHEVIQQTFNLFSTEDSSAAWEQTLLEKFSTELYQQLNDLEACVMQEERVGETPLMNADSILAVRKYFQRITLYLTKKKYSPCSWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 56 1A1 CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQFQKAQAISAFHEMMQQTFNLFSTEDSSAAWEQSLLEKFSTELHQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 57 1D10 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFRFPQEEFDGNQLQKTQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQGVGVEETPPMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 58 1F6 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQEAGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 59 2A10 CDLPQTHSLGNRRALILLAQMGRISHFSCLKDRYDFGFPQEVFDGNQFQKAQAISAFHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 60 2C3 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQSQKAQAISVLHEMIQQTFNLFSTKDSSDTWDATLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 61 2D1 CDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISAFHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 62 2D10 CDLPQTHSLGNRRALILLAQMGRVSPFSCLKDRHDFGFPQEEFDGNQFQKAQAISAFHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNLEACVIQEVGVEETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 63 2D7 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFRFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELYQQLNNLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 64 2D9 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLVNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 65 2DA2 CDLPQTHSLGNRRPLILLAQMGRISPFSCLKDRQDFGFPQEEFDGNQFQKAQAISVLHEMMQQTFNLFSTKNSSAAWEQSLLEKFSTELHQQLNELEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLIERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 66 2DH9 CDLPQTHSPGNRRALMLLAQMGRISPFSCLKDRYDFGFPQGEFDGNQFQKAQAISVLHEMMQQTFNLFSTKDSSAAWEQSLLEKFSTELYRQLNDLEACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLTEKKHSPCSWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 67 2G11 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQFQKTQAISVLHEMIQQTFNLFSTKDSSDTWEQSLLEKFYIELFQQLNDLEACVIQEVGVEETPLMNVDSILAVRKYFQRITLYLTEEKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 68 2G12 CDLPQTHSLGNRRTLMLMAQMRRISPFPRLKDRYDFGFPQEVFDGNQFQKAQAIFLFHEMMQQTFNLFSTKNSSAAWDETLLDKFYTELYQQLNDLEACVMQEGRVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEAVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 69 2H9 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 70 6BC11 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRYDFGFPQEEFDGNQLQKAQAISVLHEMIQQTFNLFSTKDSSAAWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTERKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 71 t19bb CDLPQTHSLGXXRAXXLLXQMXRXSXFSCLKDRXDFGXPPXEEFDXXXFQXXQAIXXXHEXXQQTFNXFSTKXSSXXWXXXLLXKXXTXLXQQLNXLEACVXQXVXXXXTPLMNXDXILAVXKYXQRITLYLXEXKYSPCXWEVVRAEIMRSFSFSTNLQKL SEQ ID NO: 72 CH1.1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGATGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGACAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAACAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACACTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 73 CH1.2 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGGCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCCATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTAACCAGCAGCTGAATGACCTGGAAGCCTGCGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGTGGACTCCATCCTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 74 CH1.3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATGATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACACTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATGAATGTGGACTCTATCCTGACTGTGAGAAAATACTTTCGAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 75 CH1.4 TGTGATCTGCCTCAGACCCACAGCCTGGGTAATAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTCCCCCAGGAGGAGTTTGGTGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTACATTGAACTTTTCCAGCAACTGAATGACCTGGAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAAGAAATACTTCCAAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 76 CH2.1 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATGATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTC CAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACACTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGTATGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 77 CH2.2 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGTCTGATGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGACAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAACAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACACTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATGAATGTGGACTCTATCCTGACTGTGAAGAAATACTTCCGAAGAATCACTCTTTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 78 CH2.3 TGTGATCTGCCTCAGACCCACAGCCTTGGTAACAGGAGGACTTTGATGATAATGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCCTGAAGGACAGACATGACTTTGGATTTCCTCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTACTTGGGATGAGACACTTCTAGACAAATTCTACACTGAACTTTACCAGCAGCTGAATGACCTGGAAGCCTGTATGATGCAGGAGGTTGGAGTGGAAGACACTCCTCTGATGAATGAGGACTCCATCTTGGCTGTGAAGAAATACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTCTCTTTCTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAA SEQ ID NO: 79 CH1.1 CDLPQTHSLGNRRALILLAQMGRISPFSCLMDRHDFGFPQEEFDDNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 80 CH1.2 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQGISVLHEMIQQTFHLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 81 CH1.3 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILTVRKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 82 CH1.4 CDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFGGNQFQKAQAISVLHEMIQQTFNLFSTEDSSAAWDETLLDKFYIELFQQLNDLEACVMQEERVGETPLMNADSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 83 CH2.1 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMIQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 84 CH2.2 CDLPQTHSLGNRRALILLAQMGRISPFSCLMDRHDFGFPQEEFDDNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEETPLMNVDSILTVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE SEQ ID NO: 85 CH2.3 CDLPQTHSLGNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEETPLMNEDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSFSFSTNLQKRLRRKE

