Compositions and methods for modifying regulatory T cells

Abstract

To provide a method of stabilizing regulatory T cells, and a method of actively destabilizing regulatory T cells to remove tolerogenic effects in a tumor microenvironment.SOLUTION: Provided herein are compositions and methods for modifying regulatory T cells. The inventors have identified nuclear factors that influence expression of Foxp3, a key transcriptional regulator of Treg cells. Treg cells can be modified by inhibiting and / or overexpressing one or more of these nuclear factors to produce stabilized Treg cells or destabilized Treg cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Prior Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 744,058, filed October 10, 2018, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Regulatory T cells (Treg cells) play a role in regulating immune responses. In some cases, such as in some cancers, Treg cells inhibit the immune system's ability to target and destroy cancer cells. In other cases, such as in autoimmune diseases, Treg cells are unavailable to control the immune system. Stabilizing Treg cells for the treatment of autoimmune diseases or actively destabilizing Treg cells to remove the tolerogenic effect in the tumor microenvironment has great therapeutic potential. Summary of the Invention

[0003] The present invention is directed to compositions and methods for modifying Treg cells. The inventors have identified nuclear factors that affect the expression of Foxp3, a key transcriptional regulator of Treg cells. Treg cells can be modified by inhibiting and / or overexpressing one or more of these nuclear factors to generate stabilized or destabilized Treg cells. In some examples, stabilized Treg cells are used to treat autoimmune disorders, support organ transplants, treat graft-versus-host disease or inflammation. Examples of autoimmune diseases include, but are not limited to, type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and multi-organ autoimmune syndrome. In other examples, destabilized Treg cells are used to treat cancer. For example, in some embodiments, destabilized Tregs can be used to target solid tumors, for example, where Treg cells contribute to an immunosuppressive microenvironment. Examples of such cancers include, but are not limited to, ovarian cancer.

[0004] Provided herein is a method of increasing the stability of human regulatory T (Treg) cells, the method comprising inhibiting expression of a nuclear factor listed in Table 1 and / or overexpressing a nuclear factor listed in Table 2 in human Treg cells.

[0005] Also provided is a method of reducing the stability of human Treg cells, the method comprising inhibiting expression of a nuclear factor listed in Table 2 and / or overexpressing a nuclear factor listed in Table 1 in human Treg cells.

[0006] In some embodiments, the inhibiting step comprises decreasing the expression of the nuclear factor or decreasing the expression of a polynucleotide encoding the nuclear factor in the Treg cell. In some embodiments, the overexpressing step comprises increasing the expression of the nuclear factor or increasing the expression of a polynucleotide encoding the nuclear factor in the Treg cell.

[0007] In some embodiments, the inhibiting step in Treg cells comprises contacting a polynucleotide encoding a protein with a targeting nuclease, a guide RNA (gRNA), an siRNA, an antisense RNA, a microRNA (miRNA), or a short hairpin RNA (shRNA). In some embodiments, the inhibiting step comprises contacting a polynucleotide encoding a nuclear factor with at least one gRNA and optionally a targeting nuclease, where at least one gRNA comprises a sequence selected from Table 3. In some embodiments, the inhibiting step comprises mutating the polynucleotide encoding the protein. In some embodiments, the inhibiting step comprises contacting the polynucleotide with a targeting nuclease.

[0008] In some embodiments, the targeting nuclease introduces a double-strand break at the target region in the polynucleotide. In some embodiments, the targeting nuclease is an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is a Cpf1 nuclease or a Cas9 nuclease, and the method further comprises introducing a gRNA that specifically hybridizes to the target region in the polynucleotide into the Treg cell. In some embodiments, the Cpf1 nuclease or the Cas9 nuclease and the gRNA are introduced into the Treg cell as a ribonucleoprotein (RNP) complex. In some embodiments, the inhibiting comprises clustered regularly interspaced short palindromic repeats (CRISPR) / Cas genome editing.

[0009] In some embodiments, Treg cells are administered to humans after inhibiting and / or overexpressing.In some embodiments, Treg cells are taken from humans, then treated to inhibit expression of nuclear factor and / or overexpress nuclear factor, and the treated Treg cells are reintroduced into humans.In some embodiments, nuclear factor expression is inhibited and / or nuclear factor is overexpressed in in vivo Treg cells.In some embodiments, the human has autoimmune disorder, GVHD, inflammation, or is an organ transplant recipient.In some embodiments, the human has cancer.

[0010] In another aspect, provided herein is a Treg cell generated by any of the methods described herein.In another aspect, the present invention provides a Treg cell comprising a genetically modified or heterologous polynucleotide that inhibits the expression of a nuclear factor listed in Table 1, and / or a heterologous polynucleotide that encodes a protein encoded by a nuclear factor listed in Table 2.In another aspect, the present invention provides a Treg cell comprising a genetically modified or heterologous polynucleotide that inhibits the expression of a nuclear factor listed in Table 2, and / or a heterologous polynucleotide that encodes a polypeptide encoded by a nuclear factor listed in Table 1.

[0011] In another embodiment, provided herein is a Treg comprising at least one guide RNA (gRNA) comprising a sequence selected from Table 3. In some embodiments, expression of a nuclear factor listed in Table 1 or Table 2 is decreased in said Treg cells compared to expression of the nuclear factor in a Treg cell that does not comprise a gRNA.

[0012] In another embodiment, provided herein is a method of destabilizing Treg in a subject in need thereof, comprising inhibiting expression of one or more nuclear factors listed in Table 2 and / or overexpressing one or more nuclear factors listed in Table 1 in human Treg cells of the subject. In some embodiments, Treg cells are destabilized in vivo. In other embodiments, Treg cells are destabilized ex vivo. In some embodiments, the subject has cancer.

[0013] In another embodiment, provided herein is a method of stabilizing Tregs in a subject in need thereof, comprising inhibiting expression of one or more nuclear factors listed in Table 1 and / or overexpressing one or more nuclear factors listed in Table 2 in human Treg cells of the subject. In some embodiments, the Treg cells are stabilized in vivo. In other embodiments, the Treg cells are stabilized ex vivo. In some embodiments, the subject has an autoimmune disorder.

[0014] In another aspect, provided herein is a method of treating an autoimmune disorder in a subject, comprising administering a population of stabilized Treg cells to a subject having an autoimmune disease.In another aspect, the present invention provides a method of treating cancer in a subject, comprising administering a population of destabilized Treg cells to a subject having cancer.

[0015] In another aspect, provided herein is a method of treating an autoimmune disorder, GVHD, or inflammation in a subject, or supporting an organ transplant treatment, comprising: (a) harvesting Treg cells from a subject (e.g., having an autoimmune disorder); (b) modifying Treg cells by inhibiting expression of a nuclear factor listed in Table 1 and / or overexpressing a nuclear factor listed in Table 2 in the Treg cells; and (c) administering the modified Treg cells to the subject.

[0016] In another embodiment, the invention provides a method of treating cancer in a subject, the method comprising: (a) harvesting Treg cells from a subject having cancer; (b) modifying Treg cells by inhibiting expression of a nuclear factor listed in Table 2 and / or overexpressing a nuclear factor listed in Table 1 in the Treg cells; and (c) administering the modified Treg cells to the subject. [The present invention 1001] A method for enhancing stability of human regulatory T (Treg) cells, comprising: Inhibiting the expression of one or more nuclear factors listed in Table 1; and / or overexpressing one or more nuclear factors listed in Table 2 The method comprising: [The present invention 1002] A method for reducing the stability of human Treg cells, comprising: Inhibiting the expression of one or more nuclear factors listed in Table 2; and / or overexpressing one or more nuclear factors listed in Table 1 The method comprising: [The present invention 1003] The inhibition step is decreasing the expression of a nuclear factor, or Reducing the expression of a polynucleotide encoding a nuclear factor The method of the present invention 1001 or 1002, comprising: [The present invention 1004] The step of overexpressing increasing the expression of a nuclear factor; or Increasing expression of a polynucleotide encoding a nuclear factor The method of the present invention 1001 or 1002, comprising: [The present invention 1005] The method of claim 1004, wherein the overexpressing step comprises introducing a polynucleotide encoding the nuclear factor into Treg cells. [The present invention 1006] The method of claim 1003, wherein the inhibiting step comprises contacting a polynucleotide encoding a nuclear factor with a targeting nuclease, a guide RNA (gRNA), an siRNA, an antisense RNA, a microRNA (miRNA), or a short hairpin RNA (shRNA). [The present invention 1007] The method of claim 1006, wherein the inhibiting step comprises contacting a polynucleotide encoding a nuclear factor with at least one gRNA and optionally a targeting nuclease, wherein the at least one gRNA comprises a sequence selected from Table 3. [The present invention 1008] The method according to any one of claims 1001 to 1007, wherein the inhibiting step comprises mutating a polynucleotide encoding the nuclear factor. [The present invention 1009] The method of any one of claims 10 to 15, wherein the inhibiting step comprises contacting the polynucleotide with a targeted nuclease. [The present invention 1010] The method of any one of claims 10 to 15, wherein the targeted nuclease introduces a double-stranded break at a target region within the polynucleotide. [The present invention 1011] The method of any one of claims 1006 to 1010, wherein the targeted nuclease is an RNA-guided nuclease. [The present invention 1012] The method of claim 1011, wherein the RNA-guided nuclease is Cpf1 nuclease or Cas9 nuclease, and the method further comprises the step of introducing a gRNA that specifically hybridizes to a target region within the polynucleotide into the Treg cell. [The present invention 1013] The method of the present invention, wherein the Cpf1 nuclease or Cas9 nuclease and the gRNA are introduced into Treg cells as a ribonucleoprotein (RNP) complex. [The present invention 1014] Any of the methods of claims 1009 to 1013, wherein the inhibiting step comprises performing clustered regularly interspaced short palindromic repeats (CRISPR) / Cas genome editing. [The present invention 1015] The method according to any of claims 1001 to 1014, wherein the Treg cells are administered to a human after the inhibiting and / or overexpressing steps. [The present invention 1016] Any of the methods of claims 1001 to 1015, comprising collecting Treg cells from a human, treating the Treg cells to inhibit expression of a nuclear factor and / or overexpress a nuclear factor, and reintroducing the treated Treg cells into the human. [The present invention 1017] The method of the present invention, wherein inhibition of expression and / or overexpression results in Treg cells with improved stability. [The present invention 1018] The method of claim 1017, wherein the human has an autoimmune disorder. [The present invention 1019] The method of the present invention, wherein inhibition of expression and / or overexpression results in Treg cells with reduced stability. [The present invention 1020] The method of claim 1019, wherein the human has cancer. [The present invention 1021] A Treg cell produced by any of the methods of the present inventions 1001 to 1014. [The present invention 1022] A Treg cell comprising a genetically modified or heterologous polynucleotide that inhibits expression of a nuclear factor listed in Table 1 and / or a heterologous polynucleotide that encodes a nuclear factor listed in Table 2. [The present invention 1023] A Treg cell comprising a genetically modified or heterologous polynucleotide that inhibits expression of a nuclear factor listed in Table 2 and / or a heterologous polynucleotide that encodes a nuclear factor listed in Table 1. [The present invention 1024] A Treg comprising at least one guide RNA (gRNA) comprising a sequence selected from Table 3. [The present invention 1025] The Treg cell of the present invention, wherein the expression of a nuclear factor listed in Table 1 or Table 2 is decreased in said Treg cell compared to the expression of the nuclear factor in a Treg cell not containing a gRNA. [The present invention 1026] 1. A method of destabilizing Tregs in a subject in need thereof, comprising: Inhibiting the expression of one or more nuclear factors listed in Table 2; and / or overexpressing one or more nuclear factors listed in Table 1 The method comprising: [The present invention 1027] The method of the present invention 1026, wherein the step of inhibiting expression of one or more nuclear factors listed in Table 2 and / or the step of overexpressing one or more nuclear factors listed in Table 1 are carried out in vivo. [The present invention 1028] The method for destabilizing Treg cells (a) obtaining Treg cells from a subject; (b) destabilizing the Treg cells by inhibiting expression of a nuclear factor listed in Table 2 and / or overexpressing a nuclear factor listed in Table 1 in the Treg cells; and (c) administering the destabilized Treg cells to a subject. The method of the present invention 1026, comprising: [The present invention 1029] The method of any one of claims 1026 to 1027, wherein the subject has cancer. [The present invention 1030] 1. A method for stabilizing Tregs in a subject in need thereof, comprising: Inhibiting the expression of one or more nuclear factors listed in Table 1; and / or overexpressing one or more nuclear factors listed in Table 2 The method comprising: [The present invention 1031] The method of the present invention 1030, wherein the step of inhibiting expression of one or more nuclear factors listed in Table 1 and / or the step of overexpressing one or more nuclear factors listed in Table 2 is carried out in vivo. [The present invention 1032] The method for stabilizing Treg cells (a) obtaining Treg cells from a subject; (b) stabilizing the Treg cells by inhibiting expression of a nuclear factor listed in Table 1 and / or overexpressing a nuclear factor listed in Table 2 in the Treg cells; and (c) administering the destabilized Treg cells to a subject. The method of the present invention 1030, comprising: [The present invention 1033] The method of any of claims 1030 to 1032, wherein the subject has an autoimmune disorder. [The present invention 1034] 20. A method of treating an autoimmune disorder in a subject, comprising administering to a subject having an autoimmune disease a population of Treg cells of the present invention. [The present invention 1035] 20. A method of treating cancer in a subject, comprising administering to a subject having cancer a population of Treg cells of the present invention. [Brief description of the drawings]

[0017] This application includes the following figures. The figures are intended to illustrate certain aspects and / or features of the compositions and methods, and to supplement any descriptions of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the specification expressly indicates otherwise. [Figure 1]FIG. 13 is a schematic of the Treg fate reporter mice used to identify Foxp3+ and Foxp3- ex Tregs upon inhibition of nuclear factors in a CRISPR screen. [Figure 2-1] Figure 2a is a schematic of the pooled CRISPR screening strategy used to identify nuclear factors that affect Foxp3 stability. Figure 2b is a volcano plot of hits from the screen. X-axis shows gene-level log2 fold change (LFC) Z-score; scale-adjusted median LFC of all single guide RNAs (sgRNAs) per gene. Y-axis shows p-values ​​calculated by MAGeCK. Red indicates negative regulators (depleted in Foxp3-low cells) and blue dots indicate all positive regulators (enriched in Foxp3-low cells) defined by FDR<0.5 and Z-score>0.5. Figure 2c (top panel) shows the distribution of sgRNA-level log2 fold change (LFC) values ​​in Foxp3-low cells relative to Foxp3-high cells for 2,000 guides. Figure 2c (bottom panel) shows the LFCs of all four individual sgRNAs targeting genes enriched (blue lines) and depleted (red lines) in Foxp3 low cells overlaid on a grey gradient depicting the overall distribution. [Figure 2-2] Figure 2d shows a schematic diagram of experimentally determined and predicted protein-protein interactions between the top hits, 16 negative regulators (red) and 25 positive regulators (red), generated by STRING-db. Black lines connect interacting proteins, and dotted lines indicate known protein complexes. Figure 2e shows Foxp3 expression 5 days after electroporation of Cas9 RNP in mouse Tregs, measured by flow cytometry, of the top screen hits. Figure 2f shows the mean fluorescence intensity (MFI) of Foxp3 from the data in Figure 2e. Figure 2g shows a representative histogram showing the MFI of FOXP3 and CD25 from human Tregs. Figure 2h shows a statistical analysis of FOXP3 MFI from human Tregs in six biological replicates. [Figure 2-3]Figure 2i shows the sigmoidal curve of hits from the screen. The X-axis shows the gene-level LFC rank score; rank 1 is the top negative hit (Sp1) and rank 493 is the top positive hit (Foxp3). The Y-axis shows the gene-level LFC calculated by MAGeCK. Red dots indicate selected negative hits (depleted in Foxp3 low cells) and blue dots indicate selected positive hits (enriched in Foxp3 low cells) among the top 20 ranked hits. Figure 2j shows that sgRNAs targeting Foxp3 and Usp22 were enriched in Foxp3 low cells in a targeting screen of over 2000 gRNAs. Non-targeting sgRNAs were evenly distributed throughout the cell population (black). [Diagram 3]Figure 3a-g shows the design and quality control of the targeted pooled CRISPR screen in primary mouse Tregs. (a) Design strategy for the selection of genes for an unbiased targeted library. Genes were selected based on gene ontology (GO) annotation and then subselected based on highest expression across any CD4 T cell subset, resulting in a total of 2,000 sgRNAs; (b) MSCV expression vector with Thy1.1 reporter used for retroviral transduction of the sgRNA library; (c) Schematic of a detailed timeline of the 12-day targeted screen pipeline. Arrows indicate time points when cells were split and medium replenished; (d) Retroviral transduction efficiency of the targeted library in primary mouse Tregs, as shown by Thy1.1 surface expression measured by flow cytometry. Infection was increased to achieve a high-efficiency multiplicity of infection; (e) Foxp3 expression from screen input, output, and control cells measured by flow cytometry. Top: Foxp3 expression from input Foxp3+ purified Tregs measured by GFP expression at day 0. Middle: Foxp3 expression from control Tregs (not transduced with library) measured by endogenous intracellular staining at day 12. Bottom: Foxp3 expression from screened Tregs (transduced with library) measured by endogenous intracellular staining at day 12; (f) Targeting screen (2,000 guides) shows that sgRNAs targeting Foxp3 and Usp22 were enriched in Foxp3-low cells (blue). Non-targeting control (NT Ctrl) sgRNAs were evenly distributed across the cell population (black). (g) Distribution of read numbers after next-generation sequencing of sgRNAs in sorted cell populations, Foxp3-high and Foxp3-low. [Figure 4]Figure 4a-g shows validation of gene targets regulating Foxp3 expression in primary mouse and human Tregs using Cas9 RNP arrays. (a) Overview of the orthogonal validation strategy using arrayed electroporation of Cas9 RNPs. (b) Representative flow plots showing FOXP3 and CD25 expression 7 days after electroporation of Cas9 RNPs in human Tregs. The Foxp3hiCD25hi subpopulation is highlighted with a red gate. (c) Percentage of FOXP3+ cells from human Tregs in six biological replicates. (d) Percentage of Foxp3hiCD25hi cells from human Tregs in six biological replicates. (e) RNP controls in mouse Tregs collected 5 days after electroporation; Left: CD4 expression from CD4 RNPs (cut control) compared to NT control. Right: Foxp3 expression from CD4 knockout cells (left panel) compared to NT control. (f) Foxp3 expression 6 days after electroporation of Cas9 RNP measured by flow cytometry. Cells were pre-gated for lymphocytes, live cells, CD4+, and CD25hi cells; (g) Statistical analysis of the mean fluorescence intensity (MFI) of Foxp3 from the data in panel g. Statistical analysis was performed using two-way ANOVA with Holm-Sidak multiple comparison test. **p≦0.01, ****p≦0.0001. [Diagram 5] Figure 5a-b shows validation of Rnf20 in primary mouse Tregs using Cas9 RNP arrays. (a) Flow cytometry histograms of two gRNAs targeting Rnf20 show that knockout of Rnf20 maintains stable Foxp3 expression. (b) Bar graph of Foxp3 MFI data from Figure 5a. [Figure 6] Validation of USP22 regulation of Foxp3 expression in primary human Tregs using RNP arrays. (a) Foxp3 expression 7 days after electroporation of Cas9 RNPs measured by flow cytometry. Cells were pre-gated on lymphocytes, live cells, CD4+, and CD25hi cells. (b) Foxp3 MFI from data in panel a. [Figure 7] We show that knockout of Usp22 and Atxn7l3 in mouse Tregs reduces Foxp3 expression, whereas knockdown of Rnf20 maintains stable Foxp3 expression. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0019] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA encoded by a gene, and mRNA.