SEQUENCE LISTING <110> Volker, Heinrichs (HEINRICHS, VOLKER) Teddy, Chen (CHEN, TEDDY) Philip, A, Patten (PATTEN, PHILLIP A.) <120> IFN-α homologue< 130>02-101510/0140.002<140><141><150>09/415,183<151>1999-10-07<160>88<170>PatentIn Ver.2.0<211>498<212>DNA<213>Artificial序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2DH12<400>1tgtgatctgc ctcagaccca cagccttggc aacaggaggg ccttgatgct cctggcacaa  60atgggacgaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagacc  240ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg  300atacaggagg taggggtgaa agagactccc ctgatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>2<211>498<212>DNA<213>人工序列<220><223 >人工序列的描述：合成的DNA<220><223>克隆编号2CA3<400>2tgtgatctgc ctcagaccca cagccttggt gacaggaggg ccatgatact cctggcacaa  60atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcagctga atgaactgga agcatgtgtg  300atacaggagg ttggggtggg agagactccc ctgatgaatg gggactccat cctggctgtg  360aagaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>3<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号4AB9<400>3tgtgatctgc ctcagaccca cagccttggc aacaggaggg ccttgatact cctggcacaa  60atgggacgaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccgg  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactttac cagcaactga atgaactgga agcatgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>4<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220> <223>克隆编号2DA4<400>4tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag  120gaggagtttg atagcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca aaggactcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>5<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号3DA11 <400>5tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttggtact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactttac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>6<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2DB11<400>6tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactttac cagcagctga atgacttgga agcctgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210 >7<211>498<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<220><223>Clone number 2CA5<400>7tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60atgggacgaa tcctctccttt gcct aaggacagac aagactttgg attcccccag  120gaggagtttg atggcaaccg gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>8<211>498 <212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2G6<400>8tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggaccccat cctggctgtg  360aagaaatact tccaaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>9<211>498<212>DNA<213 >人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号3AH7<400>9tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgcgaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atagcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaacttcac cagcaactga atgaactgga agcatgtgta  300gtacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aagaaatacc tccaaagaat cactctttat ctgacagaga agaagtatag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>10<211>498<212>DNA<213>人工序列<220> <223>人工序列的描述：合成的DNA<220><223>克隆编号2G5<400>10tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>11<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2BA8<400>11tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccctgatact cctggcacaa  60atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctaatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>12<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA< 220><223>克隆编号1F3<400>12tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctgggacaa  60atgggaagaa tctctcattt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaacct cttcagcaca aaggactcat ctgttgcttg ggatgagagg  240cttctagaca aactctatac tgaactttac cagcagctga atgacctgga agcctgtgtg  300atgcaggagg tgtgggtggg agggactccc ctgatgaatg aggactccat cctggctgtg  360agaaaatact tccaaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>13<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号4BE10<400>13tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacag  60atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagata  180atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactttac cagcaactga atgaactgga agcatgtgtg  300atacaggggg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg  360aggaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc  420tgggaggttg tcagagcaga aatcatgaga TCTTTCTTT TTTCAAAAAAAAAAAAA CTTGCAAAAAAAAA 480AGAAGGA GGAAGGAA 498 <210> 14 <211> 498 <212> DNA <213 <220> <223C DNA <220> <223> Klong Number 2dd9 400 2dd9 ctcagaccca cagccttggt aacaggaggg ccttgatgct cctggcacaa  60atgggaagaa tctccccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tggactctac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498 <210>15<211>498<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<220><223>Clone number 3CA1<400>15tgtgatctgc ctcagaccca cagccttggc aacaggaggg ccttgatact cctggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attaccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg  300atacaggagg ttgggatgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>16<211 >498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2F8<400>16tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc  240ctcctagaaa aattttccac tgaactttac cagcaactga atgaactgga agcatgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaagtatag cccttgttcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号6CG3<400>17tgtgatctgc ctcagaccca cagccttggt aacaagaggg ccatgatgct cctggcacaa  60atgggaagaa cctctccttt ctcctgtctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagagg gctcaagcca tctttgtcct ccatgagatg  180atccagcaga ccttcaattt cttcagcaca aaggactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggaag ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>18<211>498<212>DNA<213>人工序列<220><223 >人工序列的描述：合成的DNA<220><223>克隆编号3CG7<400>18tgtgatctgc ctcagaccca cagccttggt aacagtaggg ccttgatgct cctggcacaa  60atgggaagaa tctccccttt ctcctgcctg aaggacagac atgatttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagaac  240ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg  300atacaggagg ttgggatgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>19<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号1D3<400>19tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctcattt ctcctgcctg aaggacagac atgatttcgg attcccccag  120gaggagtttg atggccacca gttccagaag actcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgatggaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>20<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220> <223>克隆编号2G4<400>20tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccatgatgct cctggcacaa  60atgagcagaa tctctccttc ctcctgtctg atggacagac atgactttga atttccccag  120gaggaatttg atgataaaca gttccagaag gctccagcca tctctgtcct ccatgaggtg  180attcagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggaacagacc  240ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg  300atgcaggagg agagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg  360aggaaatact tccaaagaat cactctttat ctgacaaaga agaagtatag cccttgttcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>21<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号1A1 <400>21tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctcattt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggtgtttg atggcaacca gttccagaag gcccaagcca tctctgcctt ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaacttcac cagcaactga atgacctgga agcctgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aggaaatact ttcaaagaat cactctttat ctaatggaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>22<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号1D10<400>22tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctcattt ctcctgcctg aaggacagac atgatttcgg attcccccag  120gaggagtttg atggccacca gttccagaag actcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcaactga atgacctgga agcatgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgatggaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210 >23<211>498<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<220><223>Clone number 1F6<400>23tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa 60atgggaagacca gctctcctcct aaggacagac atgactttgg atttccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ctggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctaacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>24<211>498 <212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2A10<400>24tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctcattt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggtgtttg atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcatgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cctggctgtg  360aggaaatact ttcaaagaat cactctttat ctgatggaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>25<211>498<212>DNA<213 >人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2C3<400>25tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag  120gaggagtttg atggcaacca gtcccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgatacttg ggatgcgacc  240cttttagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>26<211>498<212>DNA<213>人工序列<220> <223>人工序列的描述：合成的DNA<220><223>克隆编号2D1<400>26tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggacgaa tctctccttt ctcctgcctg aaggacagac aagactttgg attcccccag  120gaggagtttg atggcaaccg gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactctac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg aggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>27<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2D10<400>27tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagag tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgcctt ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcctgcgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>28<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA< 220><223>克隆编号2D7<400>28tgtgatctgc ctcagaccca cagccttggt aacaggcggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgtctg aaggacagac atgacttcag atttccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaactttac cagcaactga ataacctgga agcttgcgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactctat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga ggaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>29<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2D9<400>29tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ctttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagactccc ctggtgaatg tggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>30<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2DA2<400>30tgtgatctgc ctcagaccca cagccttggt aacaggaggc ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac aggacttcgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactccac cagcaactga atgaactgga agcatgtgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctaatagaga ggaaatacag cccttgtgca  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498 <210>31<211>498<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<220><223>Clone number 2DH9<400>31tgtgatctgc ctcagaccca cagccctggt aacaggaggg ccttgatgct cctggcacaa 60atgggacgaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120ggggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaactctac cggcagctga atgacctgga agcctgtgtg  300atacaggagg ttggggtgga agagaccccc ctgatgaatg tggactccat cctggctgtg  360aggaagtact tccaaagaat cactctttat ctgacagaga agaagcatag cccttgttcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>32<211 >498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2G11<400>32tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg acttccccag  120gaggagtttg atggcaacca gttccagaag actcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgatacttg ggaacagagc  240ctcctagaaa aattctacat tgaacttttc cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360agaaaatact tccaaagaat cactctttat ctgacagagg agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>33<211>498<212>DNA <213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号2G12<400>33tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgct catggcacaa  60atgaggagaa tctctccttt cccccgcctg aaggacagat atgatttcgg attcccccag  120gaggtgtttg atggcaacca gttccagaag gctcaagcta tcttcctttt ccatgagatg  180atgcagcaga ccttcaatct cttcagcaca aagaactcat ctgctgcttg ggatgagacc  240ctcctagaca aattctacac tgaactctac cagcagctga atgacttgga agcctgtgtg  300atgcaggagg ggagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg  360aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggctg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>34<211>498<212>DNA<213>人工序列< 220><223>人工序列的描述：合成的DNA<220><223>克隆编号2H9<400>34tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctaga agcctgtgtg  300acacaggagg ttggggtgga agagactccc ctgatgaatg aggactctat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>35<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号6BC11<400>35tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagat atgatttcgg attcccccag  120gaggagtttg atggcaacca gctccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggattcat ctgctgcttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggagtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga ggaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>36<211>166<212>PRT<213>人工序列<220><223>人工序列的描述：合成的Amino acid <220> <223> Cell No. 2DH12 <400> 36CYS ASP Leu Pro Gln Thr His Seru Gly Asn ARG ARG Ala Leu Met1 5 10 15LEU Ala Gln Met GLY Ile Serle Ser Cys Leu Lys ASP

20 25 25 30Arg Gln Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Trp Gln THR65 75 80leu Leu Leu Lys Phe Ser THR Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Lys Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>37<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2CA3<400>37Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asp Arg Arg Ala Met Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys Les

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 75 80leu Leu LYS PHE Ser Gln Gln Gln Leu asn Glu Leu Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Gly Glu Thr Pro Leu Met

100 105 110Asn Gly Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>38<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 4AB9<400>38Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Arg Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr

 50                  55                  60Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr65                  70                  75                  80Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Glu Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Ash Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>39<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2DA4<400>39Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Gln Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Ser Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr

50 55 60phe asn leu pher leys asp seer ala ala trp asp Glu ThR65 75 80leu leu leu lys pher glu leu tyr gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>40<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 3DA11<400>40Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Val1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys Lesp

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asp seer ala ala trp asp Glu ThR65 75 80leu leu leu lys pher glu leu tyr gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>41<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2DB11<400>41Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asp seer ala ala trp asp Glu ThR65 75 80leu leu leu lys pher glu leu tyr gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>42<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2CA5<400>42Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Gln Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Arg Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asn seer ala ala ala trp glu gln seer65 75 80leu leu leu lys pher gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>43<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2G6<400>43Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Ser Ser's Ala THR TRP GLN Ser65 75 80leu Leu LYS PHE Series Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Pro Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>44<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 3AH7<400>44Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Ser Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 75 80leu Leu LYS PHE Series Gln Gln Leu asn Glu Leu Leu Leu

85 90 95Glu Ala Cys Val Val Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Leu Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>45<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2G5<400>45Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Gln Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 75 80leu Leu LYS PHE Serteu Leu Tyr Gln Leu Asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

L30 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>46<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2BA8<400>46Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 75 80leu Leu LYS PHE Serteu Leu Tyr Gln Leu Asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>47<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 1F3<400>47Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Gly Gln Met Gly Arg Ile Ser His Phesp u Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Ser Val Val Ala TRP GLU ARG65 75 80leu Leu as Leu Tyr Tyr Gln Gln Leu asn asp leu

85 90 95Glu Ala Cys Val Met Gln Glu Val Trp Val Gly Gly Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>48<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 4BE10<400>48Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Ile Met Gln Gln Thr

 50                  55                  60Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr65                  70                  75                  80Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Glu Leu

85 90 95Glu Ala Cys Val Ile Gln Gly Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>49<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2DD9<400>49Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher lys aser ala Ala Ala Trp Gln Ser65 70 80leu Leu LYS PHE Ser Gln Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>50<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 3CA1<400>50Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Leu Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asn seer ala ala trp asp Glu ThR65 75 80leu leu leu lys pher glu leu tyr gln leu asn leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>51<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2F8<400>51Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr

 50                  55                  60Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr65                  70                  75                  80Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Glu Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>52<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 6CG3<400>52Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Lys Arg Ala Met Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Thr Ser Pro Phe Lys Cys Les

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Arg Ala Gln Ala Ile Phe Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn phe the ser leys asp seer ala ala trp glu gln seer65 75 80leu leu la lys pher glu leu asn gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>53<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 3CG7<400>53Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Ser Arg Ala Leu Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Ala Ala Phe His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Trp Gln ASN65 75 80leu Leu Leu Lys Phe Ser Tyr Gln Leu asn Leu asn Leu asn Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>54<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 1D3<400>54Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser His Phesp u Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly His Gln Phe

35 40 45Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 75 80leu Leu LYS PHE Serteu Leu Tyr Gln Leu Asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>55<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2G4<400>55Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Met Met1 5 5 et A 10 15Leu Leu Ala Gln Met Ser Arg Ile Ser Pro My Ser Ser spu Cys

20 25 25 30Arg His Asp Phe Glu Phe Pro Gln Glu Glu Phe Asp Asp Lys Gln Phe

35 40 45Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Val Ile Gln Gln Thr

50 55 60phe asn leu pher glu asp sela ala ala trp Gln THR65 70 80leu leu leu lys pher glu leu tyr gln leu asn asp leu

85 90 95Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met

100 105 110Asn Ala Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Thr Lys Lys Lys Tyr Ser Pro Cys Ser Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>56<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 1A1<400>56Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser His Phesp u Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Val Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Ser Ala Ala Phe His Glu Met Met Gln Gln Thr

50 55 60phe asn leu pher glu asp ser ala ala ala trp glu gln seer65 75 80leu leu leu lys pher gln gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>57<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 1D10<400>57Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Leu

35 40 45Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Ser Ser's Ala THR TRP GLN Ser65 75 80leu Leu LYS PHE Series Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Gly Val Gly Val Glu Glu Thr Pro Pro Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>58<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 1F6<400>58Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met1 5 5 A 10 15Ile Met Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Ser Ser's Ala THR TRP GLN Ser65 75 80leu Leu LYS PHE Series Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Ala Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>59<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2Al0<400>59Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser His Phesp u Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Val Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Ala Ala Phe His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys aser sela THR TRP GLU GLN Ser65 75 80leu Leu LYS PHE Serteu Leu Tyr Gln Leu asn Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>60<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2C3<400>60Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Ser

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asp ser ser trp Ala THR65 75 80leu leu leu lys pher glu leu asn leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>61<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2D1<400>61Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Ala Ala Phe His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 70 80leu Leu LYS PHE Series PHR Gln Gln Leu asn Leu asn Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Met Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>62<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2D10<400>62Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ilel 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Val Ser Pro Phe Ser Lys Cys Lesp

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Ala Ala Phe His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys aser sela THR TRP GLU GLN Ser65 75 80leu Leu LYS PHE Serteu Leu Tyr Gln Leu asn Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>63<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2D7<400>63Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys aser sela THR TRP GLU GLN Ser65 75 80leu Leu LYS PHE Serteu Leu Tyr Gln Leu asn Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Thr Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>64<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2D9<400>64Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Ser Ser's Ala THR TRP GLN Ser65 75 80leu Leu LYS PHE Series Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Val

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>65<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2DA2<400>65Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Pro Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys Lesp

20 25 25 30Arg Gln Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr

50 55 60phe asn leu pher leys asn seer ala ala ala trp glu gln seer65 75 80leu leu leu leu leu leu his gln leu asn glu leu leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Ile Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>66<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2DH9<400>66Cys Asp Leu Pro Gln Thr His Ser Pro Gly Asn Arg Arg Ala Leu Met1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys Lesp

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Gly Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 70 80leu Leu Leu LYS PHE Serg Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Thr Glu Lys Lys His Ser Pro Cys Ser Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>67<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2G11<400>67Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Leu Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Thr Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher lys asp seer ser tRP GLU glu gln seer65 75 80leu leu lys pher Ile gln leu asn asp leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>68<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2G12<400>68Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met1 5 5 A 10 15Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe Pro Arg Lesp

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Val Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Phe Leu Phe His Glu Met Met Gln Gln Thr

50 55 60phe asn leu pher leys asn sela ala ala trp asp Glu ThR65 75 80leu leu as phete tyr glu leu tyr gln leu asn asp leu leu

85 90 95Glu Ala Cys Val Met Gln Glu Gly Arg Val Gly Glu Thr Pro Leu Met

100 105 110Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Ala Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>69<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 2H9<400>69Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asp seer ala ThR TRP GLN Ser 65 70 75 80leu Leu LYS PHE Serteu Leu asn Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Thr Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>70<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number 6BC11<400>70Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Lys Cys

20 25 25 30Arg Tyr Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Leu

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher Lysp Sero Ala Ala Ala Trp Gln Ser65 75 80leu Leu LYS PHE Series Gln Gln Leu asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 125Leu Tyr Leu Thr Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>71<211>166<212>PRT<213>artificial sequence<220><223>Description of artificial sequence: synthetic amino acid <220><223>clone number t19bb<220><221>MOD_RES<222 >(11)<223>N or D<220><221>MOD_RES<222>(12)<223>R, S, or K<220><221>MOD_RES<222>(15)<223>L or M<220><221>MOD_RES<222>(16)<223>I, M or V<220><221>MOD_RES<222>(19)<223>A or G<220><221>MOD_RES<222 >(22)<223>G or R<220><221>MOD_RES<222>(24)<223>I or T<220><221>MOD_RES<222>(26)<223>P or H<220 ><221>MOD_RES<222>(34)<223>H, Y or Q<220><221>MOD_RES<222>(38)<223>F or L<220><221>MOD_RES<222>(40 )<223>Q or R<220><221>MOD_RES<222>(45)<223>G or S<220><221>MOD_RES<222>(46)<223>N or H<220><221 >MOD_RES<222>(47)<223>Q or R<220><221>MOD_RES<222>(50)<223>K or R<220><221>MOD_RES<222>(51)<223>A or T<220><221>MOD_RES<222>(55)<223>S or F<220><221>MOD_RES<222>(56)<223>V or A<220><221>MOD_RES<222> (57)<223>L or F<220><221>MOD_RES<222>(60)<223>M or I<220><221>MOD_RES<222>(61)<223>I or M<220> <221>MOD_RES<222>(67)<223>L or F<220><221>MOD_RES<222>(72)<223>D or N<220><221>MOD_RES<222>(75)<223 >A or V<220><221>MOD_RES<222>(76)<223>A or T<220><221>MOD_RES<222>(78)<223>E or D<220><221>MOD_RES< 222>(79)<223>Q or E<220><221>MOD_RES<222>(80)<223>S, R, T, or N<220><221>MOD_RES<222>(83)<223 >E or D<220><221>MOD_RES<222>(85)<223>F or L<220><221>MOD_RES<222>(86)<223>S or Y<220><221>MOD_RES< 222>(88)<223>E or G<220><221>MOD_RES<222>(90)<223>Y, H, N<220><221>MOD_RES<222>(95)<223>D, E, or N<220><221>MOD_RES<222>(101)<223>I, M, or V<220><221>MOD_RES<222>(103)<223>E or G<220><220 ><221>MOD_RES<222>(105)<223>G or W<220><221>MOD_RES<222>(106)<223>V or M<220><221>MOD_RES<222>(107)< 223>E, G, or K<220><221>MOD_RES<222>(108)<223>E or G<220><221>MOD_RES<222>(114)<223>V, E, or G< 220><221>MOD_RES<222>(116)<223>S or P<220><221>MOD_RES<222>(121)<223>K or R<220><221>MOD_RES<222>(124) <223>F or L<220><221>MOD_RES<222>(132)<223>T, I, or M<220><221>MOD_RES<222>(134)<223>K or R<220> <221> MOD_RES <222> (140) <223> A or S <400> 71CYS ASP Leu Gln THR His Serou Gly XAA XAA ARG ALA XAA XAA XAA 1 5 10 15LEU XAA ARG XAA Serg XAA PHE SER Cys Leu Lys Asp