[0020] The term "gene" can refer to a segment of DNA involved in producing or encoding a polypeptide chain. It can include regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0021] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0022] The term "inhibiting expression" refers to inhibiting or reducing expression of a gene product, such as an RNA or protein. As used throughout, the term "nuclear factor" refers to a protein, such as a transcription factor, that directly or indirectly alters the expression of Foxp3. To inhibit or reduce expression of a gene, the sequence and / or structure of the gene can be modified so that the gene is not transcribed (in the case of DNA) or translated (in the case of RNA), or is not transcribed or translated to produce a functional protein, such as a polypeptide or protein encoded by the gene listed in Table 1 or Table 2. Various methods for inhibiting or reducing expression are described in more detail herein. Some methods may introduce nucleic acid substitutions, additions, and / or deletions in the wild-type gene. Some methods may also introduce single-stranded or double-stranded breaks in the gene. To inhibit or reduce expression of a protein, the expression of a gene or polynucleotide encoding the protein can be inhibited or reduced. In other embodiments, the protein can be directly targeted to inhibit or reduce expression of the protein, for example, using an antibody or a protease. "Inhibited" expression refers to a reduction of at least 10% compared to a reference control level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% reduction (i.e., the level is absent compared to the reference sample).

[0023] The term "overexpressing" or "overexpression" refers to increasing the expression of a gene or protein. "Overexpression" refers to an increase in expression of at least 10% compared to a reference control level, for example, an increase in the amount of mRNA or protein expressed in Treg cells, or an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%. Various methods for overexpression are known to those skilled in the art, including, but not limited to, stably or transiently introducing into cells a heterologous polynucleotide that encodes the protein to be overexpressed (i.e., the nuclear factor listed in Table 1 or Table 2), or inducing in cells the overexpression of an endogenous gene that encodes the protein.

[0024] As used herein, the term "heterologous" refers to something that is not found in nature. The term "heterologous sequence" refers to a sequence that is not normally found in a given cell in nature. Thus, a heterologous nucleotide or protein sequence may be (a) foreign to the host cell (i.e., exogenous to the cell); (b) naturally found in the host cell (i.e., endogenous), but present in the cell in a non-natural amount (i.e., greater or less than naturally found in the host cell); or (c) naturally found in the host cell, but located outside its natural locus.

[0025] "Treating" refers to any indicia of success in treating or ameliorating or preventing a disease, condition, or disorder, including any objective or subjective parameter, such as alleviation; remission; reduction in symptoms or making the disease state more tolerable to the patient; slowing the rate of degeneration or decline; or making the end point of degeneration less debilitating.

[0026] "Promoter" is defined as one or more nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, promoter includes the necessary nucleic acid sequences near the transcription start site, such as the TATA element in the case of a polymerase II type promoter. Promoter also optionally includes distal enhancer or repressor elements, which may be located as far away as several thousand base pairs from the transcription start site.

[0027] As used herein, the term "complementary" or "complementarity" refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U) or G and C. Guide RNAs described herein can include sequences that are fully complementary or substantially complementary (e.g., with 1-4 mismatches) to genomic sequences, e.g., DNA targeting sequences.

[0028] As used throughout, subject means an individual. For example, subject is a mammal, such as a primate, and more specifically a human. Non-human primates are also subjects. The term subject includes domestic animals, such as cats, dogs, etc., livestock animals (e.g., cows, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., ferrets, chinchillas, mice, rabbits, rats, gerbils, guinea pigs, etc.). Thus, veterinary and medical uses and formulations are contemplated herein. The term does not indicate a specific age or sex. Thus, adult or newborn subjects, both male and female, are intended to be covered. As used herein, patient or subject may be used interchangeably and may refer to a subject suffering from a disease or disorder.

[0029] As used throughout, the term "targeting nuclease" refers to a nuclease that targets a specific DNA sequence in the genome of a cell and creates strand breaks in the specific DNA sequence. Strand breaks can be single-stranded or double-stranded. Targeting nucleases include, but are not limited to, Cas nucleases, TAL-effector nucleases, and zinc finger nucleases.

[0030] "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II, and type III subtypes. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease, e.g., Cas9, complexed with guide and activator RNA to recognize and cleave foreign nucleic acids. Guide RNAs with both guide RNA and activator RNA activity are also known in the art. In some cases, such dual-activity guide RNAs are referred to as single guide RNAs (sgRNAs).

[0031] Cas9 homologs are found in a wide variety of eubacteria, including but not limited to bacteria from the following taxa: Actinomycetes, Aquifex, Bacteroidetes-Chlorobium, Chlamydia-Verrucomicrobium, Chloroflexus, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotoga. An exemplary Cas9 protein is the Cas9 protein of Streptococcus pyogenes. Additional Cas9 proteins and their homologues are described, for example, in Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci US A. 2013 Sep 24; 110(39): 15644-9; Sampson et al., Nature. 2013 May 9; 497(7448): 254-7; and Jinek, et al., Science. 2012 Aug 17; 337(6096): 816-21. Any variant of the Cas9 nuclease provided herein can be optimized for efficient activity or enhanced stability in host cells. Thus, engineered Cas9 nucleases are also contemplated.

[0032] As used throughout, a guide RNA (gRNA) sequence is a sequence that interacts with a site-specific or targeting nuclease and specifically binds or hybridizes to a target nucleic acid in the genome of a cell, such that the gRNA and the targeting nuclease co-localize to the target nucleic acid in the genome of the cell. Each gRNA contains a DNA targeting sequence or protospacer sequence of about 10-50 nucleotides in length that specifically binds or hybridizes to a target DNA sequence in the genome. For example, the targeting sequence may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence. Table 3 shows exemplary gRNA sequences used in the methods of the present disclosure.

[0033] As used herein, the term "Cas9" refers to an RNA-mediated nuclease (e.g., of or derived from bacterial or archaeal origin). Exemplary RNA-mediated nucleases include the Cas9 protein and its homologs described above. Other RNA-mediated nucleases include Cpf1 (see, e.g., Zetsche et al., Cell, Volume 163, Issue 3, p759-771, 22 October 2015) and its homologs. Similarly, as used herein, terms such as "Cas9 ribonucleoprotein" complexes refer to a complex of Cas9 protein and guide RNA, a complex of Cas9 protein and crRNA, a complex of Cas9 protein and trans-activating crRNA (tracrRNA), or combinations thereof (e.g., a complex comprising Cas9 protein, tracrRNA, and crRNA guide RNA). It is understood that in any of the embodiments described herein, Cas9 nuclease can be replaced with Cpf1 nuclease or any other guide nuclease.

[0034] As used herein, the phrase "modifying" refers to inducing structural changes in the sequence of genome in the target genomic region of Treg cells. For example, modification can take the form of inserting a nucleotide sequence into the genome of the cell. Such modification can be performed, for example, by inducing a double-stranded break in the target genomic region or by introducing a pair of single-stranded nicks that flank the target genomic region on opposite strands. Methods for inducing single-stranded or double-stranded breaks in or within the target genomic region include the use of Cas9 nuclease domain or its derivatives and a guide RNA or a pair of guide RNAs directed to the target genomic region. "Modifying" can also refer to changing the expression of nuclear factors in Treg cells, for example, inhibiting the expression of nuclear factors or overexpressing nuclear factors in Treg cells.

[0035] As used herein, the phrase "introducing" in the context of introducing a nucleic acid or a complex containing nucleic acid, such as an RNP complex, refers to the transfer of a nucleic acid sequence or an RNP complex from outside the cell to inside the cell.In some cases, introducing refers to the transfer of a nucleic acid or a complex from outside the cell to inside the nucleus of the cell.Various methods of such transfer are contemplated, including but not limited to electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, cell-penetrating peptide-mediated transfer, liposome-mediated transfer, etc.

[0036] Detailed Description of the Invention The following description lists various aspects and embodiments of the compositions and methods of the present invention.Specific embodiments are not intended to define the scope of the compositions and methods.Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods.The description should be read from the perspective of those skilled in the art; therefore, it does not necessarily include information that is known to those skilled in the art.

[0037] I. Introduction Treg cells are a specialized subset of CD4+ T cells that suppress inflammation to maintain homeostasis and prevent autoimmunity. The development and function of Treg cells depend on the expression of the master transcription factor Foxp3. Although Treg cells have been thought to be irreversibly committed to suppressive functions, lineage tracing studies have revealed that Treg cells can exhibit plasticity. Treg cells that have lost Foxp3 expression, termed "exTreg," have been shown to acquire the cytokine-producing capacity of inflammatory effector T cells and exacerbate autoimmunity. However, the gene regulatory programs that promote or interfere with the stability of Foxp3 in Treg cells under various physiological conditions are not well understood. We have identified nuclear factors that regulate the expression of Foxp3, thereby altering the stability of Treg cells.

[0038] II. Methods and Compositions As described herein, the disclosure provides compositions and methods directed to modifying regulatory T (Treg) cell stability by inhibiting expression of one or more nuclear factors and / or overexpressing one or more nuclear factors in Treg cells. The disclosure also features compositions comprising Treg cells with modified stability. A population of destabilized modified Treg cells may provide therapeutic benefits in the treatment of cancer. A population of stabilized modified Treg cells may provide therapeutic benefits in the treatment of autoimmune diseases.

[0039] The present invention is directed to compositions and methods for modifying the stability of regulatory T cells (also referred to as "Treg cells"). The inventors have discovered that the stability of Treg cells can be altered by inhibiting the expression of one or more nuclear factors and / or overexpressing one or more nuclear factors. In some embodiments, Treg cells can be destabilized by inhibiting the expression of one or more nuclear factors and / or overexpressing one or more nuclear factors, so that Treg cells have a lower immunosuppressive effect and may have improved therapeutic benefits for the treatment of cancer. A population of destabilized Treg cells can be used to enhance or improve various cancer treatments, or to target Treg cells of individuals with cancer to destabilize Treg cells. In other embodiments, Treg cells can be stabilized by inhibiting the expression of one or more nuclear factors and / or overexpressing one or more nuclear factors, so that Treg cells have a higher immunosuppressive effect and may have improved therapeutic benefits for the treatment of autoimmune diseases. Populations of stabilized Treg cells can be used to treat or alleviate autoimmune diseases, or Treg cells in individuals with autoimmune diseases can be targeted to stabilize Treg cells.

[0040] In the methods described herein, examples of nuclear factors whose expression can be altered to modify the stability of Treg cells include, but are not limited to, the nuclear factors listed in Table 1 and Table 2. In some embodiments, the present invention provides a method of increasing the stability of regulatory T (Treg) cells, comprising inhibiting expression of one or more nuclear factors listed in Table 1 and / or overexpressing one or more nuclear factors listed in Table 2 in Treg cells. Inhibiting one or more nuclear factors listed in Table 1 and / or overexpressing one or more nuclear factors listed in Table 2 can increase Foxp3 expression or stabilize Foxp3 expression in Treg cells (e.g., in an inflammatory environment that would otherwise result in decreased Foxp3 expression), thereby increasing the stability of Treg cells.

[0041] In another embodiment, the present invention provides a method of reducing the stability of Treg cells, comprising inhibiting expression of one or more nuclear factors listed in Table 2 and / or overexpressing one or more nuclear factors listed in Table 1 in Treg cells. Inhibiting one or more nuclear factors listed in Table 2 and / or overexpressing one or more nuclear factors listed in Table 1 can reduce the expression of Foxp3 in Treg cells, thereby reducing the stability of Treg cells. Table 1 provides nuclear factors that, when inhibited, increase Foxp3 expression. Overexpressing a nuclear factor listed in Table 1 can reduce Foxp3 expression. In some embodiments, expression of an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence listed in Table 1 is inhibited. In some embodiments, an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence listed in Table 1 is overexpressed. Table 2 provides nuclear factors that, when inhibited, reduce Foxp3 expression. Overexpression of the nuclear factors listed in Table 2 can increase Foxp3 expression. In some embodiments, expression of an amino acid sequence with at least about 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence listed in Table 2 is inhibited. In some embodiments, an amino acid sequence with at least about 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence listed in Table 2 is overexpressed. When referring to one or more nuclear factors listed in Table 1 or Table 2, it is understood that this can be a protein, i.e., a nuclear factor, or a polynucleotide that encodes a nuclear factor.

[0042] Table 1. Nuclear factors that can be inhibited to increase Foxp3 expression or overexpressed to decrease Foxp3 expression TIFF0007672006000001.tif217126TIFF0007672006000002.tif218141TIFF00076720060 00003.tif217144TIFF0007672006000004.tif215139TIFF0007672006000005.tif218110

[0043] Table 2. Nuclear factors that can be inhibited to decrease Foxp3 expression or overexpressed to increase Foxp3 expression. TIFF0007672006000006.tif222130TIFF0007672006000007.tif220145TIFF0007672006000008.tif221146TIFF0007672006 000009.tif221146TIFF0007672006000010.tif219144TIFF0007672006000011.tif221148TIFF0007672006000012.tif22185

[0044] The stability of Treg cells can be evaluated using FACS markers. Some of the FACS markers used are standard Treg cell signature proteins. For example, when certain genes are knocked out or inhibited in Treg cells, if these modified cells show acquisition or maintenance of Treg cell standard markers such as FOXP3, CTLA4, CD25, IL-10, and / or IKZF2, this can indicate that Treg cells are more stable. In some embodiments, the disappearance of Treg cell standard markers and / or the acquisition of inflammatory markers (e.g., IL-17a, IL-4, IFNγ, and IL-2) can indicate that Treg cells are destabilized. In another example, when certain nuclear factors are overexpressed in Treg cells, if these modified cells show acquisition or maintenance of Treg cell standard markers such as FOXP3, CTLA4, CD25, IL-10, and / or IKZF2, this can indicate that Treg cells are more stable. In some embodiments, when certain nuclear factors are overexpressed in Treg cells, these modified cells show the loss of Treg cell standard markers and / or the acquisition of inflammatory markers (e.g., IL-17a, IL-4, IFNγ and IL-2), which may indicate that Treg cells are destabilized.For the method of detecting and enriching Treg, see, for example, International Patent Application Publication No. WO2007140472.

[0045] In some embodiments of the methods described herein, inhibiting expression of a nuclear factor listed in Table 1 or Table 2 may include decreasing expression of the nuclear factor or decreasing expression of a polynucleotide, e.g., mRNA, encoding the nuclear factor in Treg cells. In some embodiments, expression of one or more nuclear factors listed in Table 1 or Table 2 is inhibited in Treg cells. As described in more detail herein, expression of one or more nuclear factors listed in Table 1 or Table 2 can be inhibited using one or more available methods.

[0046] In some embodiments of the methods described herein, overexpressing a nuclear factor listed in Table 1 or a nuclear factor listed in Table 2 may include introducing a polynucleotide encoding the nuclear factor into the Treg cell. In other embodiments of the methods described herein, overexpressing a nuclear factor listed in Table 1 or a nuclear factor listed in Table 2 may include introducing an agent that induces expression of an endogenous gene encoding the nuclear factor into the Treg cell. For example, expression of an endogenous gene can be induced using RNA activation, in which short double-stranded RNA targets promoter sequences to induce expression of the endogenous gene. See, for example, Wang et al. "Inducing gene expression by targeting promoter sequences using small activating RNAs," J. Biol. Methods 2(1): e14 (2015). In another example, an artificial transcription factor containing a zinc finger binding domain can be used to activate or repress expression of an endogenous gene. See, e.g., Dent et al., "Regulation of endogenous gene expressing using small molecule-controlled engineered zinc-finger protein transcription factors," Gene Ther. 14(18): 1362-9 (2007).

[0047] In some embodiments, inhibiting expression may include contacting the polynucleotide encoding the nuclear factor with a targeting nuclease, guide RNA (gRNA), siRNA, antisense RNA, microRNA (miRNA), or short hairpin RNA (shRNA). In certain embodiments, when a gRNA and a targeting nuclease (e.g., Cas9) are used to inhibit expression of a polynucleotide encoding a human nuclear factor listed in Table 1 or Table 2, the gRNA may include a sequence listed in Table 3, a sequence complementary to a sequence listed in Table 3, or a portion thereof. Table 3 provides the gene ID number, Genbank accession number of the mRNA, genomic sequence, location in the genome after nuclease cleavage, sgRNA target sequence, target context sequence, PAM sequence, and exon targeted by the sgRNA for each nuclear factor listed in Tables 1 and 2. ZNF281 is the human homolog of mouse Zfp281.

[0048] Table 3. gRNA target sequences and related information for targeting nuclear factors TIFF0007672006000013.tif233139TIFF0007672006000014.tif231150TIFF0007672006000015.tif231150 TIFF0007672006000016.tif232149TIFF0007672006000017.tif232150TIFF0007672006000018.tif231137

[0049] As described herein, the stability of Treg cells can be modified by inhibiting the expression of one or more nuclear factors listed in Table 1 or Table 2. The stability of Treg cells can also be modified by overexpressing one or more nuclear factors listed in Table 1 or Table 2. Then, once the modified Treg cells are generated, the modified Treg cells can be administered to a human. Depending on whether the Treg cells are stabilized or destabilized, the modified Treg cells can be used to treat different indications. For example, Treg cells can be isolated from a human whole blood sample and expanded ex vivo. The expanded Treg cells can then be treated to inhibit the expression of the nuclear factors listed in Table 1 or Table 2, thus generating modified Treg cells. The modified Treg cells can be reintroduced into a human to treat an indication. In some embodiments, the destabilized Treg cells with lower immunosuppressive effects can be used to treat cancer. In some embodiments, the stabilized Treg cells with improved immunosuppressive effects can be used to treat autoimmune diseases. Certain nuclear factors in Treg cells, when their expression is inhibited, increase Foxp3 expression (Table 1), having a stabilizing effect, while other nuclear factors, when their expression is inhibited, decrease Foxp3 expression in Treg cells (Table 2), having a destabilizing effect. Cell stability can be determined by a multicolor FACS panel based on effectors such as Treg cell markers, such as Foxp3, Helios, CTLA-4, CD25, IL-10, and cytokines typically associated with effector T cell subsets, such as IL-2, IFNγ, IL-17a, and IL-4. Assays for measuring Treg cell stability can be found, for example, in McClymont, et al., "Plasticity of Human Regulatory T Cells in Healthy Subjects and Patients with Type 1 Diabetes" J. immunol. 186 (2011).Depending on the indication and therapeutic need, one can choose to target one or more nuclear factors to generate destabilized or stabilized modified Treg cells.

[0050] In other cases, the subject's Treg cells can be modified in vivo, for example, by using a targeting vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector. In vivo delivery of a targeting nuclease that modifies the genome of Treg cells can also be used. See, for example, U.S. Patent No. 9,737,604, and Zhang et al. "Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy," NPG Asia Materials Volume 9, page e441 (2017).

[0051] Also provided are Treg cells in which expression of one or more nuclear factors listed in Table 1 or Table 2 is inhibited. Further provided are Treg cells in which one or more nuclear factors listed in Table 1 or Table 2 are overexpressed. The present disclosure also features Treg cells comprising a genetically modified or heterologous polynucleotide that inhibits expression of one or more nuclear factors listed in Table 1 and / or a heterologous polynucleotide that encodes a nuclear factor listed in Table 2. Also provided are Treg cells comprising a genetically modified or heterologous polynucleotide that inhibits expression of a nuclear factor listed in Table 2 and / or a heterologous polynucleotide that encodes a nuclear factor listed in Table 1.

[0052] The genetic modification may be a nucleotide mutation or any sequence change in the polynucleotide encoding the nuclear factor, which results in the inhibition of the expression of the nuclear factor. A heterologous polynucleotide may refer to a polynucleotide that originally encodes a nuclear factor but is modified, i.e., contains one or more nucleotide mutations or sequence changes. In some embodiments, the heterologous polynucleotide is inserted into the genome of Treg cells by introducing a vector, such as a viral vector, that contains the polynucleotide. Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, retroviral vectors, or lentiviral vectors. In some embodiments, the lentiviral vector is an integrase-deficient lentiviral vector.

[0053] Also provided herein is a Treg cell comprising at least one guide RNA (gRNA) comprising a sequence selected from Table 3. The expression of one or more nuclear factors listed in Table 1 or Table 2 in a Treg cell comprising a gRNA may be decreased in the Treg cell compared to the expression of one or more nuclear factors in a Treg cell not comprising a gRNA. In another example, an endogenous nuclear factor listed in Table 1 or Table 2 can be inhibited by targeting a non-activated targeting nuclease fused to a transcription repressor, such as dCAs9, to the promoter region of the endogenous nuclear factor gene. In another example, an endogenous nuclear factor listed in Table 1 or Table 2 can be upregulated or overexpressed by targeting a non-activated targeting nuclease fused to a transcription activator, such as dCAs9, to the promoter region of the endogenous nuclear factor gene. See, e.g., Qi et al. "The New State of the Art: Cas9 for Gene Activation and Repression," Mol. and Cell. Biol., 35(22): 3800-3809 (2015).

[0054] III. Methods of Inhibiting Expression CRISPR / Cas genome editing The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated proteins) nuclease system is an engineered nuclease system based on a bacterial system that can be used for genome manipulation. It is based in part on the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, a segment of the invader's DNA is converted into CRISPR RNA (crRNA) by the "immune" response. The crRNA then associates with another type of RNA, called tracrRNA, through a region of partial complementarity, and guides the Cas (e.g., Cas9) nuclease to a region of homology to the crRNA in the target DNA, called the "protospacer." The Cas (e.g., Cas9) nuclease cleaves DNA to generate blunt ends at the double-stranded break at a site specified by a 20-nucleotide guide sequence contained within the crRNA transcript. The Cas (e.g., Cas9) nuclease may require both the crRNA and tracrRNA for site-specific DNA recognition and cleavage. This system has now been engineered such that crRNA and tracrRNA can be combined into one molecule ("guide RNA" or "gRNA"), and the crRNA equivalent portion of the single guide RNA can be engineered to direct a Cas (e.g., Cas9) nuclease to target any desired sequence (see, e.g., Jinek et al. (2012) Science 337:816-821; Jinek et al. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563). Thus, the CRISPR / Cas system can be engineered to generate double-stranded breaks at desired targets in a cell's genome, and utilize the cell's endogenous machinery to repair the induced breaks by homology-directed repair (HDR) or non-homologous end joining (NHEJ).

[0055] In some embodiments of the methods described herein, CRISPR / Cas genome editing can be used to inhibit expression of one or more nuclear factors listed in Table 1 or Table 2.

[0056] In some embodiments, the Cas nuclease has DNA cleavage activity. The Cas nuclease can direct the cleavage of one or both strands at a position in a target DNA sequence, i.e., at a position in a polynucleotide encoding a nuclear factor listed in Table 1 or Table 2. In some embodiments, the Cas nuclease can be a nickase with one or more inactivating catalytic domains that cleaves a single strand of a target DNA sequence.

[0057] Non-limiting examples of Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm Cas nucleases include 4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, their variants, their mutants, and their derivatives. There are three major types of Cas nucleases (type I, type II, and type III), with ten subtypes including five type I, three type II, and two type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(1):58-66). Type II Cas nucleases include Cas1, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is described, for example, in NBCI reference sequence number NP_269215, and the amino acid sequence of the Streptococcus thermophilus wild-type Cas9 polypeptide is described, for example, in NBCI reference sequence number WP_011681470. Some CRISPR-associated endonucleases that can be used in the methods described herein are disclosed, for example, in U.S. Patent Application Publication Nos. 2014 / 0068797, 2014 / 0302563, and 2014 / 0356959.

[0058] Cas nucleases, such as Cas9 polypeptides, can be derived from a variety of bacterial species, including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius. Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis canis, Mycoplasma synoviae, Eubacteriumrectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum (Rhodospirillum rubrum), Candidatus Puniceispirillum marinum (Candidatus Puniceispirillummarinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. multocida, Sutterella wadsworthensis, Proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0059] Wild-type Cas9 nuclease has two functional domains, such as RuvC and HNH, that can cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active. Cas9 enzyme can include one or more catalytic domains of Cas9 protein from bacteria belonging to the group consisting of Corynebacter, Stellera, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivora, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratiphracta, and Campylobacter. In some embodiments, Cas9 can be a fusion protein, for example, the two catalytic domains are from different bacterial species.

[0060] A useful variant of the Cas9 nuclease is RuvC. - or HNH -Like an enzyme or nickase, it may contain a single inactive catalytic domain. Cas9 nickase has only one active functional domain and can only cut one strand of target DNA, thereby generating a single-strand break or nick. In some embodiments, Cas9 nuclease may be a mutant Cas9 nuclease with one or more amino acid mutations. For example, a mutant Cas9 with at least D10A mutation is a Cas9 nickase. In other embodiments, a mutant Cas9 nuclease with at least H840A mutation is a Cas9 nickase. Other examples of mutations present in Cas9 nickase include, but are not limited to, N854A and N863A. When at least two DNA-targeting RNAs that target opposite DNA strands are used, Cas9 nickase can be used to introduce double-strand breaks. The double-nicked induced double-strand break can be repaired by NHEJ or HDR (Ran et al., 2013, Cell, 154:1380-1389). This gene editing strategy favors HDR and reduces the frequency of indel mutations at off-target DNA sites. Non-limiting examples of Cas9 nuclease or nickase are described in, for example, U.S. Patent Nos. 8,895,308; 8,889,418; and 8,865,406, and U.S. Patent Application Publication Nos. 2014 / 0356959, 2014 / 0273226, and 2014 / 0186919. Cas9 nuclease or nickase can be codon-optimized for target cells or target organisms.

[0061] In some embodiments, the Cas nuclease may be a Cas9 polypeptide that contains two silencing mutations (D10A and H840A) in the RuvC1 and HNH nuclease domains, which is referred to as dCas9 (Jinek et al., Science, 2012, 337:816-821; Qi et al., Cell, 152(5):1173-1183). In one embodiment, the dCas9 polypeptide from Streptococcus pyogenes contains at least one mutation at positions D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, A987, or any combination thereof. Descriptions of such dCas9 polypeptides and their variants are provided, for example, in International Patent Publication No. WO 2013 / 176772. The dCas9 enzyme may comprise a mutation at D10, E762, H983, or D986, and a mutation at H840 or N863. In some cases, the dCas9 enzyme may comprise a D10A or D10N mutation. Similarly, the dCas9 enzyme may comprise H840A, H840Y, or H840N. In some embodiments, the dCas9 enzyme may comprise the following substitutions: D10A and H840A; D10A and H840Y; D10A and H840N; D10N and H840A; D10N and H840Y; or D10N and H840N. The substitution may be a conservative or non-conservative substitution that renders the Cas9 polypeptide catalytically inactive and capable of binding to target DNA.

[0062] In some embodiments, the Cas nuclease may be a high-fidelity or enhanced specificity Cas9 polypeptide variant with reduced off-target effects and robust on-target cleavage. Non-limiting examples of Cas9 polypeptide variants with improved on-target specificity include the SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (also referred to as eSpCas9(1.0)), and SpCas9 (K848A / K1003A / R1060A) (also referred to as eSpCas9(1.1)) variants described in Slaymaker et al., Science, 351(6268):84-8 (2016), as well as SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (also referred to as eSpCas9(1.0)), SpCas9 (K848A / K1003A / R1060A) (also referred to as eSpCas9(1.1)) variants described in Kleinstiver et al., Nature, 529(7587):490-5 (2016), which include one, two, three, or four of the following mutations: N497A, R661A, Q695A, and Q926A. (e.g., SpCas9-HF1 contains all four mutations).

[0063] As described above, gRNAs may include crRNAs and tracrRNAs. gRNAs may be configured to form stable and active complexes with gRNA-mediated nucleases (e.g., Cas9 or dCas9). gRNAs include binding regions that provide specific binding to target genetic elements. Exemplary gRNAs that may be used to target regions within polynucleotides encoding nuclear factors listed in Table 1 or Table 2 are listed in Table 3 below. gRNAs used to target regions within polynucleotides encoding nuclear factors listed in Table 1 or Table 2 may include sequences selected from Table 3 below or portions thereof.

[0064] In some embodiments, the targeting nuclease, such as Cpf1 nuclease or Cas9 nuclease, and the gRNA are introduced into the Treg cells as a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex is about 1×10 5 ~Approx. 2×10 6 cells (e.g., 1 × 105 Individual cells ~ approx. 5×10 5 Individual cells, approximately 1 x 10 5 Individual cells ~ approx. 1×10 6 Individual cells, 1×10 5 Individual cells ~ approx. 1.5×10 6 Individual cells, 1×10 5 Individual cells ~ approx. 2×10 6 Individual cells, approximately 1 x 10 6 Individual cells ~ approx. 1.5×10 6 individual cells, or approximately 1 x 10 6 Individual cells ~ approx. 2×10 6 In some embodiments, the Treg cells are cultured under conditions effective to expand the population of modified Treg cells. Also disclosed herein is a population of Treg cells in which at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the genome of the cells comprises a genetically modified or heterologous polynucleotide that inhibits expression of one or more nuclear factors set forth in Table 1 or Table 2.

[0065] In some embodiments, the RNP complex is introduced into Treg cells by electroporation.Methods, compositions and devices for electroporating cells to introduce RNP complex are available in the art, for example, see WO 2016 / 123578, WO / 2006 / 001614 and Kim, JA et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions, and devices for electroporating cells to introduce RNP complexes include those described in U.S. Patent Application Publication Nos. 2006 / 0094095, 2005 / 0064596; or 2006 / 0087522; Li, LH et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Patent Nos. 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6,485,961; 7,029,916; and U.S. Patent Application Publication Nos. 2014 / 0017213; and 2012 / 0088842; Geng, T. et al., J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010).

[0066] In some embodiments, the sequence of the gRNA or a portion thereof is designed to be complementary (e.g., fully complementary) or substantially complementary (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% complementary) to a target region within a polynucleotide encoding a protein. In some embodiments, the portion of the gRNA that is complementary to and binds to a target region within a polynucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length or more, or approximately such length. In some cases, the portion of the gRNA that complements and binds to the target region within the polynucleotide is about 19 to about 21 nucleotides in length. In some cases, the gRNA may incorporate wobble or degenerate bases to bind to the target region. In some cases, the gRNA may be modified to increase stability. For example, non-natural nucleotides may be incorporated to increase the resistance of the RNA to degradation. In some cases, the gRNA may be modified or designed to avoid or reduce secondary structure formation. In some cases, the gRNA may be designed to optimize GC content. In some cases, the GC content is about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some cases, the binding region may contain modified nucleotides, such as, but not limited to, methylated or phosphorylated nucleotides.

[0067] In some embodiments, gRNAs can be optimized for expression by substitution, deletion, or addition of one or more nucleotides. In some cases, nucleotide sequences that result in inefficient transcription from a coding template nucleic acid can be deleted or replaced. For example, in some cases, gRNAs are transcribed from a nucleic acid operably linked to an RNA polymerase III promoter. In such cases, gRNA sequences that cause inefficient transcription by RNA polymerase III, as described in Nielsen et al., Science. 2013 Jun 28;340(6140):1577-80, can be deleted or replaced. For example, one or more consecutive uracils can be deleted or replaced from the gRNA sequence. In some cases, the gRNA sequence can be altered to exchange adenine for uracil when uracil is hydrogen bonded with the corresponding adenine. This "AU flip" can maintain the overall structure and function of the gRNA molecule while improving expression by reducing the number of consecutive uracil nucleotides.

[0068] In some embodiments, gRNA can be optimized for stability. Stability can be enhanced by optimizing the stability of gRNA:nuclease interaction, optimizing the association of gRNA:nuclease complex, removing or modifying RNA destabilizing sequence elements, or adding RNA stabilizing sequence elements. In some embodiments, gRNA contains a 5' stem-loop structure proximal to or adjacent to the region that interacts with gRNA-mediated nuclease. Optimizing the 5' stem-loop structure can result in enhanced stability or association of gRNA:nuclease complex. In some cases, the 5' stem-loop structure is optimized by increasing the length of the stem portion of the stem-loop structure.

[0069] gRNA can be modified by methods known in the art. In some cases, modification can include, but is not limited to, the addition of one or more of the following sequence elements: 5' cap (e.g., 7-methylguanylic acid cap); 3' polyadenylation tail: riboswitch sequence; stability control sequence: hairpin; intracellular localization sequence; detection sequence or label; or binding site for one or more proteins. Modification can also include the introduction of non-natural nucleotides, including, but not limited to, one or more of fluorescent nucleotides and methylated nucleotides.

[0070] Also described herein are expression cassettes and vectors for producing gRNA in a host cell. The expression cassette may include a promoter (e.g., a heterologous promoter) operably linked to the polynucleotide encoding the gRNA. The promoter may be inducible or constitutive. The promoter may be tissue specific. In some cases, the promoter is a U6, H1, or spleen focus forming virus (SFFV) long terminal repeat promoter. In some cases, the promoter is a weak mammalian promoter compared to the human elongation factor 1 promoter (EF1A). In some cases, the weak mammalian promoter is a ubiquitin C promoter or a phosphoglycerate kinase 1 promoter (PGK). In some cases, the weak mammalian promoter is a TetOn promoter in the absence of an inducer. In some cases, when utilizing a TetOn promoter, the host cell is also contacted with a tetracycline transactivator. In some embodiments, the strength of the gRNA promoter selected is selected to express an amount of gRNA in an amount proportional to the amount of Cas9 or dCas9. The expression cassette may be present in a vector, such as a plasmid, a viral vector, a lentiviral vector, etc. In some cases, the expression cassette is present in a host cell. The gRNA expression cassette may be episomal or integrated into the host cell.

[0071] Zinc finger nucleases (ZFNs) "Zinc finger nuclease" or "ZFN" is a fusion of a FokI cleavage domain and a DNA recognition domain that contains three or more zinc finger motifs. Heterodimerization of two individual ZFNs at specific positions in DNA with precise orientation and spacing results in double-stranded breaks in DNA. In some embodiments of the methods described herein, ZFNs can be used to inhibit the expression of one or more nuclear factors listed in Table 1 or Table 2, i.e., by cleaving a polynucleotide that encodes a protein.