20 25 25 30Arg Xaa Asp Phe Gly Xaa Pro Xaa Glu Glu Phe Asp Xaa Xaa Xaa Phe

35 40 45Gln Xaa Xaa Gln Ala Ile Xaa Xaa Xaa His Glu Xaa Xaa Gln Gln Thr

50 55 60phe asn XAA PHE Ser THR LYS XAA Ser XAA XAA TRP XAA XAA XAA XAA65 70 80leu XAA LYS XAA THR XAA Leu's X

85 90 95Glu Ala Cys Val Xaa Gln Xaa Val Xaa Xaa Xaa Xaa Thr Pro Leu Met

100 105 110Asn Xaa Asp Xaa Ile Leu Ala Val Xaa Lys Tyr Xaa Gln Arg Ile Thr

115 120 125Leu Tyr Leu Xaa Glu Xaa Lys Tyr Ser Pro Cys Xaa Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>72<211>498<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<220><223>Clone number CH1.1<400>72tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgtctg atggacagac atgactttgg atttccccag  120gaggagtttg atgacaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccaacaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca  240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg  360aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210> 73<211>498<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<220><223>Clone number CH1.2<400>73tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa 60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg atggcaacca gttccagaag gctcaaggca tctctgtcct ccatgagatg  180atccagcaga ccttccatct cttcagcaca aaggactcat ctgctacttg ggaacagagc  240ctcctagaaa aattttccac tgaacttaac cagcagctga atgacctgga agcctgcgtg  300atacaggagg ttggggtgga agagactccc ctgatgaatg tggactccat cctggctgtg  360aagaaatact tccgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>74<211> 498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号CH1.3<400>74tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag 120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ceatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca  240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg  300atgcaggagg ttggagtgga agacactcct ctgatgaatg tggactctat cctgactgtg  360agaaaatact ttcgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>75<211>498<212> DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号CH1.4<400>75tgtgatctgc ctcagaccca cagcctgggt aataggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg attcccccag  120gaggagtttg gtggcaacca gttccagaag gctcaagcca tctctgtcct ceatgagatg  180atccagcaga ccttcaatct cttcagcaca gaggactcat ctgctgcttg ggatgagacc  240ctcctagaca aattctacat tgaacttttc cagcaactga atgacctgga agcctgtgtg  300atgcaggagg agagggtggg agaaactccc ctgatgaatg cggactccat cttggctgtg  360aagaaatact tccaaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>76<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号CH2.1tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggacteat ctgctacttg ggatgagaca 240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg  300atacaggagg ttggggtgga agagactccc ctgatgaatg aggactccat cttggctgtg  360aagaaatact tccgaagaat GaGtctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>77<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号CH2.2<400>77tgtgatctgc ctcagaccca cagccttggt aacaggaggg ccttgatact cctggcacaa  60atgggaagaa tctctccttt ctcctgtctg atggacagac atgactttgg atttccccag  120gaggagtttg atgacaacca gttccagaag gctcaagcca tctctgtcct ccatgagatg  180atccaacaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca  240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg  300atgcaggagg ttggagtgga agacactcct ctgatgaatg tggactctat cctgactgtg  360aagaaatact tccgaagaat cactctttat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tttcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>78<211>498<212>DNA<213>人工序列<220><223>人工序列的描述：合成的DNA<220><223>克隆编号CH2.3<400>78tgtgatctgc ctcagaccca cagccttggt aacaggagga ctttgatgat aatggcacaa  60atgggaagaa tctctccttt ctcctgcctg aaggacagac atgactttgg atttcctcag  120gaggagtttg atggcaacca gttccagaag gctcaagcca tctctgtcct ceatgagatg  180atccagcaga ccttcaatct cttcagcaca aaggactcat ctgctacttg ggatgagaca  240cttctagaca aattctacac tgaactttac cagcagctga atgacctgga agcctgtatg  300atgcaggagg ttggagtgga agacactcct ctgatgaatg aggactceat cttggctgtg  360aagaaatact tccgaagaat cactctctat ctgacagaga agaaatacag cccttgtgcc  420tgggaggttg tcagagcaga aatcatgaga tctttctctt tctcaacaaa cttgcaaaaa  480agattaagga ggaaggaa                                                498<210>79<211>166<212>PRT<213>人工序列<220><223>人工序列的描述：合成的氨基酸<220> <223> CH1.1 <400> 79CYS ASP Leu Pro Gln Thr His Seru Gly Asn ARG Ala Leu Ileu Leu Ala Gln Met GLY ILE Serite Serte Seru Met ASP

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Asp Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher leys asp seer ala ThR TRP ASP GLU THR65 75 80leu Leu as PHE TYR GLU TYR Gln Gln Leu Asn Asn Asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>80<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number CH1.2<400>80Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Le Ser Lys Pro Cys Phe

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Gly Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe his leu pher leys asp seer ala THR TRP GLN Ser65 70 75 80leu Leu Leu LYS PHE Series Gln Gln Leu Asn ASP Leu

85 90 95Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>81<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number CH1.3<400>81Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met1 5 5 A 10 15Ile Met Ala Gln Met Gly Arg Ile Ser u spey Ser Cys Phe

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher leys asp seer ala ThR TRP ASP GLU THR65 75 80leu Leu as PHE TYR GLU TYR Gln Gln Leu Asn Asn Asn ASP Leu

85 90 95Glu Ala Cys Met Met Gln Glu Val Gly Val Glu Asp Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Thr Val Arg Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>82<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number CH1.4<400>82Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A 10 15Leu Leu Ala Gln Met Gly Arg Ile Le Ser Lys Pro Cys Phe

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher glu asp ser ala ala trp ASP GLU ThR65 75 80leu leu as pher Ile gln gln leu asn asp leu

85 90 95Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met

100 105 110Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>83<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number CH2.1<400>83Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met1 5 5 A 10 15Ile Met Ala Gln Met Gly Arg Ile Ser u spey Ser Cys Phe

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher leys asp seer ala ThR TRP ASP GLU THR65 75 80leu Leu as PHE TYR GLU TYR Gln Gln Leu Asn Asn Asn ASP Leu

85 90 95Glu Ala Cys Met Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>84<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number CH2.2<400>84Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile1 5 5 A et 10 15Leu Leu Ala Gln Met Gly Arg Ile M Ser Pro C ys Phe

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Asp Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 60phe asn leu pher leys asp seer ala THR TRP ASP GLU THR 65 75 80leu Leu as PHE TYR GLU TYR Gln Gln Leu Asn Asn ASP Leu