[0072] In some cases, ZFNs fuse a cleavage domain to the C-terminus of each zinc finger domain. To allow the two cleavage domains to dimerize and cleave the DNA, two individual ZFNs bind to opposite strands of DNA with their C-terminus separated by a certain distance. In some cases, the linker sequence between the zinc finger domain and the cleavage domain requires that the 5' ends of each binding site are about 5-7 bp apart. Exemplary ZFNs that can be used in the methods described herein include Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Nat Methods, 2012, 9(8):805-7; U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,7 Nos. 7,585,849; 7,595,376; 6,903,185; 6,479,626; and those described in U.S. Patent Application Publication Nos. 2003 / 0232410 and 2009 / 0203140.

[0073] ZFNs can generate double-stranded breaks in the target DNA, resulting in DNA break repair that allows for the introduction of genetic modifications. DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template can be provided that contains homologous arms flanking the site of the target DNA.

[0074] In some embodiments, ZFN is zinc finger nickase, which can be engineered ZFN, which induces site-specific single-stranded DNA break or nick, thus resulting in HDR.The description of zinc finger nickase can be found, for example, in Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8;Kim et al., Genome Res, 2012, 22(7):1327-33.

[0075] TALEN TALENs can also be used to inhibit the expression of one or more nuclear factors listed in Table 1 or Table 2. A "TALEN" or "TAL effector nuclease" is an engineered transcription activator-like effector nuclease that includes a central domain of DNA-binding tandem repeats, a nuclear localization signal, and a C-terminal transcription activation domain. In some cases, the DNA-binding tandem repeats include 33-35 amino acids in length and include two hypervariable amino acid residues at positions 12 and 13 that can recognize one or more specific DNA base pairs. TALENs can be made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. For example, a TALE protein can be fused to a nuclease such as a wild-type or mutant FokI endonuclease or the catalytic domain of FokI. For example, several mutations to FokI that improve cleavage specificity or activity have been made for its use in TALENs. Such TALENs can be engineered to bind to any desired DNA sequence.

[0076] TALEN can be used to create double-stranded breaks in target DNA sequences, which then undergo NHEJ or HDR, resulting in genetic modification. In some cases, a single-stranded donor DNA repair template is provided to facilitate HDR.

[0077] Detailed descriptions of TALENs and their use for gene editing can be found, for example, in U.S. Pat. Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49.

[0078] Meganuclease A "meganuclease" is a rare-cutting endonuclease or homing endonuclease that may have high specificity and recognize a DNA target site that spans at least 12 base pairs in length, for example 12-40 base pairs in length or 12-60 base pairs in length. Meganucleases may be modular DNA-binding nucleases, such as any fusion protein that includes at least one catalytic domain of an endonuclease and at least one DNA-binding domain or protein that specifies a nucleic acid target sequence. The DNA-binding domain may include at least one motif that recognizes single-stranded or double-stranded DNA. Meganucleases may be monomeric or dimeric.

[0079] In some embodiments of the methods described herein, meganucleases can be used to inhibit expression of one or more nuclear factors listed in Table 1 or Table 2, i.e., by cleaving a target region within a polynucleotide encoding the nuclear factor. In some examples, the meganuclease is naturally occurring (found in nature) or wild-type, while in other examples, the meganuclease is non-natural, artificial, engineered, synthetic, or rationally designed. In certain embodiments, meganucleases that can be used in the methods described herein include, but are not limited to, I-CreI meganuclease, I-CeuI meganuclease, I-MsoI meganuclease, I-SceI meganuclease, variants thereof, mutants thereof, and derivatives thereof.

[0080] Detailed descriptions of useful meganucleases and their application in gene editing can be found, for example, in Silva et al., Curr Gene Ther, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061-4066, as well as U.S. Patent Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.

[0081] RNA-based technologies Various RNA-based technologies can also be used in the methods described herein to inhibit expression of one or more nuclear factors listed in Table 1 or Table 2. Examples of RNA-based technologies include, but are not limited to, small interfering RNA (siRNA), antisense RNA, microRNA (miRNA), and short hairpin RNA (shRNA).

[0082] RNA-based technology can use siRNA, antisense RNA, miRNA, or shRNA to target the sequence that encodes transcription factor or a part thereof.In some embodiments, one or more genes that are regulated by transcription factor can also be targeted by siRNA, antisense RNA, miRNA, or shRNA.siRNA, antisense RNA, miRNA, or shRNA can target the sequence that comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides.

[0083] siRNA can be produced from short hairpin RNA (shRNA). shRNA is an artificial RNA molecule with a hairpin turn that can be used to silence the expression of a target gene through the siRNA it produces in cells. See, for example, Fire et. al., Nature 391:806-811, 1998;Elbashir et al., Nature 411:494-498, 2001;Chakraborty et al., Mol Ther Nucleic Acids 8:132-143, 2017;and Bouard et al., Br. J. Pharmacol. 157:153-165, 2009. Expression of shRNA in cells is typically achieved by delivery of a plasmid or via a viral or bacterial vector. Suitable bacterial vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and lentivirus. After the vector is integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III (depending on the promoter used). The resulting pre-shRNA is exported from the nucleus and then processed by a protein called Dicer and loaded into the RNA-induced silencing complex (RISC). The sense strand is degraded by the RISC, and the antisense strand guides the RISC to the mRNA with the complementary sequence. A protein called Ago2 in the RISC then cleaves the mRNA or, in some cases, inhibits the translation of the mRNA, resulting in the destruction of the mRNA and ultimately the reduction of the protein encoded by the mRNA. Thus, the shRNA results in the silencing of the targeted gene.

[0084] The shRNA or siRNA may be encoded in a vector. In some embodiments, the vector further comprises appropriate expression control elements known in the art, including, for example, a promoter (e.g., an inducible or tissue-specific promoter), an enhancer, and a transcription terminator.

[0085] IV. Treatment Method Any of the methods described herein can be used to modify Treg cells in or taken from a human subject. Any of the methods and compositions described herein can be used to modify Treg cells taken from a human subject to treat or prevent disease (e.g., cancer, autoimmune disease, infectious disease, transplant rejection, graft-versus-host disease, or other inflammatory disorder in a subject).

[0086] Provided herein is a method of treating an autoimmune disorder in a subject, the method comprising administering to a subject having an autoimmune disorder a population of Treg cells comprising a genetically modified or heterologous polynucleotide that inhibits expression of a nuclear factor listed in Table 1, and / or a heterologous polynucleotide that encodes a nuclear factor listed in Table 2.

[0087] Also provided is a method of treating cancer in a subject comprising administering to a subject having cancer a population of Treg cells comprising a genetically modified or heterologous polynucleotide that inhibits expression of a nuclear factor listed in Table 2 and / or a heterologous polynucleotide that encodes a nuclear factor listed in Table 1.

[0088] Provided herein is a method for treating cancer in a human subject, the method comprising: a) harvesting Treg cells from the subject; b) modifying Treg cells using any of the methods provided herein to reduce the stability of Treg cells; and c) administering the modified Treg cells to the subject, where the human subject has cancer.Also provided herein is a method for treating autoimmune disease in a human subject, the method comprising: a) harvesting Treg cells from the subject; b) modifying Treg cells using any of the methods provided herein to increase the stability of Treg cells; and c) administering the modified Treg cells to the subject, where the human subject has autoimmune disease.

[0089] In some embodiments, Treg cells taken from a cancer subject can be expanded ex vivo. The characteristics of the subject's cancer can determine a tailored set of cell modifications (i.e., which nuclear factors from Table 1 and / or Table 2 should be targeted), and these modifications can be applied to Treg cells using any of the methods described herein. The modified Treg cells can then be reintroduced into the subject. This strategy leverages the subject's natural cancer-specific T cell repertoire and enhances its function, providing a versatile weapon to rapidly eliminate mutagenic cancer cells. A similar strategy may also be applicable to the treatment of autoimmune diseases, where modified Treg cells will have improved stability.

[0090] In other cases, Treg cells in a subject can be targeted for in vivo modification. See, for example, U.S. Patent No. 9,737,604 and Zhang et al. "Lipid nanoparticle-mediated efficient delivery of CRISPR / Cas9 for tumor therapy," NPG Asia Materials Volume 9, page e441 (2017).

[0091] Disclosed are materials, compositions, and components that can be used in, can be used in conjunction with, can be used in the preparation of, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, although specific references to various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a method is disclosed and discussed, and several modifications that can be made to one or more molecules included in the method are discussed, any and all combinations and permutations of the method and possible modifications are specifically contemplated, unless specifically indicated otherwise. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to steps in methods that use the disclosed compositions. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered as specifically contemplated and disclosed.

[0092] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety. EXAMPLES

[0093] The following examples are offered by way of illustration only, and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same or similar results.

[0094] mouse As previously described (Bailey-Bucktrout et al., “Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response,” Immunity. 39, 949-62 (2013)), B6 ​​Foxp3-GFP-Cre mice (Zhou et al., “Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity,” J Exp Med. 205, 1983-91 (2008)) were crossed with B6 Rosa26-RFP reporter mice (Luche et al., “Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies,” Eur. J. Immunol. 37, 43-53 (2007)) to generate Foxp3 fate reporter mice (Figure 1). These mice were then crossed with B6 constitutive Cas9 expressing mice (Platt et al., "CRISPR-Cas9 knockin mice for genome editing and cancer modeling," Cell. 159, 440-455 (2014)) to generate Foxp3-GFP-Cre / Rosa26-RFP / Cas9 mice used in the CRISPR screen. For array validation experiments, B6 Foxp3-EGFP knockin mice (strain no. 006772) obtained from Jackson Laboratories were used.All mice were housed in the UCSF specific pathogen-free animal facility in accordance with guidelines established by the Institutional Animal Care and Use Committee and the Laboratory Animal Resource Center.

[0095] Isolation and culture of primary mouse Tregs Spleens and peripheral lymph nodes were removed from mice and dissociated in 1x PBS containing 2% FBS and 1 mM EDTA. The mixture was then passed through a 70μm filter. CD4+ T cells were isolated using a CD4+ negative selection kit (StemCell Technologies, Cat# 19752) followed by fluorescence activated cell sorting. For pre-screen sorting, Tregs were gated on lymphocytes, live cells, CD4+, CD62L+, RFP+, Foxp3-GFP+ cells. For arraying validation experiments, Tregs were gated on lymphocytes, live cells, CD4+, Foxp3-GFP+ cells. Sorted Tregs were cultured at 1 million cells / mL in complete DMEM, 10% FBS, 1% pen / strep+2000U hIL-2 in 24-well plates. Tregs were stimulated for 48 h using CD3 / CD28 Mouse T Activator Dynabeads (Thermo Fisher, Cat# 11456D) at a bead-to-cell ratio of 3:1. Cells were split and medium was renewed every 2–3 days.

[0096] Design and construction of pooled sgRNA libraries For targeting library cloning, we followed a custom sgRNA library cloning protocol previously described (Joung et al., "Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening," Nat Protocols. 12, 828-863 (2017)). We utilized the MSCV-U6-sgRNA-IRES-Thy1.1 backbone. To optimize this plasmid for library cloning, we first replaced the sgRNA with a 1.9 kb stuffer from the lentiGuide-Puro plasmid (Addgene, plasmid #52963) with flanking BsgI cleavage sites. The stuffer was excised using BsgI restriction enzyme (NEB, Cat# R0559) and the linear backbone was gel purified (Qiagen, Cat# 28706). A targeting library was designed to include all genes with gene ontology matches for "nucleic acid-binding transcription factor," "protein-binding transcription factor," "involved in chromatin assembly," and "involved in epigenetic regulation." Genes were then selected based on those with the highest expression levels across any mouse CD4 T cell subset, as defined by Stubbington et al. (Stubbington et al., "An atlas of mouse CD4+ T cell transcriptomes," Biol Direct. 10. 14 (2015)). In total, 493 targets with four guides per gene and 28 non-targeting controls were included. Guides were subset from the Brie sgRNA library (Doench et al., "Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9," Nature biotechnology. 34(2), 184-191 (2016)), and pooled oligo libraries were ordered from Twist Bioscience to match the vector backbone.Oligos were PCR amplified and cloned into a modified MSCV backbone by Gibson assembly as described by Joung et al.. The library was amplified using Endura electrocompetent cells (Endura, Cat #60242-1) according to the manufacturer's protocol.

[0097] Retrovirus production Sixteen hours prior to transfection, Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, Inc., Cat# RV-101) were seeded at 10 million cells in 15 cm poly-L-lysine coated dishes and cultured in complete DMEM, 10% FBS, 1% pen / strep, 1 μg / mL puromycin, and 10 μg / mL blasticidin. Just prior to transfection, the medium was replaced with complete DMEM without antibiotics, 10% FBS. Cells were transfected with sgRNA delivery plasmid (MSCV-U6-sgRNA-IRES-Thy1.1) using TransIT-293 transfection reagent (Mirus, Cat# MIR 2700) according to the manufacturer's protocol. The following morning, the medium was replaced with complete DMEM, 10% FBS, 1% pen / strep. Viral supernatants were collected 48 hours after transfection and filtered through a 0.45 μm polyethersulfone sterile syringe filter (Whatman, Cat# 6780-2504) to remove cellular debris. Viral supernatants were divided into aliquots and stored at −80° C. until use.

[0098] Retroviral transduction Tregs were stimulated as above for 48–60 h. Cells were counted and plated at 3 million cells in 1 mL of medium containing 2× hIL-2 into each well of a 6-well plate that had been coated with 15 μg / mL RetroNectin (Takara, Cat# T100A) for 3 h at room temperature and subsequently washed with 1× PBS. Retrovirus was added (1 mL) at a 1:1 v / v ratio, the plate was centrifuged at 2000 g at 30°C for 1 h, and placed in a 37°C incubator overnight. The next day, half of the 1:1 retrovirus-medium mixture (1 mL) was removed from the plate and 1 mL of fresh retrovirus was added. The plate was immediately centrifuged at 2000 g at 30°C for 1 h. After the second spinfection, cells were pelleted, washed, and cultured in fresh medium.

[0099] Foxp3 intracellular staining and cell collection after screen Tregs were harvested from their culture vessels 8 days after the second transduction and centrifuged at 300g for 5 min. Cells were first stained with a viability dye diluted 1:1,000 in 1x PBS for 20 min at 4°C and then washed with EasySep buffer (1x PBS, 2% FBS, 1 mM EDTA). Cells were then resuspended in the appropriate surface staining antibody cocktail and incubated for 30 min at 4°C before being washed with EasySep buffer. Cells were then fixed, permeabilized, and stained for transcription factors using Foxp3 Transcription Factor Staining Buffer Set (eBioscience, Cat# 00-5523-00) according to the manufacturer's instructions. For the CRISPR screen, Foxp3 high and Foxp3 low populations were isolated using fluorescence-activated cell sorting by gating on lymphocytes, live cells, CD4+, and on the top 40% of Foxp3 expressing cells (Foxp3 high) and bottom 40% of Foxp3 expressing cells (Foxp3 low) by endogenous Foxp3 intracellular staining. To maintain library coverage of at least 1,000 cells per sgRNA, over 2 million cells were collected for both sorted populations.

[0100] Isolation of genomic DNA from fixed cells After sorting and harvesting the cells, genomic DNA (gDNA) was isolated using a protocol specific for fixed cells. The cell pellet was resuspended in cell lysis buffer (0.5% SDS, 50 mM Tris, pH 8, 10 mM EDTA) containing 5M NaCl at 1:25 v / v to reverse crosslinks and incubated at 66°C overnight. RNase A (10 mg / mL) was added at 1:50 v / v and incubated at 37°C for 1 h. Proteinase K (20 mg / mL) was added at 1:50 v / v and incubated at 45°C for 1 h. Phenol:chloroform:isoamyl alcohol (25:24:1) was added at 1:1 v / v to the samples, transferred to phase lock gel light tubes (QuantaBio, Cat# 2302820), vigorously inverted, and centrifuged at 20,000g for 5 min. The aqueous phase was then transferred to a clean tube and 1:10 v / v NaAc, 1 μl GeneElute-LPA (Sigma, Cat#56575), and 2.5:1 v / v isopropanol were added. Samples were vortexed and incubated at -80°C until frozen solid. They were then thawed and centrifuged at 20,000g for 30 min. The cell pellet was washed with 500 μl 75% EtOH, centrifuged at 20,000g for 5 min with gentle inversion, aspirated, dried, and resuspended in 20 μl TE buffer.

[0101] Preparation of genomic DNA for next generation sequencing Amplification and barcoding of sgRNAs for the cell surface sublibraries was performed as previously described with some modifications (Gilbert et al., "Genome scale CRISPR-mediated control of gene repression and activation," Cell. 159, 647-661 (2014)). Briefly, after gDNA isolation, sgRNAs were amplified using one-step PCR and barcoded with TruSeq Single Index. TruSeq Adaptor Index 12 (CTTGTA) was used for the Foxp3 low population, and TrueSeq Adaptor Index 14 (AGTTCC) was used for the Foxp3 high population. Each PCR reaction consisted of 50 μL of NEBNext Ultra II Q5 Master Mix (NEB #M0544), 1 μg of gDNA, 2.5 μL each of 10 μM forward and reverse primers, and water to make a total of 100 μL. PCR cycling conditions were 98°C for 3 min, followed by 26 cycles of 98°C for 10 s, 62°C for 10 s, 72°C for 25 s; and a final 2 min extension at 72°C. After PCR, samples were purified using Agencourt AMPure XPSPRI beads (Beckman Coulter, cat #A63880) according to the manufacturer's protocol, quantified using Qubit ssDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, cat #Q32854), and then analyzed on a 2100 Bioanalyzer instrument. Samples were then sequenced on an Illumina MiniSeq using custom sequencing primers.

[0102] Pooled CRISPR screen pipeline Primary Tregs were isolated from the spleens and lymph nodes of three 5-7 month old male Foxp3-GFP-Cre / Rosa26-RFP / Cas9 mice, pooled together, and stimulated for 60 h. Cells were then retrovirally transduced with the sgRNA library and cultured at a density of 1 million cells / ml while continuing to maintain library coverage of at least 1,000 cells per sgRNA. Eight days after the second transduction, cells were sorted based on Foxp3 expression defined by intracellular staining. Genomic DNA was harvested from each population, and the sgRNA coding regions were then amplified by PCR and sequenced on an Illumina MiniSeq using custom sequencing primers. From this data, we quantified the frequency of cells expressing each different sgRNA in each population (Foxp3 high and Foxp3 low) and quantified the sgRNA phenotype, defined as Foxp3 stabilizing (enriched in Foxp3 high) or Foxp3 destabilizing (enriched in Foxp3 low) (Figure 2).

[0103] Analysis of pooled CRISPR screens Analyses were performed as previously described (Shifrut et al., Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Biorxiv. (2018)doi: https: / / doi.org / 10.1101 / 384776)). To identify hits from the screen, MAGeCK software was used to quantify and examine guide enrichment (Li et al., "MAGeCK enables robust identification of essential genes from genome-scale CRISPR / Cas9 knockout screens," Genome Biol.15, 554 (2014)). Guide abundance was first determined by using the MAGeCK "count" module on the raw fastq files. In the targeted libraries, a constant 5' trim was automatically detected by MAGeCK. To examine robust guide and gene-level enrichment, the MAGeCK "test" module was used with default parameters. This stage includes median ratio normalization to account for various read depths. A size factor for normalization was estimated using non-targeted control guides, and a mean-variance model of the null distribution was also constructed, which is used to find significant guide enrichment. MAGeCK produced guide-level enrichment scores for each direction (i.e., positive and negative), which were then used for α-robust rank aggregation (RRA) to obtain gene-level scores. The p-value for each gene was determined by a permutation test randomizing the guide assignment and adjusted for false discovery rate by the Benjamini-Hochberg method. Log2 fold change (LFC) was also calculated for each gene, defined as the median LFC of all guides per gene target across all. Where indicated, LFC was normalized to have a mean of 0 and standard deviation of 1 to obtain an LFC Z-score.

[0104] Preparation and electroporation of arrayed Cas9 ribonucleoproteins (RNPs) RNPs were generated by complexing the binary gRNA with Cas9 as previously described (Schumann et al., "Generation of knock-in primary human T cells using Cas9 ribonucleoproteins," Proc. Natl Acad. Sci.USA. 112, 10437-10442 (2015)). Briefly, crRNA and tracrRNA were chemically synthesized (IDT), and recombinant Cas9-NLS was produced and purified (QB3 Macrolab). Lyophilized RNA was resuspended in nuclease-free Duplex buffer (IDT, Cat# 1072570) at a concentration of 160 μM and stored in aliquots at -80°C. Aliquots of crRNA and tracrRNA were thawed, mixed 1:1 by volume, and annealed by incubation at 37°C for 30 min to form an 80 μM gRNA solution. Recombinant Cas9 was stored at 40 μM in 20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT, and then mixed with 80 μM gRNA 1:1 by volume (2:1 gRNA to Cas9 molar ratio) for 15 min at 37°C to form 20 μM RNPs. RNPs were electroporated immediately after complex formation. RNPs were electroporated 3 days after the first stimulation. Tregs were collected from their culture vessels, centrifuged at 300g for 5 min, aspirated, and resuspended in Lonza electroporation buffer P3 using 20 μl of buffer per 200,000 cells. 200,000 Tregs were electroporated per well using a Lonza 4D 96-well electroporation system with pulse code EO148. Immediately after electroporation, 80 μL of pre-warmed medium was added to each well and the cells were incubated for 15 minutes at 37° C. The cells were then transferred to round-bottom 96-well tissue culture plates and cultured at 200,000 cells / well in 200 μl of medium in complete DMEM, 10% FBS, 1% pen / strep+2000U hIL-2.

[0105] Isolation and culture of human Treg cells Primary human Treg cells for all experiments were obtained from the residue from the leukapheresis chamber after Trima apheresis (Blood Centers of the Pacific) under a protocol approved by the UCSF Committee on Human Research (CHR#13-11950). Peripheral blood mononuclear cells (PBMCs) were isolated from samples by Lymphoprep centrifugation (StemCell, Cat #07861) using SepMate tubes (StemCell, Cat #85460). CD4+ T cells were isolated from PBMCs by magnetic negative selection using the EasySep Human CD4+ T Cell Isolation Kit (StemCell, Cat #17952), and Tregs were then isolated using fluorescence-activated cell sorting by gating on CD4+, CD25+, CD127 low cells. After isolation, cells were stimulated with ImmunoCult human CD3 / CD28 / CD2 T cell activator (StemCell, Cat# 10970) according to the manufacturer's protocol and expanded for 9 days. Cells were cultured at 1 million cells / mL in complete RPMI medium containing 300U / mL hIL-2, 10% FBS, 50 mM 2-mercaptoethanol, and 1% pen / strep. After expansion, Tregs were restimulated in the same way for 24 hours before RNP electroporation.

[0106] result Using the above method, a pooled CRISPR screen of transcription factors identified transcription factors that increase Foxp3 expression (Foxp3 high), including Sp1, Rnf20, Smarcb1, Satb1, Sp3, and Nsd1, as shown in Figures 2a-j and Table 1. The screen also identified transcription factors that decrease Foxp3 expression (Foxp3 low), including Cbfb, Myc, Atxn713, Runx1, Usp22, and Stat5b, as shown in Figures 2a-j and Table 2. Figures 3a-3g show the design and results of the pooled CRISPR screen in primary mouse Tregs.

[0107] Additional studies were performed to validate the role of previously undescribed candidate genes from the CRISPR screen, including Rnf20, as well as members of the SAGA deubiquitination module, Usp22 and Atxn7l3. We used CRISPR-Cas9 ribonucleoprotein (RNP) to knock out the candidate genes in both human and mouse primary Tregs and identified changes in several Treg-specific markers and inflammatory cytokines by flow cytometry. Five of the top-ranked positive regulators were assessed by individual CRISPR knockout with Cas9 RNP. All guides tested resulted in a reduction in Foxp3 expression, recapitulating the screen data (Figures 2e and 2f).

[0108] It was also found that knockout of Usp22 and Atxn7l3 in mouse Tregs reduced Foxp3 expression (Figures 4a, 4f, and 4g), while knockdown of Rnf20 maintained stable Foxp3 expression (Figures 5a, 5b, and 7). Figure 4e shows the RNP control in mouse Tregs collected 5 days after electroporation. Knockout of Usp22 in human Tregs was also found to reduce Foxp3 expression (Figure 6). Additional studies showed that RNP knockout of USP22 reduced FOXP3 and CD25 mean fluorescence intensity (MFI) (Figures 2g and 2h) as well as FOXP3 expression in USP22-deficient human Tregs across six biological replicates. hi CD25 hi The frequency of Treg cells was significantly decreased in the IL-16 / ...