85 90 95Glu Ala Cys Met Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Val Asp Ser Ile Leu Thr Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>85<211>166<212>PRT<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic amino acid <220><223>Clone number CH2.3<400>85Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Thr Leu Met1 5 5 A 10 15Ile Met Ala Gln Met Gly Arg Ile Ser u spey Ser Cys Phe

20 25 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe

35 40 45Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr

50 55 55phe asn leu pher leys asp seer ala ThR TRP ASP GLU THR65 75 80leu Leu as PHE TYR GLU TYR Gln Gln Leu Asn Asn Asn ASP Leu

85 90 95Glu Ala Cys Met Met Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met

100 105 110Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr

115 120 Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val

130 135 140ARG ALU ILE MET ARG Serg Serg Serg Serge Ser THR ASN Leu Gln Lys145 150 155RG Leu ARG LYS GLU

165<210>86<211>15<212>DNA<213>Artificial sequence<220><223>Description of artificial sequence: Synthetic DNA<400>86tgcgacttac cacaa 2 <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic amino acid <400> 87TRP Glu Val Val ARG Serg Serg Serg Serg Serg Sergr Serg ARG Leu ARG ARG LYS Asp

20 25<210>88<211>26<212>PRT<213>artificial sequence <220><223>description of artificial sequence: synthetic amino acid <223>description of artificial sequence: synthetic amino acid <400>88Trp Glu Leu Val Arg Ala Glu Ile Val Arg Ser Phe Ser Phe Ser Thr1 5 10 15Asn Leu Asn Lys Arg Leu Arg Lys Lys Glu

20 25

Claims (123)