[0109] Array information SEQUENCE LISTING <110> The Regents of the University of California <120> Compositions and Methods for Modifying Regulatory T Cells <150> US 62 / 744,058 <151> 2018-10-10 <160> 271 <170> PatentIn version 3.5 <210> 1 <211> 737 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 1 Met Ser Asp Gln Asp His Ser Met Asp Glu Met Thr Ala Val Val Lys 1 5 10 15 Ile Glu Lys Gly Val Gly Gly Asn Asn Gly Gly Asn Gly Asn Gly Gly 20 25 30 Gly Ala Phe Ser Gln Ala Arg Ser Ser Ser Thr Gly Ser Ser Ser Ser 35 40 45 Thr Gly Gly Gly Gly Gln Gly Ala Asn Gly Trp Gln Ile Ile Ser Ser 50 55 60 Ser Ser Gly Ala Thr Pro Thr Ser Lys Glu Gln Ser Gly Ser Ser Thr 65 70 75 80 Asn Gly Ser Asn Gly Ser Glu Ser Ser Lys Asn Arg Thr Val Ser Gly 85 90 95 Gly Gln Tyr Val Val Ala Ala Ala Pro Asn Leu Gln Asn Gln Gln Val 100 105 110 Leu Thr Gly Leu Pro Gly Val Met Pro Asn Ile Gln Tyr Gln Val Ile 115 120 125 Pro Gln Phe Gln Thr Val Asp Gly Gln Gln Leu Gln Phe Ala Ala Thr 130 135 140 Gly Ala Gln Val Gln Gln Asp Gly Ser Gly Gln Ile Gln Ile Ile Pro 145 150 155 160 Gly Ala Asn Gln Gln Ile Ile Thr Asn Arg Gly Ser Gly Gly Asn Ile 165 170 175 Ile Ala Ala Met Pro Asn Leu Leu Gln Gln Ala Val Pro Leu Gln Gly 180 185 190 Leu Ala Asn Asn Val Leu Ser Gly Gln Thr Gln Tyr Val Thr Asn Val 195 200 205 Pro Val Ala Leu Asn Gly Asn Ile Thr Leu Leu Pro Val Asn Ser Val 210 215 220 Ser Ala Ala Thr Leu Thr Pro Ser Ser Gln Ala Val Thr Ile Ser Ser 225 230 235 240 Ser Gly Ser Gln Glu Ser Gly Ser Gln Pro Val Thr Ser Gly Thr Thr 245 250 255 Ile Ser Ser Ala Ser Leu Val Ser Ser Gln Ala Ser Ser Ser Ser Phe 260 265 270 Phe Thr Asn Ala Asn Ser Tyr Ser Thr Thr Thr Thr Thr Ser Asn Met 275 280 285 Gly Ile Met Asn Phe Thr Thr Ser Gly Ser Ser Gly Thr Asn Ser Gln 290 295 300 Gly Gln Thr Pro Gln Arg Val Ser Gly Leu Gln Gly Ser Asp Ala Leu 305 310 315 320 Asn Ile Gln Gln Asn Gln Thr Ser Gly Gly Ser Leu Gln Ala Gly Gln 325 330 335 Gln Lys Glu Gly Glu Gln Asn Gln Gln Thr Gln Gln Gln Gln Ile Leu 340 345 350 Ile Gln Pro Gln Leu Val Gln Gly Gly Gln Ala Leu Gln Ala Leu Gln 355 360 365 Ala Ala Pro Leu Ser Gly Gln Thr Phe Thr Thr Gln Ala Ile Ser Gln 370 375 380 Glu Thr Leu Gln Asn Leu Gln Leu Gln Ala Val Pro Asn Ser Gly Pro 385 390 395 400 Ile Ile Ile Arg Thr Pro Thr Val Gly Pro Asn Gly Gln Val Ser Trp 405 410 415 Gln Thr Leu Gln Leu Gln Asn Leu Gln Val Gln Asn Pro Gln Ala Gln 420 425 430 Thr Ile Thr Leu Ala Pro Met Gln Gly Val Ser Leu Gly Gln Thr Ser 435 440 445 Ser Ser Asn Thr Thr Leu Thr Pro Ile Ala Ser Ala Ala Ser Ile Pro 450 455 460 Ala Gly Thr Val Thr Val Asn Ala Ala Gln Leu Ser Ser Met Pro Gly 465 470 475 480 Leu Gln Thr Ile Asn Leu Ser Ala Leu Gly Thr Ser Gly Ile Gln Val 485 490 495 His Pro Ile Gln Gly Leu Pro Leu Ala Ile Ala Asn Ala Pro Gly Asp 500 505 510 His Gly Ala Gln Leu Gly Leu His Gly Ala Gly Gly Asp Gly Ile His 515 520 525 Asp Asp Thr Ala Gly Gly Glu Glu Gly Glu Asn Ser Pro Asp Ala Gln 530 535 540 Pro Gln Ala Gly Arg Arg Thr Arg Arg Glu Ala Cys Thr Cys Pro Tyr 545 550 555 560 Cys Lys Asp Ser Glu Gly Arg Gly Ser Gly Asp Pro Gly Lys Lys Lys 565 570 575 Gln His Ile Cys His Ile Gln Gly Cys Gly Lys Val Tyr Gly Lys Thr 580 585 590 Ser His Leu Arg Ala His Leu Arg Trp His Thr Gly Glu Arg Pro Phe 595 600 605 Met Cys Thr Trp Ser Tyr Cys Gly Lys Arg Phe Thr Arg Ser Asp Glu 610 615 620 Leu Gln Arg His Lys Arg Thr His Thr Gly Glu Lys Lys Phe Ala Cys 625 630 635 640 Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp His Leu Ser Lys His 645 650 655 Ile Lys Thr His Gln Asn Lys Lys Gly Gly Pro Gly Val Ala Leu Ser 660 665 670 Val Gly Thr Leu Pro Leu Asp Ser Gly Ala Gly Ser Glu Gly Ser Gly 675 680 685 Thr Ala Thr Pro Ser Ala Leu Ile Thr Thr Asn Met Val Ala Met Glu 690 695 700 Ala Ile Cys Pro Glu Gly Ile Ala Arg Leu Ala Asn Ser Gly Ile Asn 705 710 715 720 Val Met Gln Val Ala Asp Leu Gln Ser Ile Asn Ile Ser Gly Asn Gly 725 730 735 Phe <210> 2 <211> 975 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 2 Met Ser Gly Ile Gly Asn Lys Arg Ala Ala Gly Glu Pro Gly Thr Ser 1 5 10 15 Met Pro Pro Glu Lys Lys Ala Ala Val Glu Asp Ser Gly Thr Thr Val 20 25 30 Glu Thr Ile Lys Leu Gly Gly Val Ser Ser Thr Glu Glu Leu Asp Ile 35 40 45 Arg Thr Leu Gln Thr Lys Asn Arg Lys Leu Ala Glu Met Leu Asp Gln 50 55 60 Arg Gln Ala Ile Glu Asp Glu Leu Arg Glu His Ile Glu Lys Leu Glu 65 70 75 80 Arg Arg Gln Ala Thr Asp Asp Ala Ser Leu Leu Ile Val Asn Arg Tyr 85 90 95 Trp Ser Gln Phe Asp Glu Asn Ile Arg Ile Ile Leu Lys Arg Tyr Asp 100 105 110 Leu Glu Gln Gly Leu Gly Asp Leu Leu Thr Glu Arg Lys Ala Leu Val 115 120 125 Val Pro Glu Pro Glu Pro Asp Ser Asp Ser Asn Gln Glu Arg Lys Asp 130 135 140 Asp Arg Glu Arg Gly Glu Gly Gln Glu Pro Ala Phe Ser Phe Leu Ala 145 150 155 160 Thr Leu Ala Ser Ser Ser Ser Glu Glu Met Glu Ser Gln Leu Gln Glu 165 170 175 Arg Val Glu Ser Ser Arg Arg Ala Val Ser Gln Ile Val Thr Val Tyr 180 185 190 Asp Lys Leu Gln Glu Lys Val Glu Leu Leu Ser Arg Lys Leu Asn Ser 195 200 205 Gly Asp Asn Leu Ile Val Glu Glu Ala Val Gln Glu Leu Asn Ser Phe 210 215 220 Leu Ala Gln Glu Asn Met Arg Leu Gln Glu Leu Thr Asp Leu Leu Gln 225 230 235 240 Glu Lys His Arg Thr Met Ser Gln Glu Phe Ser Lys Leu Gln Ser Lys 245 250 255 Val Glu Thr Ala Glu Ser Arg Val Ser Val Leu Glu Ser Met Ile Asp 260 265 270 Asp Leu Gln Trp Asp Ile Asp Lys Ile Arg Lys Arg Glu Gln Arg Leu 275 280 285 Asn Arg His Leu Ala Glu Val Leu Glu Arg Val Asn Ser Lys Gly Tyr 290 295 300 Lys Val Tyr Gly Ala Gly Ser Ser Leu Tyr Gly Gly Thr Ile Thr Ile 305 310 315 320 Asn Ala Arg Lys Phe Glu Glu Met Asn Ala Glu Leu Glu Glu Asn Lys 325 330 335 Glu Leu Ala Gln Asn Arg Leu Cys Glu Leu Glu Lys Leu Arg Gln Asp 340 345 350 Phe Glu Glu Val Thr Thr Gln Asn Glu Lys Leu Lys Val Glu Leu Arg 355 360 365 Ser Ala Val Glu Gln Val Val Lys Glu Thr Pro Glu Tyr Arg Cys Met 370 375 380 Gln Ser Gln Phe Ser Val Leu Tyr Asn Glu Ser Leu Gln Leu Lys Ala 385 390 395 400 His Leu Asp Glu Ala Arg Thr Leu Leu His Gly Thr Arg Gly Thr His 405 410 415 Gln His Gln Val Glu Leu Ile Glu Arg Asp Glu Val Ser Leu His Lys 420 425 430 Lys Leu Arg Thr Glu Val Ile Gln Leu Glu Asp Thr Leu Ala Gln Val 435 440 445 Arg Lys Glu Tyr Glu Met Leu Arg Ile Glu Phe Glu Gln Thr Leu Ala 450 455 460 Ala Asn Glu Gln Ala Gly Pro Ile Asn Arg Glu Met Arg His Leu Ile 465 470 475 480 Ser Ser Leu Gln Asn His Asn His Gln Leu Lys Gly Glu Val Leu Arg 485 490 495 Tyr Lys Arg Lys Leu Arg Glu Ala Gln Ser Asp Leu Asn Lys Thr Arg 500 505 510 Leu Arg Ser Gly Ser Ala Leu Leu Gln Ser Gln Ser Ser Thr Glu Asp 515 520 525 Pro Lys Asp Glu Pro Ala Glu Leu Lys Pro Asp Ser Glu Asp Leu Ser 530 535 540 Ser Gln Ser Ser Ala Ser Lys Ala Ser Gln Glu Asp Ala Asn Glu Ile 545 550 555 560 Lys Ser Lys Arg Asp Glu Glu Glu Arg Glu Arg Glu Arg Arg Glu Lys 565 570 575 Glu Arg Glu Arg Glu Arg Glu Arg Glu Lys Glu Lys Glu Arg Glu Arg 580 585 590 Glu Lys Gln Lys Leu Lys Glu Ser Glu Lys Glu Arg Asp Ser Ala Lys 595 600 605 Asp Lys Glu Lys Gly Lys His Asp Asp Gly Arg Lys Lys Glu Ala Glu 610 615 620 Ile Ile Lys Gln Leu Lys Ile Glu Leu Lys Lys Ala Gln Glu Ser Gln 625 630 635 640 Lys Glu Met Lys Leu Leu Leu Asp Met Tyr Arg Ser Ala Pro Lys Glu 645 650 655 Gln Arg Asp Lys Val Gln Leu Met Ala Ala Glu Lys Lys Ser Lys Ala 660 665 670 Glu Leu Glu Asp Leu Arg Gln Arg Leu Lys Asp Leu Glu Asp Lys Glu 675 680 685 Lys Lys Glu Asn Lys Lys Met Ala Asp Glu Asp Ala Leu Arg Lys Ile 690 695 700 Arg Ala Val Glu Glu Gln Ile Glu Tyr Leu Gln Lys Lys Leu Ala Met 705 710 715 720 Ala Lys Gln Glu Glu Glu Ala Leu Leu Ser Glu Met Asp Val Thr Gly 725 730 735 Gln Ala Phe Glu Asp Met Gln Glu Gln Asn Ile Arg Leu Met Gln Gln 740 745 750 Leu Arg Glu Lys Asp Asp Ala Asn Phe Lys Leu Met Ser Glu Arg Ile 755 760 765 Lys Ser Asn Gln Ile His Lys Leu Leu Lys Glu Glu Lys Glu Glu Leu 770 775 780 Ala Asp Gln Val Leu Thr Leu Lys Thr Gln Val Asp Ala Gln Leu Gln 785 790 795 800 Val Val Arg Lys Leu Glu Glu Lys Glu His Leu Leu Gln Ser Asn Ile 805 810 815 Gly Thr Gly Glu Lys Glu Leu Gly Leu Arg Thr Gln Ala Leu Glu Met 820 825 830 Asn Lys Arg Lys Ala Met Glu Ala Ala Gln Leu Ala Asp Asp Leu Lys 835 840 845 Ala Gln Leu Glu Leu Ala Gln Lys Lys Leu His Asp Phe Gln Asp Glu 850 855 860 Ile Val Glu Asn Ser Val Thr Lys Glu Lys Asp Met Phe Asn Phe Lys 865 870 875 880 Arg Ala Gln Glu Asp Ile Ser Arg Leu Arg Arg Lys Leu Glu Thr Thr 885 890 895 Lys Lys Pro Asp Asn Val Pro Lys Cys Asp Glu Ile Leu Met Glu Glu 900 905 910 Ile Lys Asp Tyr Lys Ala Arg Leu Thr Cys Pro Cys Cys Asn Met Arg 915 920 925 Lys Lys Asp Ala Val Leu Thr Lys Cys Phe His Val Phe Cys Phe Glu 930 935 940 Cys Val Lys Thr Arg Tyr Asp Thr Arg Gln Arg Lys Cys Pro Lys Cys 945 950 955 960 Asn Ala Ala Phe Gly Ala Asn Asp Phe His Arg Ile Tyr Ile Gly 965 970 975 <210> 3 <211> 1460 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 3 Met Ala Glu Glu Gln Gln Gln Pro Pro Pro Gln Gln Pro Asp Ala His 1 5 10 15 Gln Gln Leu Pro Pro Ser Ala Pro Asn Ser Gly Val Ala Leu Pro Ala 20 25 30 Leu Val Pro Gly Leu Pro Gly Thr Glu Ala Ser Ala Leu Gln His Lys 35 40 45 Ile Lys Asn Ser Ile Cys Lys Thr Val Gln Ser Lys Val Asp Cys Ile 50 55 60 Leu Gln Glu Val Glu Lys Phe Thr Asp Leu Glu Lys Leu Tyr Leu Tyr 65 70 75 80 Leu Gln Leu Pro Ser Gly Leu Ser Asn Gly Glu Lys Ser Asp Gln Asn 85 90 95 Ala Met Ser Ser Ser Arg Ala Gln Gln Met His Ala Phe Ser Trp Ile 100 105 110 Arg Asn Thr Leu Glu Glu His Pro Glu Thr Ser Leu Pro Lys Gln Glu 115 120 125 Val Tyr Asp Glu Tyr Lys Ser Tyr Cys Asp Asn Leu Gly Tyr His Pro 130 135 140 Leu Ser Ala Ala Asp Phe Gly Lys Ile Met Lys Asn Val Phe Pro Asn 145 150 155 160 Met Lys Ala Arg Arg Leu Gly Thr Arg Gly Lys Ser Lys Tyr Cys Tyr 165 170 175 Ser Gly Leu Arg Lys Lys Ala Phe Val His Met Pro Thr Leu Pro Asn 180 185 190 Leu Asp Phe His Lys Thr Gly Asp Gly Leu Glu Gly Ala Glu Pro Ser 195 200 205 Gly Gln Leu Gln Asn Ile Asp Glu Glu Val Ile Ser Ser Ala Cys Arg 210 215 220 Leu Val Cys Glu Trp Ala Gln Lys Val Leu Ser Gln Pro Phe Asp Thr 225 230 235 240 Val Leu Glu Leu Ala Arg Phe Leu Val Lys Ser His Tyr Ile Gly Thr 245 250 255 Lys Ser Met Ala Ala Leu Thr Val Met Ala Ala Ala Pro Ala Gly Met 260 265 270 Lys Gly Ile Thr Gln Pro Ser Ala Phe Ile Pro Thr Ala Glu Ser Asn 275 280 285 Ser Phe Gln Pro Gln Val Lys Thr Leu Pro Ser Pro Ile Asp Ala Lys 290 295 300 Gln Gln Leu Gln Arg Lys Ile Gln Lys Lys Gln Gln Glu Gln Lys Leu 305 310 315 320 Gln Ser Pro Leu Pro Gly Glu Ser Ala Ala Lys Lys Ser Glu Ser Ala 325 330 335 Thr Ser Asn Gly Val Thr Asn Leu Pro Asn Gly Asn Pro Ser Ile Leu 340 345 350 Ser Pro Gln Pro Ile Gly Ile Val Val Ala Ala Val Pro Ser Pro Ile 355 360 365 Pro Val Gln Arg Thr Arg Gln Leu Val Thr Ser Pro Ser Pro Met Ser 370 375 380 Ser Ser Asp Gly Lys Val Leu Pro Leu Asn Val Gln Val Val Thr Gln 385 390 395 400 His Met Gln Ser Val Lys Gln Ala Pro Lys Thr Pro Gln Asn Val Pro 405 410 415 Ala Ser Pro Gly Gly Asp Arg Ser Ala Arg His Arg Tyr Pro Gln Ile 420 425 430 Leu Pro Lys Pro Ala Asn Thr Ser Ala Leu Thr Ile Arg Ser Pro Thr 435 440 445 Thr Val Leu Phe Thr Ser Ser Pro Ile Lys Thr Ala Val Val Pro Ala 450 455 460 Ser His Met Ser Ser Leu Asn Val Val Lys Met Thr Thr Ile Ser Leu 465 470 475 480 Thr Pro Ser Asn Ser Asn Thr Pro Leu Lys His Ser Ala Ser Val Ser 485 490 495 Ser Ala Thr Gly Thr Thr Glu Glu Ser Arg Ser Val Pro Gln Ile Lys 500 505 510 Asn Gly Ser Val Val Ser Leu Gln Ser Pro Gly Ser Arg Ser Ser Ser 515 520 525 Ala Gly Gly Thr Ser Ala Val Glu Val Lys Val Glu Pro Glu Thr Ser 530 535 540 Ser Asp Glu His Pro Val Gln Cys Gln Glu Asn Ser Asp Glu Ala Lys 545 550 555 560 Ala Pro Gln Thr Pro Ser Ala Leu Leu Gly Gln Lys Ser Asn Thr Asp 565 570 575 Gly Ala Leu Gln Lys Pro Ser Asn Glu Gly Val Ile Glu Ile Lys Ala 580 585 590 Thr Lys Val Cys Asp Gln Arg Thr Lys Cys Lys Ser Arg Cys Asn Glu 595 600 605 Met Leu Pro Gly Thr Ser Thr Gly Asn Asn Gln Ser Thr Ile Thr Leu 610 615 620 Ser Val Ala Ser Gln Asn Leu Thr Phe Thr Ser Ser Ser Ser Pro Pro 625 630 635 640 Asn Gly Asp Ser Ile Asn Lys Asp Pro Lys Leu Cys Thr Lys Ser Pro 645 650 655 Arg Lys Arg Leu Ser Ser Thr Leu Gln Glu Thr Gln Val Pro Pro Val 660 665 670 Lys Lys Pro Ile Val Glu Gln Leu Ser Ala Ala Thr Ile Glu Gly Gln 675 680 685 Lys Gln Gly Ser Val Lys Lys Asp Gln Lys Val Pro His Ser Gly Lys 690 695 700 Thr Glu Gly Ser Thr Ala Gly Ala Gln Ile Pro Ser Lys Val Ser Val 705 710 715 720 Asn Val Ser Ser His Ile Gly Ala Asn Gln Pro Leu Asn Ser Ser Ala 725 730 735 Leu Val Ile Ser Asp Ser Ala Leu Glu Gln Gln Thr Thr Pro Ser Ser 740 745 750 Ser Pro Asp Ile Lys Val Lys Leu Glu Gly Ser Val Phe Leu Leu Asp 755 760 765 Ser Asp Ser Lys Ser Val Gly Ser Phe Asn Pro Asn Gly Trp Gln Gln 770 775 780 Ile Thr Lys Asp Ser Glu Phe Ile Ser Ala Ser Cys Glu Gln Gln Gln 785 790 795 800 Asp Ile Ser Val Met Thr Ile Pro Glu His Ser Asp Ile Asn Asp Leu 805 810 815 Glu Lys Ser Val Trp Glu Leu Glu Gly Met Pro Gln Asp Thr Tyr Ser 820 825 830 Gln Gln Leu His Ser Gln Ile Gln Glu Ser Ser Leu Asn Gln Ile Gln 835 840 845 Ala His Ser Ser Asp Gln Leu Pro Leu Gln Ser Glu Leu Lys Glu Phe 850 855 860 Glu Pro Ser Val Ser Gln Thr Asn Glu Ser Tyr Phe Pro Phe Asp Asp 865 870 875 880 Glu Leu Thr Gln Asp Ser Ile Val Glu Glu Leu Val Leu Met Glu Gln 885 890 895 Gln Met Ser Met Asn Asn Ser His Ser Tyr Gly Asn Cys Leu Gly Met 900 905 910 Thr Leu Gln Ser Gln Ser Val Thr Pro Gly Ala Pro Met Ser Ser His 915 920 925 Thr Ser Ser Thr His Phe Tyr His Pro Ile His Ser Asn Gly Thr Pro 930 935 940 Ile His Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro 945 950 955 960 Thr Pro Thr Pro Thr Ser Glu Met Ile Ala Gly Ser Gln Ser Leu Ser 965 970 975 Arg Glu Ser Pro Cys Ser Arg Leu Ala Gln Thr Thr Pro Val Asp Ser 980 985 990 Ala Leu Gly Ser Ser Arg His Thr Pro Ile Gly Thr Pro His Ser Asn 995 1000 1005 Cys Ser Ser Ser Val Pro Pro Ser Pro Val Glu Cys Arg Asn Pro 1010 1015 1020 Phe Ala Phe Thr Pro Ile Ser Ser Ser Met Ala Tyr His Asp Ala 1025 1030 1035 Ser Ile Val Ser Ser Ser Pro Val Lys Pro Met Gln Arg Pro Met 1040 1045 1050 Ala Thr His Pro Asp Lys Thr Lys Leu Glu Trp Met Asn Asn Gly 1055 1060 1065 Tyr Ser Gly Val Gly Asn Ser Ser Val Ser Gly His Gly Ile Leu 1070 1075 1080 Pro Ser Tyr Gln Glu Leu Val Glu Asp Arg Phe Arg Lys Pro His 1085 1090 1095 Ala Phe Ala Val Pro Gly Gln Ser Tyr Gln Ser Gln Ser Arg His 1100 1105 1110 His Asp Thr Asn Phe Gly Arg Leu Thr Pro Val Ser Pro Val Gln 1115 1120 1125 His Gln Gly Ala Thr Val Asn Asn Thr Asn Lys Gln Glu Gly Phe 1130 1135 1140 Ala Val Pro Ala Pro Leu Asp Asn Lys Gly Thr Asn Ser Ser Ala 1145 1150 1155 Ser Ser Asn Phe Arg Cys Arg Ser Val Ser Pro Ala Val His Arg 1160 1165 1170 Gln Arg Asn Leu Ser Gly Ser Thr Leu Tyr Pro Val Ser Asn Ile 1175 1180 1185 Pro Arg Ser Asn Val Thr Pro Phe Gly Ser Pro Val Thr Pro Glu 1190 1195 1200 Val His Val Phe Thr Asn Val His Thr Asp Ala Cys Ala Asn Asn 1205 1210 1215 Ile Ala Gln Arg Ser Gln Ser Val Pro Leu Thr Val Met Met Gln 1220 1225 1230 Thr Ala Phe Pro Asn Ala Leu Gln Lys Gln Ala Asn Ser Lys Lys 1235 1240 1245 Ile Thr Asn Val Leu Leu Ser Lys Leu Asp Ser Asp Asn Asp Asp 1250 1255 1260 Ala Val Arg Gly Leu Gly Met Asn Asn Leu Pro Ser Asn Tyr Thr 1265 1270 1275 Ala Arg Met Asn Leu Thr Gln Ile Leu Glu Pro Ser Thr Val Phe 1280 1285 1290 Pro Ser Ala Asn Pro Gln Asn Met Ile Asp Ser Ser Thr Ser Val 1295 1300 1305 Tyr Glu Phe Gln Thr Pro Ser Tyr Leu Thr Lys Ser Asn Ser Thr 1310 1315 1320 Gly Gln Ile Asn Phe Ser Pro Gly Asp Asn Gln Ala Gln Ser Glu 1325 1330 1335 Ile Gly Glu Gln Gln Leu Asp Phe Asn Ser Thr Val Lys Asp Leu 1340 1345 1350 Leu Ser Gly Asp Ser Leu Gln Thr Asn Gln Gln Leu Val Gly Gln 1355 1360 1365 Gly Ala Ser Asp Leu Thr Asn Thr Ala Ser Asp Phe Ser Ser Asp 1370 1375 1380 Ile Arg Leu Ser Ser Glu Leu Ser Gly Ser Ile Asn Asp Leu Asn 1385 1390 1395 Thr Leu Asp Pro Asn Leu Leu Phe Asp Pro Gly Arg Gln Gln Gly 1400 1405 1410 Gln Asp Asp Glu Ala Thr Leu Glu Glu Leu Lys Asn Asp Pro Leu 1415 1420 1425 Phe Gln Gln Ile Cys Ser Glu Ser Met Asn Ser Met Thr Ser Ser 1430 1435 1440 Gly Phe Glu Trp Ile Glu Ser Lys Asp His Pro Thr Val Glu Met 1445 1450 1455 Leu Gly 1460 <210> 4 <211> 508 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 4 Met Leu Pro Thr Gln Ala Gly Ala Ala Ala Ala Leu Gly Arg Gly Ser 1 5 10 15 Ala Leu Gly Gly Ser Leu Asn Arg Thr Pro Thr Gly Arg Pro Gly Gly 20 25 30 Gly Gly Gly Thr Arg Gly Ala Asn Gly Gly Arg Val Pro Gly Asn Gly 35 40 45 Ala Gly Leu Gly Pro Gly Arg Leu Glu Arg Glu Ala Ala Ala Ala Ala 50 55 60 Ala Thr Thr Pro Ala Pro Thr Ala Gly Ala Leu Tyr Ser Gly Ser Glu 65 70 75 80 Gly Asp Ser Glu Ser Gly Glu Glu Glu Glu Leu Gly Ala Glu Arg Arg 85 90 95 Gly Leu Lys Arg Ser Leu Ser Glu Met Glu Ile Gly Met Val Val Gly 100 105 110 Gly Pro Glu Ala Ser Ala Ala Ala Thr Gly Gly Tyr Gly Pro Val Ser 115 120 125 Gly Ala Val Ser Gly Ala Lys Pro Gly Lys Lys Thr Arg Gly Arg Val 130 135 140 Lys Ile Lys Met Glu Phe Ile Asp Asn Lys Leu Arg Arg Tyr Thr Thr 145 150 155 160 Phe Ser Lys Arg Lys Thr Gly Ile Met Lys Lys Ala Tyr Glu Leu Ser 165 170 175 Thr Leu Thr Gly Thr Gln Val Leu Leu Leu Val Ala Ser Glu Thr Gly 180 185 190 His Val Tyr Thr Phe Ala Thr Arg Lys Leu Gln Pro Met Ile Thr Ser 195 200 205 Glu Thr Gly Lys Ala Leu Ile Gln Thr Cys Leu Asn Ser Pro Asp Ser 210 215 220 Pro Pro Arg Ser Asp Pro Thr Thr Asp Gln Arg Met Ser Ala Thr Gly 225 230 235 240 Phe Glu Glu Thr Asp Leu Thr Tyr Gln Val Ser Glu Ser Asp Ser Ser 245 250 255 Gly Glu Thr Lys Asp Thr Leu Lys Pro Ala Phe Thr Val Thr Asn Leu 260 265 270 Pro Gly Thr Thr Ser Thr Ile Gln Thr Ala Pro Ser Thr Ser Thr Thr 275 280 285 Met Gln Val Ser Ser Gly Pro Ser Phe Pro Ile Thr Asn Tyr Leu Ala 290 295 300 Pro Val Ser Ala Ser Val Ser Pro Ser Ala Val Ser Ser Ala Asn Gly 305 310 315 320 Thr Val Leu Lys Ser Thr Gly Ser Gly Pro Val Ser Ser Gly Gly Leu 325 330 335 Met Gln Leu Pro Thr Ser Phe Thr Leu Met Pro Gly Gly Ala Val Ala 340 345 350 Gln Gln Val Pro Val Gln Ala Ile Gln Val His Gln Ala Pro Gln Gln 355 360 365 Ala Ser Pro Ser Arg Asp Ser Ser Thr Asp Leu Thr Gln Thr Ser Ser 370 375 380 Ser Gly Thr Val Thr Leu Pro Ala Thr Ile Met Thr Ser Ser Val Pro 385 390 395 400 Thr Thr Val Gly Gly His Met Met Tyr Pro Ser Pro His Ala Val Met 405 410 415 Tyr Ala Pro Thr Ser Gly Leu Gly Asp Gly Ser Leu Thr Val Leu Asn 420 425 430 Ala Phe Ser Gln Ala Pro Ser Thr Met Gln Val Ser His Ser Gln Val 435 440 445 Gln Glu Pro Gly Gly Val Pro Gln Val Phe Leu Thr Ala Ser Ser Gly 450 455 460 Thr Val Gln Ile Pro Val Ser Ala Val Gln Leu His Gln Met Ala Val 465 470 475 480 Ile Gly Gln Gln Ala Gly Ser Ser Ser Asn Leu Thr Glu Leu Gln Val 485 490 495 Val Asn Leu Asp Thr Ala His Ser Thr Lys Ser Glu 500 505 <210> 5 <211> 891 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 5 Met Val Ala Pro Val Leu Glu Thr Ser His Val Phe Cys Cys Pro Asn 1 5 10 15 Arg Val Arg Gly Val Leu Asn Trp Ser Ser Gly Pro Arg Gly Leu Leu 20 25 30 Ala Phe Gly Thr Ser Cys Ser Val Val Leu Tyr Asp Pro Leu Lys Arg 35 40 45 Val Val Val Thr Asn Leu Asn Gly His Thr Ala Arg Val Asn Cys Ile 50 55 60 Gln Trp Ile Cys Lys Gln Asp Gly Ser Pro Ser Thr Glu Leu Val Ser 65 70 75 80 Gly Gly Ser Asp Asn Gln Val Ile His Trp Glu Ile Glu Asp Asn Gln 85 90 95 Leu Leu Lys Ala Val His Leu Gln Gly His Glu Gly Pro Val Tyr Ala 100 105 110 Val His Ala Val Tyr Gln Arg Arg Thr Ser Asp Pro Ala Leu Cys Thr 115 120 125 Leu Ile Val Ser Ala Ala Ala Asp Ser Ala Val Arg Leu Trp Ser Lys 130 135 140 Lys Gly Pro Glu Val Met Cys Leu Gln Thr Leu Asn Phe Gly Asn Gly 145 150 155 160 Phe Ala Leu Ala Leu Cys Leu Ser Phe Leu Pro Asn Thr Asp Val Thr 165 170 175 Trp Lys Thr Gly Gln Val Glu Arg Gly Arg Ala Trp Lys Pro Pro Ala 180 185 190 Ser Leu Ala Leu Cys Ser Arg Ser Cys Asp Ser Met Val Ser Cys Tyr 195 200 205 Ala Ser Ile Leu Cys Lys Ala Leu Trp Lys Glu Lys Leu His Thr Phe 210 215 220 Trp His His Asn Arg Ile Ser Phe Leu Pro Ser Ala Phe Arg Pro Ile 225 230 235 240 Pro Ile Leu Ala Cys Gly Asn Asp Asp Cys Arg Ile His Ile Phe Ala 245 250 255 Gln Gln Asn Asp Gln Phe Gln Lys Val Leu Ser Leu Cys Gly His Glu 260 265 270 Asp Trp Ile Arg Gly Val Glu Trp Ala Ala Phe Gly Arg Asp Leu Phe 275 280 285 Leu Ala Ser Cys Ser Gln Asp Cys Leu Ile Arg Ile Trp Lys Leu Tyr 290 295 300 Ile Lys Ser Thr Ser Leu Glu Thr Gln Asp Asp Asp Asn Ile Arg Leu 305 310 315 320 Lys Glu Asn Thr Phe Thr Ile Glu Asn Glu Ser Val Lys Ile Ala Phe 325 330 335 Ala Val Thr Leu Glu Thr Val Leu Ala Gly His Glu Asn Trp Val Asn 340 345 350 Ala Val His Trp Gln Pro Val Phe Tyr Lys Asp Gly Val Leu Gln Gln 355 360 365 Pro Val Arg Leu Leu Ser Ala Ser Met Asp Lys Thr Met Ile Leu Trp 370 375 380 Ala Pro Asp Glu Glu Ser Gly Val Trp Leu Glu Gln Val Arg Val Gly 385 390 395 400 Glu Val Gly Gly Asn Thr Leu Gly Phe Tyr Asp Cys Gln Phe Asn Glu 405 410 415 Asp Gly Ser Met Ile Ile Ala His Ala Phe His Gly Ala Leu His Leu 420 425 430 Trp Lys Gln Asn Thr Val Asn Pro Arg Glu Trp Thr Pro Glu Ile Val 435 440 445 Ile Ser Gly His Phe Asp Gly Val Gln Asp Leu Val Trp Asp Pro Glu 450 455 460 Gly Glu Phe Ile Ile Thr Val Gly Thr Asp Gln Thr Thr Arg Leu Phe 465 470 475 480 Ala Pro Trp Lys Arg Lys Asp Gln Ser Gln Val Thr Trp His Glu Ile 485 490 495 Ala Arg Pro Gln Ile His Gly Tyr Asp Leu Lys Cys Leu Ala Met Ile 500 505 510 Asn Arg Phe Gln Phe Val Ser Gly Ala Asp Glu Lys Val Leu Arg Val 515 520 525 Phe Ser Ala Pro Arg Asn Phe Val Glu Asn Phe Cys Ala Ile Thr Gly 530 535 540 Gln Ser Leu Asn His Val Leu Cys Asn Gln Asp Ser Asp Leu Pro Glu 545 550 555 560 Gly Ala Thr Val Pro Ala Leu Gly Leu Ser Asn Lys Ala Val Phe Gln 565 570 575 Gly Asp Ile Ala Ser Gln Pro Ser Asp Glu Glu Glu Leu Leu Thr Ser 580 585 590 Thr Gly Phe Glu Tyr Gln Gln Val Ala Phe Gln Pro Ser Ile Leu Thr 595 600 605 Glu Pro Pro Thr Glu Asp His Leu Leu Gln Asn Thr Leu Trp Pro Glu 610 615 620 Val Gln Lys Leu Tyr Gly His Gly Tyr Glu Ile Phe Cys Val Thr Cys 625 630 635 640 Asn Ser Ser Lys Thr Leu Leu Ala Ser Ala Cys Lys Ala Ala Lys Lys 645 650 655 Glu His Ala Ala Ile Ile Leu Trp Asn Thr Thr Ser Trp Lys Gln Val 660 665 670 Gln Asn Leu Val Phe His Ser Leu Thr Val Thr Gln Met Ala Phe Ser 675 680 685 Pro Asn Glu Lys Phe Leu Leu Ala Val Ser Arg Asp Arg Thr Trp Ser 690 695 700 Leu Trp Lys Lys Gln Asp Thr Ile Ser Pro Glu Phe Glu Pro Val Phe 705 710 715 720 Ser Leu Phe Ala Phe Thr Asn Lys Ile Thr Ser Val His Ser Arg Ile 725 730 735 Ile Trp Ser Cys Asp Trp Ser Pro Asp Ser Lys Tyr Phe Phe Thr Gly 740 745 750 Ser Arg Asp Lys Lys Val Val Val Trp Gly Glu Cys Asp Ser Thr Asp 755 760 765 Asp Cys Ile Glu His Asn Ile Gly Pro Cys Ser Ser Val Leu Asp Val 770 775 780 Gly Gly Ala Val Thr Ala Val Ser Val Cys Pro Val Leu His Pro Ser 785 790 795 800 Gln Arg Tyr Val Val Ala Val Gly Leu Glu Cys Gly Lys Ile Cys Leu 805 810 815 Tyr Thr Trp Lys Lys Thr Asp Gln Val Pro Glu Ile Asn Asp Trp Thr 820 825 830 His Cys Val Glu Thr Ser Gln Ser Gln Ser His Thr Leu Ala Ile Arg 835 840 845 Lys Leu Cys Trp Lys Asn Cys Ser Gly Lys Thr Glu Gln Lys Glu Ala 850 855 860 Glu Gly Ala Glu Trp Leu His Phe Ala Ser Cys Gly Glu Asp His Thr 865 870 875 880 Val Lys Ile His Arg Val Asn Lys Cys Ala Leu 885 890 <210> 6 <211> 2427 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 6 Met Pro Leu Lys Thr Arg Thr Ala Leu Ser Asp Asp Pro Asp Ser Ser 1 5 10 15 Thr Ser Thr Leu Gly Asn Met Leu Glu Leu Pro Gly Thr Ser Ser Ser 20 25 30 Ser Thr Ser Gln Glu Leu Pro Phe Cys Gln Pro Lys Lys Lys Ser Thr 35 40 45 Pro Leu Lys Tyr Glu Val Gly Asp Leu Ile Trp Ala Lys Phe Lys Arg 50 55 60 Arg Pro Trp Trp Pro Cys Arg Ile Cys Ser Asp Pro Leu Ile Asn Thr 65 70 75 80 His Ser Lys Met Lys Val Ser Asn Arg Arg Pro Tyr Arg Gln Tyr Tyr 85 90 95 Val Glu Ala Phe Gly Asp Pro Ser Glu Arg Ala Trp Val Ala Gly Lys 100 105 110 Ala Ile Val Met Phe Glu Gly Arg His Gln Phe Glu Glu Leu Pro Val 115 120 125 Leu Arg Arg Arg Gly Lys Gln Lys Glu Lys Gly Tyr Arg His Lys Val 130 135 140 Pro Gln Lys Ile Leu Ser Lys Trp Glu Ala Ser Val Gly Leu Ala Glu 145 150 155 160 Gln Tyr Asp Val Pro Lys Gly Ser Lys Asn Arg Lys Cys Ile Pro Gly 165 170 175 Ser Ile Lys Leu Asp Ser Glu Glu Asp Met Pro Phe Glu Asp Cys Thr 180 185 190 Asn Asp Pro Glu Ser Glu His Asp Leu Leu Leu Asn Gly Cys Leu Lys 195 200 205 Ser Leu Ala Phe Asp Ser Glu His Ser Ala Asp Glu Lys Glu Lys Pro 210 215 220 Cys Ala Lys Ser Arg Ala Arg Lys Ser Ser Asp Asn Pro Lys Arg Thr 225 230 235 240 Ser Val Lys Lys Gly His Ile Gln Phe Glu Ala His Lys Asp Glu Arg 245 250 255 Arg Gly Lys Ile Pro Glu Asn Leu Gly Leu Asn Phe Ile Ser Gly Asp 260 265 270 Ile Ser Asp Thr Gln Ala Ser Asn Glu Leu Ser Arg Ile Ala Asn Ser 275 280 285 Leu Thr Gly Ser Asn Thr Ala Pro Gly Ser Phe Leu Phe Ser Ser Cys 290 295 300 Gly Lys Asn Thr Ala Lys Lys Glu Phe Glu Thr Ser Asn Gly Asp Ser 305 310 315 320 Leu Leu Gly Leu Pro Glu Gly Ala Leu Ile Ser Lys Cys Ser Arg Glu 325 330 335 Lys Asn Lys Pro Gln Arg Ser Leu Val Cys Gly Ser Lys Val Lys Leu 340 345 350 Cys Tyr Ile Gly Ala Gly Asp Glu Glu Lys Arg Ser Asp Ser Ile Ser 355 360 365 Ile Cys Thr Thr Ser Asp Asp Gly Ser Ser Asp Leu Asp Pro Ile Glu 370 375 380 His Ser Ser Glu Ser Asp Asn Ser Val Leu Glu Ile Pro Asp Ala Phe 385 390 395 400 Asp Arg Thr Glu Asn Met Leu Ser Met Gln Lys Asn Glu Lys Ile Lys 405 410 415 Tyr Ser Arg Phe Ala Ala Thr Asn Thr Arg Val Lys Ala Lys Gln Lys 420 425 430 Pro Leu Ile Ser Asn Ser His Thr Asp His Leu Met Gly Cys Thr Lys 435 440 445 Ser Ala Glu Pro Gly Thr Glu Thr Ser Gln Val Asn Leu Ser Asp Leu 450 455 460 Lys Ala Ser Thr Leu Val His Lys Pro Gln Ser Asp Phe Thr Asn Asp 465 470 475 480 Ala Leu Ser Pro Lys Phe Asn Leu Ser Ser Ser Ile Ser Ser Glu Asn 485 490 495 Ser Leu Ile Lys Gly Gly Ala Ala Asn Gln Ala Leu Leu His Ser Lys 500 505 510 Ser Lys Gln Pro Lys Phe Arg Ser Ile Lys Cys Lys His Lys Glu Asn 515 520 525 Pro Val Met Ala Glu Pro Pro Val Ile Asn Glu Glu Cys Ser Leu Lys 530 535 540 Cys Cys Ser Ser Asp Thr Lys Gly Ser Pro Leu Ala Ser Ile Ser Lys 545 550 555 560 Ser Gly Lys Val Asp Gly Leu Lys Leu Leu Asn Asn Met His Glu Lys 565 570 575 Thr Arg Asp Ser Ser Asp Ile Glu Thr Ala Val Val Lys His Val Leu 580 585 590 Ser Glu Leu Lys Glu Leu Ser Tyr Arg Ser Leu Gly Glu Asp Val Ser 595 600 605 Asp Ser Gly Thr Ser Lys Pro Ser Lys Pro Leu Leu Phe Ser Ser Ala 610 615 620 Ser Ser Gln Asn His Ile Pro Ile Glu Pro Asp Tyr Lys Phe Ser Thr 625 630 635 640 Leu Leu Met Met Leu Lys Asp Met His Asp Ser Lys Thr Lys Glu Gln 645 650 655 Arg Leu Met Thr Ala Gln Asn Leu Val Ser Tyr Arg Ser Pro Gly Arg 660 665 670 Gly Asp Cys Ser Thr Asn Ser Pro Val