1. An isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 1 to SEQ ID NO: 35, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO: 36 to SEQ ID NO: 70, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence that hybridizes to substantially the full-length polynucleotide sequence (a) or (b) under highly stringent hybridization conditions; and (d) A polynucleotide sequence comprising a fragment of (a), (b) or (c) encoding a polypeptide having anti-proliferative activity in an assay based on the human Daudi cell line. 2. An isolated or recombinant nucleic acid comprising a polynucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 72 to SEQ ID NO: 78, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO: 79 to SEQ ID NO: 85, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence that hybridizes to substantially the full-length polynucleotide sequence (a) or (b) under highly stringent hybridization conditions; and (d) A polynucleotide sequence comprising a fragment of (a), (b) or (c) encoding a polypeptide having antiviral activity in a murine cell line/EMCV based assay. 3. An isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising the following amino acid sequence: CDLPQTHSLG-X ₁₁ -X ₁₂ -RA-X ₁₅ -X ₁₆ -LL-X ₁₉ -QM-X ₂₂ -RX ₂₄ -SX ₂₆ -FSCLKDR-X ₃₄ -DFG-X ₃₈ -PX ₄₀ -EEFD-X ₄₅ -X ₄₆ -X ₄₇ -FQ-X ₅₀ -X ₅₁ -QAI-X ₅₅ -X ₅₆ -X ₅₇ -HE-X ₆₀ -X ₆₁ -QTFN-X ₆₇ -FSTK-X ₇₂ -SS-X ₇₅ -X ₇₆ -WX ₇₈ -X ₇₉ -X ₈₀ -LL-X ₈₃ -KX ₈₅ -X ₈₆ -TX ₈₈ - LX ₉₀ -QQLN-X ₉₅ -LEACV-X ₁₀₁ -QX ₁₀₃ -VX ₁₀₅ -X ₁₀₆ -X ₁₀₇ -X ₁₀₈ -TPLMN-X ₁₁₄ -DX ₁₁₆ -ILAV-X ₁₂₁ -KY-X ₁₂₄ -QRITLYL-X ₁₃₂ -EX ₁₃₄ -KYSPC-X ₁₄₀ -WEVVRAEIMRSFSFSTNLQKRLRRKE, or conservatively substituted variants thereof, wherein X ₁₁ is N or D; X ₁₂ is R, S or K; X ₁₅ is L or M; X ₁₆ is I, M or V; X ₁₉ is A or G; X ₂₂ is G or R; X ₂₄ is I or T; X ₂₆ is P or H; X ₃₄ is H, Y or Q; X ₃₈ is F or L; X ₄₀ is Q or R; X ₄₅ is G or S; X ₄₆ is N or H; X ₄₇ is Q or R; X ₅₀ is K or R; X ₅₁ is A or T; X ₅₅ is S or F; X ₅₆ is V or A; X ₅₇ is L or F; X ₆₀ is M or I; X ₆₁ is I or M; X ₆₇ is L or F; X ₇₂ is D or N; X ₇₅ is A or V; X ₇₆ is A or T ; X ₇₈ is E or D; X ₇₉ is Q or E; X ₈₀ is S, R, T or N; X ₈₃ is E or D; X ₈₅ is F or L; X ₈₆ is S or Y; X ₈₈ is E or G; X ₉₀ is Y, H, N; X ₉₅ is D, E or N; X ₁₀₁ is I, M or V; X ₁₀₃ is E or G; X ₁₀₅ is G or W; X ₁₀₆ is V or M; X ₁₀₇ is E, G or K; X ₁₀₈ is E or G; X ₁₁₄ is V, E or G; X ₁₁₆ is S or P; X ₁₂₁ is K or R; X ₁₂₄ is F or L; X ₁₃₂ is T, I or M; X ₁₃₄ is K or R; and X ₁₄₀ is A or S. 4. The nucleic acid of claim 3, said polypeptide having antiproliferative activity in a cell proliferation assay based on the human Daudi cell line, or antiviral activity in an assay based on human WISH cells/EMCV. 5. The nucleic acid of claim 3, wherein the encoded polypeptide has an antiproliferative activity of at least about 8.3 x ¹⁰ units/mg in an assay based on the human Daudi cell line, or in an assay based on human WISH cells/EMCV Has an antiviral activity of at least about 2.1 x ¹⁰⁷ units/mg. 6. The nucleic acid of claim 3, wherein the encoded polypeptide contains an amino acid sequence selected from SEQ ID NO: 36 to SEQ ID NO: 54. 7. The nucleic acid of claim 3, said nucleic acid comprising a polynucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 19. 8. An isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising: at least 20 consecutive amino acids of any one of SEQ ID NO: 36-70, and Ala19, (Tyr Or one or more amino acids in Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein the numbering of the amino acid corresponds to the numbering in SEQ ID NO:36. 9. The nucleic acid of claim 8, wherein the encoded polypeptide is 166 amino acids in length. 10. The nucleic acid of claim 8, wherein the encoded polypeptide has antiproliferative activity in an assay based on the human Daudi cell line. 11. The nucleic acid of claim 8, wherein the encoded polypeptide has antiviral activity in a human WISH cell/EMCV based assay. 12. The nucleic acid of claim 8, wherein the encoded polypeptide comprises amino acids Ala19, (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166. 13. The nucleic acid of claim 8, wherein the encoded polypeptide contains at least 50 consecutive amino acid residues of any one of SEQ ID NO: 36-70. 14. The nucleic acid of claim 8, wherein the encoded polypeptide contains at least 100 consecutive amino acid residues of any one of SEQ ID NO: 36-70. 15. The nucleic acid of claim 8, wherein the encoded polypeptide contains at least 150 consecutive amino acid residues of any one of SEQ ID NO: 36-70. 16. The nucleic acid of claim 8, wherein the encoded polypeptide contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 45 and SEQ ID NO: 46. 17. The nucleic acid of claim 8, comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO : 7, SEQ ID NO: 10 and SEQ ID NO: 11. 18. An isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide comprising: at least 155 contiguous amino acids of any one of SEQ ID NO: 36-70, said amino acid sequence comprising amino acids Lys160 and Glu166 , wherein the numbering of amino acids corresponds to the numbering in SEQ ID NO:36. 19. The nucleic acid of claim 18, wherein the encoded polypeptide contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 45 and SEQ ID NO: 46. 20. A cell comprising the nucleic acid of claim 1, 2, 8 or 18. 21. The cell of claim 20, wherein said cell expresses a polypeptide encoded by said nucleic acid. 22. A vector comprising the nucleic acid of claim 1, 2, 8 or 18. 23. The vector of claim 20, wherein said vector comprises a plasmid, cosmid, phage or virus. 24. The vector of claim 22, wherein said vector is an expression vector. 25. A cell transduced with the vector of claim 22. 26. A composition comprising a nucleic acid according to claim 1, 2, 8 or 18 and an excipient. 27. The composition of claim 26, wherein the excipient is a pharmaceutically acceptable excipient. 28. A composition produced by digestion of one or more nucleic acids of claim 1, 2, 3, 8 or 18 with a restriction endonuclease, RNase or DNase. 29. A composition produced by a method comprising incubating one or more nucleic acids of claims 1, 2, 3, 8 or 18 in the presence of deoxyribonucleotide triphosphates and a nucleic acid polymerase. 30. The composition of claim 29, wherein the nucleic acid polymerase is a thermostable polymerase. 31. An isolated or recombinant polypeptide encoded by the nucleic acid of claim 1, 2, 3, 8 or 18. 32. The isolated or recombinant polypeptide of claim 31 comprising a sequence selected from SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85. 33. The polypeptide of claim 31, which has an antiproliferative activity of at least about 8.3×10 units/mg in an assay based on the human Daudi cell line, or at least about 8.3× ¹⁰ units/mg in an assay based on human WISH cells/EMCV Antiviral activity of 2.1×10 ⁷ units/mg. 34. An isolated or recombinant polypeptide comprising the following amino acid sequence: CDLPQTHSLG-X11-X12-RA-X15- _X16 -LL- _X19 -QM- _X22 - _RX24 - _SX26 -FSCLKDR- _X34 -DFG- X ₃₈ -PX ₄₀ -EEFD-X ₄₅ -X ₄₆ -X ₄₇ -FQ-X ₅₀ -X ₅₁ -QAI-X ₅₅ -X ₅₆ -X ₅₇ -HE-X ₆₀ -X ₆₁ -QTFN-X ₆₇ -FSTK -X ₇₂ -SS-X ₇₅ -X ₇₆ -WX ₇₈ -X ₇₉ -X ₈₀ -LL-X ₈₃ -KX ₈₅ -X 86 -TX _{88 -LX 90} -QQLN-X ₉₅ -LEACV-X ₁₀₁ -QX ₁₀₃ -VX ₁₀₅ -X ₁₀₆ -X ₁₀₇ -X ₁₀₈ -TPLMN-X ₁₁₄ -DX ₁₁₆ -ILAV-X ₁₂₁ -KY-X ₁₂₄ -QRITLYL-X ₁₃₂ -EX ₁₃₄ -KYSPC-X ₁₄₀ -WEVVRAEIMRSFSFSTNLQKRLRRKE, or conservative substitutions thereof wherein X ₁₁ is N or D; X ₁₂ is R, S or K; X ₁₅ is L or M; X ₁₆ is I, M or V; X ₁₉ is A or G; X ₂₂ is G or R ; X ₂₄ is I or T; X ₂₆ is P or H; X ₃₄ is H, Y or Q; X ₃₈ is F or L; X ₄₀ is Q or R; X ₄₅ is G or S; X ₄₆ is N or H; X ₄₇ is Q or R; X ₅₀ is K or R; X ₅₁ is A or T; X ₅₅ is S or F; X ₅₆ is V or A; X ₅₇ is L or F; X ₆₀ is M or I ; X ₆₁ is I or M; X ₆₇ is L or F; X ₇₂ is D or N; X ₇₅ is A or V; X ₇₆ is A or T; X ₇₈ is E or D; X ₇₉ is Q or E; X ₈₀ is S, R, T or N; X ₈₃ is E or D; X ₈₅ is F or L; X ₈₆ is S or Y; X ₈₈ is E or G; X ₉₀ is Y, H, N; X ₉₅ is D, E or N; X ₁₀₁ is I, M or V; X ₁₀₃ is E or G; X ₁₀₅ is G or W; X ₁₀₆ is V or M; X ₁₀₇ is E, G or K; X ₁₀₈ is E or G; X ₁₁₄ is V, E or G; X ₁₁₆ is S or P; X ₁₂₁ is K or R; X ₁₂₄ is F or L; X ₁₃₂ is T, I or M; X ₁₃₄ is K or R; and X ₁₄₀ is A or S. 35. The polypeptide of claim 34, which has an antiproliferative activity of at least about 8.3×10 units/mg in an assay based on the human Daudi cell line, or at least about 8.3× ¹⁰ units/mg in an assay based on human WISH cells/EMCV Antiviral activity of 2.1×10 ⁷ units/mg. 36. The polypeptide of claim 34, comprising a sequence selected from SEQ ID NO: 36 to SEQ ID NO: 54. 37. A polypeptide comprising at least 100 contiguous amino acids of a protein encoded by an encoding polynucleotide sequence, wherein said polynucleotide sequence is selected from the group consisting of: (a) SEQ ID NO: 1 to SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78; (b) an encoding polynucleotide sequence encoding a first polypeptide selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85; and (c) a complementary polynucleotide sequence that hybridizes to substantially the full-length polynucleotide sequence (a) or (b) under highly stringent conditions. 38. The polypeptide of claim 37, which has antiproliferative activity in a cell proliferation assay based on the human Daudi cell line, or antiviral activity in an assay based on human WISH cells/EMCV. 39. The polypeptide of claim 37, wherein said polypeptide specifically binds human alpha-interferon receptor. 40. The polypeptide of claim 37 comprising at least 150 contiguous amino acids of the encoded protein. 41. An isolated or recombinant polypeptide comprising an amino acid sequence comprising at least 50 contiguous amino acids of any one of SEQ ID NO: 36-70, and comprising one or more of the following amino acids: Ala19, (Tyr or Gln)34, Amino acid sequences of Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein the numbering of amino acids corresponds to the numbering in SEQ ID NO:36. 42. The polypeptide of claim 41, wherein said polypeptide binds human alpha-interferon receptor. 43. The polypeptide of claim 41 having antiproliferative activity in a cell proliferation assay based on the human Daudi cell line, or antiviral activity in an assay based on human WISH cells/EMCV. 44. The polypeptide of claim 41, which has an antiproliferative activity of at least about 8.3 x 10 units/mg in an assay based on the human Daudi cell line, or at least about 8.3 x ¹⁰ units/mg in an assay based on human WISH cells/EMCV Antiviral activity of 2.1×10 ⁷ units/mg. 45. The polypeptide of claim 41, wherein said polypeptide is 166 amino acids in length. 46. The polypeptide of claim 41, said polypeptide contains amino acids: Ala19, (Tyr or Gln) 34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ile132, Arg134, Phe152, Lys160 and Glu166, wherein the amino acid numbering of said polypeptide Corresponds to the amino acid number in SEQ ID NO:36. 47. The polypeptide of claim 41 comprising at least 100 contiguous amino acid residues of any one of SEQ ID NOs: 36-70. 48. The polypeptide of claim 41 comprising at least 150 contiguous amino acid residues of any one of SEQ ID NOs: 36-70. 49. The polypeptide of claim 41 comprising at least 155 contiguous amino acid residues of any one of SEQ ID NOs: 36-70. 50. The polypeptide of claim 41, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 , SEQ ID NO: 45 and SEQ ID NO: 46. 51. An isolated or recombinant polypeptide, which contains an amino acid sequence containing at least 155 consecutive amino acids of any one of SEQ ID NO: 36-70, the isolated or recombinant polypeptide contains amino acids Lys160 and Glu166, wherein the amino acid numbering Corresponds to the number in SEQ ID NO:36. 52. The polypeptide of claim 51 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 , SEQ ID NO: 45 and SEQ ID NO: 46. 53. The polypeptide of claim 51 having an antiproliferative activity of at least about 8.3 x ¹⁰ units/mg in an assay based on the human Daudi cell line, or at least in an assay based on human WISH cells/EMCV The antiviral activity is about 2.1×10 ⁷ units/mg. 54. The polypeptide of claim 31, 34, 37, 41 or 51, further comprising a secretion/localization sequence. 55. The polypeptide of claim 31, 34, 37, 41 or 51, further comprising a polypeptide purification subsequence. 56. The polypeptide of claim 55, wherein the purification-facilitating sequence is selected from the group consisting of epitope tags, FLAG tags, polyhistidine tags and GST fusions. 57. The polypeptide of claim 31, 34, 37, 41 or 51, further comprising a methionine at the N-terminus. 58. The polypeptide of claim 31, 34, 37, 41 or 51 comprising a modified amino acid. 59. The polypeptide of claim 58, wherein the modified amino acid is selected from the group consisting of glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids and biotinylated amino acids. 60. A composition comprising the polypeptide of claim 31, 34, 37, 41 or 51 and an excipient. 61. The composition of claim 60, wherein the excipient is a pharmaceutically acceptable excipient. 62. A composition comprising the polypeptide of claim 58 and a pharmaceutically acceptable excipient. 63. A polypeptide that specifically binds to a polyclonal antiserum raised against at least one antigen comprising SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85 At least one amino acid sequence, or a fragment thereof, wherein said antiserum is subtracted with an IFN-alpha polypeptide encoded by a nucleic acid corresponding to one or more of the following GenBank accession numbers: J00210(alpha-D) , J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1) , I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1). 64. An antibody or antiserum produced by administering the polypeptide of claim 31, 34, 37, 41 or 51 to a mammal, said antibody or antiserum specifically binding to at least one antigen, said at least one antigen comprising The polypeptide contains at least one amino acid sequence in SEQ ID NO: 36 to SEQ ID NO: 70 and SEQ ID NO: 79 to SEQ ID NO: 85, or a fragment thereof, wherein the antibody or antiserum cannot specifically bind to the corresponding IFN-α polypeptides encoded by nucleic acids of one or more of the following GenBank accession numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533 (α- H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C ), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350(α-F), M38289, V00549(α-2a) and I08313(α -Con1). 65. An antibody or antiserum capable of specifically binding a polypeptide comprising a sequence selected from SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85, wherein the antibody or the antiserum does not specifically bind the IFN-α polypeptide encoded by a nucleic acid corresponding to one or more of the following GenBank accession numbers: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955( α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350(α-F), M38289, V00549 (α-2a) and I08313 (α-Con1). 66. A method of producing a polypeptide, said method comprising: introducing into a population of cells the nucleic acid of claim 1, 2, 3, 8 or 18 operably linked to a regulatory sequence effective to produce the encoded polypeptide; and The cells are grown in culture medium to produce the polypeptide. 67. A method of producing a polypeptide, said method comprising: Introducing a recombinant expression vector containing the nucleic acid of claim 1, 2, 3, 8 or 18 into the cell population; and The cells are cultured in a culture medium under conditions suitable for production of the polypeptide encoded by the expression vector. 68. A method of inhibiting the growth of a tumor cell population, the method comprising: contacting a population of tumor cells with an effective amount of the polypeptide of claim 31, 34, 37, 41 or 51, thereby inhibiting the growth of tumor cells in said population of cells, wherein said effective amount is sufficient to inhibit the growth of tumor cells in said population of tumor cells growth. 69. The method of claim 68, wherein said tumor cells are selected from the group consisting of: human cancer cells, human leukemia cells, human T-lymphoma cells and human melanoma cells. 70. The method of claim 68, wherein said tumor cells are in culture. 71. A method of inhibiting viral replication in at least one cell infected by a virus, said method comprising: Contacting said at least one infected cell with an effective amount of the polypeptide of claim 31, 34, 37, 41 or 51, thereby inhibiting viral replication in said at least one infected cell, wherein said effective amount is sufficient to inhibit said Virus replication within at least one infected cell. 72. The method of claim 71, wherein said virus is an RNA virus. 73. The method of claim 72, wherein the virus is human immunodeficiency virus or hepatitis C virus. 74. The method of claim 71, wherein said virus is a DNA virus. 75. The method of claim 74, wherein said virus is hepatitis B virus. 76. The method of claim 71, wherein said cells are cultured. 77. A method of treating an autoimmune disease in a patient, said method comprising: administering to the patient an effective amount of the polypeptide of claim 31, 34, 37, 41 or 51. 78. The method of claim 77, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, lupus erythematosus, and type I diabetes. 79. A method of treating a disease treatable by administering interferon-alpha to a subject, said improved method comprising: administering to the subject an effective amount of the polypeptide of claim 31, 34, 37, 41 or 51. 80. The method of claim 79, wherein the disease treatable by administering interferon-alpha is selected from the group consisting of sclerosis, rheumatoid arthritis, lupus erythematosus and type I diabetes. 81. A method of preparing a modified or recombinant nucleic acid, said method comprising: Recursive recombination of the sequence of one or more nucleic acids of claim 1, 2, 3, 8 or 18 with sequences of one or more other nucleic acids, each of which encodes an interferon -α or an amino acid subsequence thereof. 82. The method of claim 81, wherein said regressive recombination produces at least one library of recombinant interferon-alpha homolog nucleic acids. 83. A nucleic acid library produced by the method of claim 82. 84. A population of cells comprising the library of claim 83. 85. A recombinant interferon-alpha homolog nucleic acid produced by the method of claim 82. 86. A cell comprising the nucleic acid of claim 85. 87. The method of claim 81, wherein the regressive recombination is performed in vitro. 88. The method of claim 81, wherein the regressive recombination is performed in vivo or ex vivo. 89. A composition comprising two or more nucleic acids of claims 1, 2, 3, 8 or 18. 90. The composition of claim 89, wherein the composition comprises a library comprising at least 10 nucleic acids. 91. A method of producing a modified or recombinant interferon-alpha homolog nucleic acid comprising mutating the nucleic acid of claim 1, 2, 3, 8 or 18. 92. A modified or recombinant interferon-alpha homolog nucleic acid produced by the method of claim 91. 93. A computer or computer readable medium containing a database containing a sequence record containing one or more sequences corresponding to a nucleic acid or protein sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 85 feature chain. 94. An integrated system comprising a computer or computer readable medium comprising a database containing one or more sequence records, each of which contains one or more sequence records corresponding to those selected from the group consisting of SEQ ID NO: 1 To the characteristic strand of the nucleic acid or protein sequence of SEQ ID NO: 85, the integrated system also includes a user input interface that enables the user to selectively view said one or more sequence records. 95. The integrated system of claim 94, wherein the computer or computer readable medium contains a set of alignment instructions for aligning the signature with one or more other signatures corresponding to nucleic acid or protein sequences. 96. The integrated system of claim 95, wherein the instruction set comprises one or more of the following: a local homology comparison assay, a homology sequence alignment assay, a similarity search assay, and a BLAST assay. 97. The integrated system of claim 95, further comprising a user-readable output element for displaying the results of the alignment generated by the set of sequence alignment instructions. 98. The integrated system of claim 94, wherein the computer or the computer readable medium also contains a set of instructions capable of translating at least one nucleic acid sequence into an amino acid sequence, said nucleic acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 1 to the sequence of SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78. 99. The integrated system of claim 94, wherein the computer or the computer-readable medium also contains a set of instructions capable of back-translating at least one amino acid sequence into a nucleic acid sequence, said amino acid sequence containing being selected from the group consisting of SEQ ID NO: 36 to the sequence of SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85. 100. The integrated system of claim 99, wherein the set of instructions is operable to select nucleic acid sequences by applying a set of codon usage instructions or a set of instructions for determining sequence identity to a test nucleic acid sequence. 101. A method of presenting information inherent in at least one of a plurality of sequence records stored in a database, each of said sequence records containing at least one feature corresponding to SEQ ID NO: 1 to SEQ ID NO: 85, using a computer system chain, the method includes: determining a list of at least one signature chain corresponding to one or more of SEQ ID NO: 1 to SEQ ID NO: 85 or a subsequence thereof; determining which of at least one feature chain of said list is selected by the user; and Displays each selected signature chain, or compares each selected signature chain to an additional signature chain. 102. The method of claim 101, further comprising displaying a sequence alignment of each selected signature with the additional signature. 103. The method of claim 101, further comprising displaying the list. 104. A nucleic acid comprising a unique subsequence in a nucleic acid selected from SEQ ID NO: 1 to SEQ ID NO: 35 or SEQ ID NO: 72 to SEQ ID NO: 78, wherein it is different from known interferon-alpha nucleic acid This unique subsequence is unique compared to the nucleic acid sequence of the sequence or the nucleic acid corresponding to any of the following GenBank accession numbers: J00210(α-D), J00207(α-A), X02958(α-6), X02956( α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α -4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350(α-F), M38289, V00549 (α-2a) and I08313 (α-Con1). 105. A polypeptide comprising a unique subsequence in a polypeptide selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 85, which is identical to a known interferon-alpha polypeptide sequence , or this unique subsequence is unique compared to the sequence of the polypeptide encoded by the nucleic acid corresponding to any of the following GenBank accession numbers: J00210(α-D), J00207(α-A), X02958(α-6) , X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350(α-F), M38289, V00549 (α-2a) and I08313 (α-Con1). 106. A target nucleic acid that hybridizes under stringent conditions to a unique encoding oligonucleotide selected from the group consisting of SEQ ID NO: 36 to SEQ ID NO: 70 or SEQ ID NO: 79 to SEQ ID NO: 79 to SEQ ID NO: A unique subsequence in a polypeptide of ID NO: 85, wherein the unique subsequence is unique compared to a known interferon-alpha polypeptide sequence, or the sequence of a polypeptide encoded by a nucleic acid corresponding to any of the following GenBank accession numbers : J00210(α-D), J00207(α-A), X02958(α-6), X02956(α-5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene) , R0067 (Gx-1), I01614, I01787, I07821, M12350 (α-F), M38289, V00549 (α-2a) and I08313 (α-Con1). 107. The nucleic acid of claim 106, wherein the stringent conditions are selected so that an oligonucleotide that is exactly complementary to a uniquely encoded oligonucleotide can hybridize to a uniquely encoded oligonucleotide with a signal-to-noise ratio that is higher than that of an exactly complementary oligonucleotide. The signal-to-noise ratio of the hybridization of the oligonucleotide to a control nucleic acid corresponding to any of the following GenBank numbers is at least about 5-fold higher: J00210 (α-D), J00207 (α-A), X02958 (α-6), X02956 (α- 5), V00533(α-H), V00542(α-14), V00545(IFN-1B), X03125(α-8), X02957(α-16), V00540(α-21), X02955(α-4b ), V00532(α-C), X02960(α-7), X02961(α-10 pseudogene), R0067(Gx-1), I01614, I01787, I07821, M12350(α-F), M38289, V00549(α -2a) and I08313(α-Con1), wherein said target nucleic acid hybridizes to said unique encoding oligonucleotide with a signal-to-noise ratio at least greater than that of a control nucleic acid hybridized to said encoding oligonucleotide about 2 times higher. 108. claims 1,2,3,8 or 18 any one nucleic acid, wherein nucleic acid coding interferon-alpha homologue, with respect to the growth inhibitory activity of human interferon-alpha 2a to cancer cell population, described The homologue has enhanced growth inhibitory activity against said cancer cell population. 109. The nucleic acid of claim 108, wherein the cancer cells of the cancer cell population contain cancer cell lines selected from the group consisting of: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; ) cancer cell lines; ovarian cancer cell lines; breast cancer cell lines; The concentration (GI50 value) of the interferon-α homologue is at least 2 times lower than the GI50 value of human interferon-α2a. 110. The nucleic acid of claim 109, wherein the encoded interferon-alpha homologue has a GI50 value at least 5-fold lower than the GI50 value of human interferon-alpha 2a. 111. The nucleic acid of claim 107, wherein the encoded interferon-alpha homologue has a GI50 value at least 10-fold lower than the GI50 value of human interferon-alpha 2a. 112. Claims 1, 2, 3, the nucleic acid of any one of 8 or 18, wherein said nucleic acid encodes an interferon-alpha homologue, with respect to the cell quiescence activity of human interferon-alpha 2a to cancer cell populations, The homologue has enhanced cytostatic activity on the cancer cell population. 113. The nucleic acid of claim 112, wherein the cancer cells contain cancer cell lines selected from the group consisting of: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; CNS cancer cell lines; ovarian cancer cell lines; Cancer cell lines; prostate cancer cell lines and kidney cancer cell lines, cell quiescence activity was determined as the concentration of interferon-α required to completely inhibit the growth of the cell line (TGI value), wherein the interferon-α homologue The TGI value is at least 2-fold lower than that of human interferon-α2a. 114. The nucleic acid of claim 112, wherein the TGI value of the encoded interferon-alpha homologue is at least 5-fold lower than the TGI value of human interferon-alpha 2a. 115. The nucleic acid of claim 112, wherein the TGI value of the encoded interferon-alpha homologue is at least 10-fold lower than the TGI value of human interferon-alpha 2a. 116. Claims 1, 2, 3, the nucleic acid of any one of 8 or 18, wherein said nucleic acid encodes an interferon-alpha homologue, with respect to the cytotoxic activity of human interferon-alpha 2a to cancer cell populations, Said homologue has enhanced cytotoxic activity against said cancer cell population. 117. The nucleic acid of claim 116, wherein the cancer cells contain cancer cell lines selected from the group consisting of: leukemia cell lines; melanoma cell lines; lung cancer cell lines; colon cancer cell lines; central nervous system (CNS) cancer cell lines; For cancer cell lines; breast cancer cell lines; prostate cancer cell lines and kidney cancer cell lines, the cytotoxic activity was determined as: the amount of interferon-alpha required to reduce the amount of cellular proteins in the cell lines measured by 50% after the incubation period Concentrations (LC50 values) in which the LC50 value of the interferon-α homologue is at least 2-fold lower than the LC50 value of human interferon-α2a. 118. The nucleic acid of claim 116, wherein the encoded interferon-alpha homologue has an LC50 value at least 5-fold lower than the LC50 value of human interferon-alpha 2a. 119. The nucleic acid of claim 116, wherein the encoded interferon-alpha homologue has an LC50 value at least 10-fold lower than the LC50 value of human interferon-alpha 2a. 120. The polypeptide of any one of claims 31, 34, 37, 41 or 51, the growth inhibitory activity of said polypeptide on a population of cancer cells relative to the inhibitory activity of human interferon-α2a on a population of cancer cells Enhanced. 121. The polypeptide of claim 120, wherein the cancer cell population comprises a cancer cell line selected from the group consisting of: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; For breast cancer cell lines; prostate cancer cell lines and kidney cancer cell lines, the growth inhibitory activity was determined as the concentration (GI50 value) of the polypeptide or human interferon-α2a required for 50% inhibition of the growth of the cell lines, wherein the polypeptide The GI50 value is at least 2-fold lower than the GI50 value of human interferon-α2a. 122. A nucleic acid produced by the method of claim 81. 123. An interferon-alpha polypeptide or amino acid subsequence thereof produced by the method of claim 81.

Images