Gly Val Ser Lys Val Leu Val 675 680 685 Ser Gly Gly Ser Thr His Asn Ser Glu Lys Lys Gly Asp Gly Thr Gln 690 695 700 Asn Ser Ala Asn Pro Ser Pro Ser Gly Gly Asp Ser Ala Leu Ser Gly 705 710 715 720 Glu Leu Ser Ala Ser Leu Pro Gly Leu Leu Ser Asp Lys Arg Asp Leu 725 730 735 Pro Ala Ser Gly Lys Ser Arg Ser Asp Cys Val Thr Arg Arg Asn Cys 740 745 750 Gly Arg Ser Lys Pro Ser Ser Lys Leu Arg Asp Ala Phe Ser Ala Gln 755 760 765 Met Val Lys Asn Thr Val Asn Arg Lys Ala Leu Lys Thr Glu Arg Lys 770 775 780 Arg Lys Leu Asn Gln Leu Pro Ser Val Thr Leu Asp Ala Val Leu Gln 785 790 795 800 Gly Asp Arg Glu Arg Gly Gly Ser Leu Arg Gly Gly Ala Glu Asp Pro 805 810 815 Ser Lys Glu Asp Pro Leu Gln Ile Met Gly His Leu Thr Ser Glu Asp 820 825 830 Gly Asp His Phe Ser Asp Val His Phe Asp Ser Lys Val Lys Gln Ser 835 840 845 Asp Pro Gly Lys Ile Ser Glu Lys Gly Leu Ser Phe Glu Asn Gly Lys 850 855 860 Gly Pro Glu Leu Asp Ser Val Met Asn Ser Glu Asn Asp Glu Leu Asn 865 870 875 880 Gly Val Asn Gln Val Val Pro Lys Lys Arg Trp Gln Arg Leu Asn Gln 885 890 895 Arg Arg Thr Lys Pro Arg Lys Arg Met Asn Arg Phe Lys Glu Lys Glu 900 905 910 Asn Ser Glu Cys Ala Phe Arg Val Leu Leu Pro Ser Asp Pro Val Gln 915 920 925 Glu Gly Arg Asp Glu Phe Pro Glu His Arg Thr Pro Ser Ala Ser Ile 930 935 940 Leu Glu Glu Pro Leu Thr Glu Gln Asn His Ala Asp Cys Leu Asp Ser 945 950 955 960 Ala Gly Pro Arg Leu Asn Val Cys Asp Lys Ser Ser Ala Ser Ile Gly 965 970 975 Asp Met Glu Lys Glu Pro Gly Ile Pro Ser Leu Thr Pro Gln Ala Glu 980 985 990 Leu Pro Glu Pro Ala Val Arg Ser Glu Lys Lys Arg Leu Arg Lys Pro 995 1000 1005 Ser Lys Trp Leu Leu Glu Tyr Thr Glu Glu Tyr Asp Gln Ile Phe 1010 1015 1020 Ala Pro Light Light Light Gln Light Light Val Glu Glu Glu Val His Light 1025 1030 1035 Val Ser Ser Arg Cys Glu Glu Glu Ser Leu Leu Ala Arg Gly Arg 1040 1045 1050 Ser Ser Ala Gln Asn Lys Gln Val Asp Glu Asn Ser Leu Ile Ser 1055 1060 1065 Thr Lys Glu Glu Pro Pro Val Leu Glu Arg Glu Ala Pro Phe Leu 1070 1075 1080 Glu Gly Pro Leu Ala Gln Ser Glu Leu Gly Gly Gly His Ala Glu 1085 1090 1095 Leu Pro Gln Leu Thr Leu Ser Val Pro Val Ala Pro Glu Val Ser 1100 1105 1110 Pro Arg Pro Ala Leu Glu Ser Glu Glu Leu Leu Val Lys Thr Pro 1115 1120 1125 Gly Asn Tyr Glu Ser Lys Arg Gln Arg Lys Pro Thr Lys Lys Leu 1130 1135 1140 Leu Glu Ser Asn Asp Leu Asp Pro Gly Phe Met Pro Lys Lys Gly 1145 1150 1155 Asp Leu Gly Leu Ser Lys Lys Cys Tyr Glu Ala Gly His Leu Glu 1160 1165 1170 Asn Gly Ile Thr Glu Ser Cys Ala Thr Ser Tyr Ser Lys Asp Phe 1175 1180 1185 Gly Gly Gly Thr Thr Lys Ile Phe Asp Lys Pro Arg Lys Arg Lys 1190 1195 1200 Arg Gln Arg His Ala Ala Ala Lys Met Gln Cys Lys Lys Val Lys 1205 1210 1215 Asn Asp Asp Ser Ser Lys Glu Ile Pro Gly Ser Glu Gly Glu Leu 1220 1225 1230 Met Pro His Arg Thr Ala Thr Ser Pro Lys Glu Thr Val Glu Glu 1235 1240 1245 Gly Val Glu His Asp Pro Gly Met Pro Ala Ser Lys Lys Met Gln 1250 1255 1260 Gly Glu Arg Gly Gly Gly Ala Ala Leu Lys Glu Asn Val Cys Gln 1265 1270 1275 Asn Cys Glu Lys Leu Gly Glu Leu Leu Leu Cys Glu Ala Gln Cys 1280 1285 1290 Cys Gly Ala Phe His Leu Glu Cys Leu Gly Leu Thr Glu Met Pro 1295 1300 1305 Arg Gly Lys Phe Ile Cys Asn Glu Cys Arg Thr Gly Ile His Thr 1310 1315 1320 Cys Phe Val Cys Lys Gln Ser Gly Glu Asp Val Lys Arg Cys Leu 1325 1330 1335 Leu Pro Leu Cys Gly Lys Phe Tyr His Glu Glu Cys Val Gln Lys 1340 1345 1350 Tyr Pro Pro Thr Val Met Gln Asn Lys Gly Phe Arg Cys Ser Leu 1355 1360 1365 His Ile Cys Ile Thr Cys His Ala Ala Asn Pro Ala Asn Val Ser 1370 1375 1380 Ala Ser Lys Gly Arg Leu Met Arg Cys Val Arg Cys Pro Val Ala 1385 1390 1395 Tyr His Ala Asn Asp Phe Cys Leu Ala Ala Gly Ser Lys Ile Leu 1400 1405 1410 Ala Ser Asn Ser Ile Ile Cys Pro Asn His Phe Thr Pro Arg Arg 1415 1420 1425 Gly Cys Arg Asn His Glu His Val Asn Val Ser Trp Cys Phe Val 1430 1435 1440 Cys Ser Glu Gly Gly Ser Leu Leu Cys Cys Asp Ser Cys Pro Ala 1445 1450 1455 Ala Phe His Arg Glu Cys Leu Asn Ile Asp Ile Pro Glu Gly Asn 1460 1465 1470 Trp Tyr Cys Asn Asp Cys Lys Ala Gly Lys Lys Pro His Tyr Arg 1475 1480 1485 Glu Ile Val Trp Val Lys Val Gly Arg Tyr Arg Trp Trp Pro Ala 1490 1495 1500 Glu Ile Cys His Pro Arg Ala Val Pro Ser Asn Ile Asp Lys Met 1505 1510 1515 Arg His Asp Val Gly Glu Phe Pro Val Leu Phe Phe Gly Ser Asn 1520 1525 1530 Asp Tyr Leu Trp Thr His Gln Ala Arg Val Phe Pro Tyr Met Glu 1535 1540 1545 Gly Asp Val Ser Ser Lys Asp Lys Met Gly Lys Gly Val Asp Gly 1550 1555 1560 Thr Tyr Lys Lys Ala Leu Gln Glu Ala Ala Ala Arg Phe Glu Glu 1565 1570 1575 Leu Lys Ala Gln Lys Glu Leu Arg Gln Leu Gln Glu Asp Arg Lys 1580 1585 1590 Asn Asp Lys Lys Pro Pro Pro Tyr Lys His Ile Lys Val Asn Arg 1595 1600 1605 Pro Ile Gly Arg Val Gln Ile Phe Thr Ala Asp Leu Ser Glu Ile 1610 1615 1620 Pro Arg Cys Asn Cys Lys Ala Thr Asp Glu Asn Pro Cys Gly Ile 1625 1630 1635 Asp Ser Glu Cys Ile Asn Arg Met Leu Leu Tyr Glu Cys His Pro 1640 1645 1650 Thr Val Cys Pro Ala Gly Gly Arg Cys Gln Asn Gln Cys Phe Ser 1655 1660 1665 Lys Arg Gln Tyr Pro Glu Val Glu Ile Phe Arg Thr Leu Gln Arg 1670 1675 1680 Gly Trp Gly Leu Arg Thr Lys Thr Asp Ile Lys Lys Gly Glu Phe 1685 1690 1695 Val Asn Glu Tyr Val Gly Glu Leu Ile Asp Glu Glu Glu Cys Arg 1700 1705 1710 Ala Arg Ile Arg Tyr Ala Gln Glu His Asp Ile Thr Asn Phe Tyr 1715 1720 1725 Met Leu Thr Leu Asp Lys Asp Arg Ile Ile Asp Ala Gly Pro Lys 1730 1735 1740 Gly Asn Tyr Ala Arg Phe Met Asn His Cys Cys Gln Pro Asn Cys 1745 1750 1755 Glu Thr Gln Lys Trp Ser Val Asn Gly Asp Thr Arg Val Gly Leu 1760 1765 1770 Phe Ala Leu Ser Asp Ile Lys Ala Gly Thr Glu Leu Thr Phe Asn 1775 1780 1785 Tyr Asn Leu Glu Cys Leu Gly Asn Gly Lys Thr Val Cys Lys Cys 1790 1795 1800 Gly Ala Pro Asn Cys Ser Gly Phe Leu Gly Val Arg Pro Lys Asn 1805 1810 1815 Gln Pro Ile Ala Thr Glu Glu Lys Ser Lys Lys Phe Lys Lys Lys 1820 1825 1830 Gln Gln Gly Lys Arg Arg Thr Gln Gly Glu Ile Thr Lys Glu Arg 1835 1840 1845 Glu Asp Glu Cys Phe Ser Cys Gly Asp Ala Gly Gln Leu Val Ser 1850 1855 1860 Cys Lys Lys Pro Gly Cys Pro Lys Val Tyr His Ala Asp Cys Leu 1865 1870 1875 Asn Leu Thr Lys Arg Pro Ala Gly Lys Trp Glu Cys Pro Trp His 1880 1885 1890 Gln Cys Asp Ile Cys Gly Lys Glu Ala Ala Ser Phe Cys Glu Met 1895 1900 1905 Cys Pro Ser Ser Phe Cys Lys Gln His Arg Glu Gly Met Leu Phe 1910 1915 1920 Ile Ser Lys Leu Asp Gly Arg Leu Ser Cys Thr Glu His Asp Pro 1925 1930 1935 Cys Gly Pro Asn Pro Leu Glu Pro Gly Glu Ile Arg Glu Tyr Val 1940 1945 1950 Pro Pro Pro Val Pro Leu Pro Pro Gly Pro Ser Thr His Leu Ala 1955 1960 1965 Glu Gln Ser Thr Gly Met Ala Ala Gln Ala Pro Lys Met Ser Asp 1970 1975 1980 Lys Pro Pro Ala Asp Thr Asn Gln Met Leu Ser Leu Ser Lys Lys 1985 1990 1995 Ala Leu Ala Gly Thr Cys Gln Arg Pro Leu Leu Pro Glu Arg Pro 2000 2005 2010 Leu Glu Arg Thr Asp Ser Arg Pro Gln Pro Leu Asp Lys Val Arg 2015 2020 2025 Asp Leu Ala Gly Ser Gly Thr Lys Ser Gln Ser Leu Val Ser Ser 2030 2035 2040 Gln Arg Pro Leu Asp Arg Pro Pro Ala Val Ala Gly Pro Arg Pro 2045 2050 2055 Gln Leu Ser Asp Lys Pro Ser Pro Val Thr Ser Pro Ser Ser Ser 2060 2065 2070 Pro Ser Val Arg Ser Gln Pro Leu Glu Arg Pro Leu Gly Thr Ala 2075 2080 2085 Asp Pro Arg Leu Asp Lys Ser Ile Gly Ala Ala Ser Pro Arg Pro 2090 2095 2100 Gln Ser Leu Glu Lys Thr Ser Val Pro Thr Gly Leu Arg Leu Pro 2105 2110 2115 Pro Pro Asp Arg Leu Leu Ile Thr Ser Ser Pro Lys Pro Gln Thr 2120 2125 2130 Ser Asp Arg Pro Thr Asp Lys Pro His Ala Ser Leu Ser Gln Arg 2135 2140 2145 Leu Pro Pro Pro Glu Lys Val Leu Ser Ala Val Val Gln Thr Leu 2150 2155 2160 Val Ala Lys Glu Lys Ala Leu Arg Pro Val Asp Gln Asn Thr Gln 2165 2170 2175 Ser Lys Asn Arg Ala Ala Leu Val Met Asp Leu Ile Asp Leu Thr 2180 2185 2190 Pro Arg Gln Lys Glu Arg Ala Ala Ser Pro His Gln Val Thr Pro 2195 2200 2205 Gln Ala Asp Glu Lys Met Pro Val Leu Glu Ser Ser Ser Trp Pro 2210 2215 2220 Ala Ser Lys Gly Leu Gly His Met Pro Arg Ala Val Glu Lys Gly 2225 2230 2235 Cys Val Ser Asp Pro Leu Gln Thr Ser Gly Lys Ala Ala Ala Pro 2240 2245 2250 Ser Glu Asp Pro Trp Gln Ala Val Lys Ser Leu Thr Gln Ala Arg 2255 2260 2265 Leu Leu Ser Gln Pro Pro Ala Lys Ala Phe Leu Tyr Glu Pro Thr 2270 2275 2280 Thr Gln Ala Ser Gly Arg Ala Ser Ala Gly Ala Glu Gln Thr Pro 2285 2290 2295 Gly Pro Leu Ser Gln Ser Pro Gly Leu Val Lys Gln Ala Lys Gln 2300 2305 2310 Met Val Gly Gly Gln Gln Leu Pro Ala Leu Ala Ala Lys Ser Gly 2315 2320 2325 Gln Ser Phe Arg Ser Leu Gly Lys Ala Pro Ala Ser Leu Pro Thr 2330 2335 2340 Glu Glu Lys Lys Leu Val Thr Thr Glu Gln Ser Pro Trp Ala Leu 2345 2350 2355 Gly Lys Ala Ser Ser Arg Ala Gly Leu Trp Pro Ile Val Ala Gly 2360 2365 2370 Gln Thr Leu Ala Gln Ser Cys Trp Ser Ala Gly Ser Thr Gln Thr 2375 2380 2385 Leu Ala Gln Thr Cys Trp Ser Leu Gly Arg Gly Gln Asp Pro Lys 2390 2395 2400 Pro Glu Gln Asn Thr Leu Pro Ala Leu Asn Gln Ala Pro Ser Ser 2405 2410 2415 His Lys Cys Ala Glu Ser Glu Gln Lys 2420 2425 <210> 7 <211> 403 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 7 Met Met Met Met Ala Leu Ser Lys Thr Phe Gly Gln Lys Pro Val Lys 1 5 10 15 Phe Gln Leu Glu Asp Asp Gly Glu Phe Tyr Met Ile Gly Ser Glu Val 20 25 30 Gly Asn Tyr Leu Arg Met Phe Arg Gly Ser Leu Tyr Lys Arg Tyr Pro 35 40 45 Ser Leu Trp Arg Arg Leu Ala Thr Val Glu Glu Arg Lys Lys Ile Val 50 55 60 Ala Ser Ser His Gly Lys Lys Thr Lys Pro Asn Thr Lys Asp His Gly 65 70 75 80 Tyr Thr Thr Leu Ala Thr Ser Val Thr Leu Leu Lys Ala Ser Glu Val 85 90 95 Glu Glu Ile Leu Asp Gly Asn Asp Glu Lys Tyr Lys Ala Val Ser Ile 100 105 110 Ser Thr Glu Pro Pro Thr Tyr Leu Arg Glu Gln Lys Ala Lys Arg Asn 115 120 125 Ser Gln Trp Val Pro Thr Leu Pro Asn Ser Ser His His Leu Asp Ala 130 135 140 Val Pro Cys Ser Thr Thr Ile Asn Arg Asn Arg Met Gly Arg Asp Lys 145 150 155 160 Lys Arg Thr Phe Pro Leu Trp Cys Gly Cys Ile Ala Ala Leu Thr Leu 165 170 175 Arg Ala Asp Ser Ala Leu Val Leu His Phe Asp Asp His Asp Pro Ala 180 185 190 Val Ile His Glu Asn Ala Ser Gln Pro Glu Val Leu Val Pro Ile Arg 195 200 205 Leu Asp Met Glu Ile Asp Gly Gln Lys Leu Arg Asp Ala Phe Thr Trp 210 215 220 Asn Met Asn Glu Lys Leu Met Thr Pro Glu Met Phe Ser Glu Ile Leu 225 230 235 240 Cys Asp Asp Leu Asp Leu Asn Pro Leu Thr Phe Val Pro Ala Ile Ala 245 250 255 Ser Ala Ile Arg Gln Gln Ile Glu Ser Tyr Pro Thr Asp Ser Ile Leu 260 265 270 Glu Asp Gln Ser Asp Gln Arg Val Ile Ile Lys Leu Asn Ile His Val 275 280 285 Gly Asn Ile Ser Leu Val Asp Gln Phe Glu Trp Asp Met Ser Glu Lys 290 295 300 Glu Asn Ser Pro Glu Lys Phe Ala Leu Lys Leu Cys Ser Glu Leu Gly 305 310 315 320 Leu Gly Gly Glu Phe Val Thr Thr Ile Ala Tyr Ser Ile Arg Gly Gln 325 330 335 Leu Ser Trp His Gln Lys Thr Tyr Ala Phe Ser Glu Asn Pro Leu Pro 340 345 350 Thr Val Glu Ile Ala Ile Arg Asn Thr Gly Asp Ala Asp Gln Trp Cys 355 360 365 Pro Leu Leu Glu Thr Leu Thr Asp Ala Glu Met Glu Lys Lys Ile Arg 370 375 380 Asp Gln Asp Arg Asn Thr Arg Arg Met Arg Arg Leu Ala Asn Thr Ala 385 390 395 400 Pro Ala Trp <210> 8 <211> 355 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 8 Met Ala Leu Ser Glu Pro Ile Leu Pro Ser Phe Ser Thr Phe Ala Ser 1 5 10 15 Pro Cys Arg Glu Arg Gly Leu Gln Glu Arg Trp Pro Arg Ala Glu Pro 20 25 30 Glu Ser Gly Gly Thr Asp Asp Asp Leu Asn Ser Val Leu Asp Phe Ile 35 40 45 Leu Ser Met Gly Leu Asp Gly Leu Gly Ala Glu Ala Ala Pro Glu Pro 50 55 60 Pro Pro Pro Pro Pro Pro Pro Ala Phe Tyr Tyr Pro Glu Pro Gly Ala 65 70 75 80 Pro Pro Pro Tyr Ser Ala Pro Ala Gly Gly Leu Val Ser Glu Leu Leu 85 90 95 Arg Pro Glu Leu Asp Ala Pro Leu Gly Pro Ala Leu His Gly Arg Phe 100 105 110 Leu Leu Ala Pro Pro Gly Arg Leu Val Lys Ala Glu Pro Pro Glu Ala 115 120 125 Asp Gly Gly Gly Gly Tyr Gly Cys Ala Pro Gly Leu Thr Arg Gly Pro 130 135 140 Arg Gly Leu Lys Arg Glu Gly Ala Pro Gly Pro Ala Ala Ser Cys Met 145 150 155 160 Arg Gly Pro Gly Gly Arg Pro Pro Pro Pro Pro Asp Thr Pro Pro Leu 165 170 175 Ser Pro Asp Gly Pro Ala Arg Leu Pro Ala Pro Gly Pro Arg Ala Ser 180 185 190 Phe Pro Pro Pro Phe Gly Gly Pro Gly Phe Gly Ala Pro Gly Pro Gly 195 200 205 Leu His Tyr Ala Pro Pro Ala Pro Pro Ala Phe Gly Leu Phe Asp Asp 210 215 220 Ala Ala Ala Ala Ala Ala Ala Leu Gly Leu Ala Pro Pro Ala Ala Arg 225 230 235 240 Gly Leu Leu Thr Pro Pro Ala Ser Pro Leu Glu Leu Leu Glu Ala Lys 245 250 255 Pro Lys Arg Gly Arg Arg Ser Trp Pro Arg Lys Arg Thr Ala Thr His 260 265 270 Thr Cys Ser Tyr Ala Gly Cys Gly Lys Thr Tyr Thr Lys Ser Ser His 275 280 285 Leu Lys Ala His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr His Cys 290 295 300 Asn Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp Glu Leu Thr 305 310 315 320 Arg His Tyr Arg Lys His Thr Gly His Arg Pro Phe Gln Cys His Leu 325 330 335 Cys Asp Arg Ala Phe Ser Arg Ser Asp His Leu Ala Leu His Met Lys 340 345 350 Arg His Met 355 <210> 9 <211> 725 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 9 Met Glu Gly Asp Ala Val Glu Ala Ile Val Glu Glu Ser Glu Thr Phe 1 5 10 15 Ile Lys Gly Lys Glu Arg Lys Thr Tyr Gln Arg Arg Arg Glu Gly Gly 20 25 30 Gln Glu Glu Asp Ala Cys His Leu Pro Gln Asn Gln Thr Asp Gly Gly 35 40 45 Glu Val Val Gln Asp Val Asn Ser Ser Val Gln Met Val Met Met Glu 50 55 60 Gln Leu Asp Pro Thr Leu Leu Gln Met Lys Thr Glu Val Met Glu Gly 65 70 75 80 Thr Val Ala Pro Glu Ala Glu Ala Ala Val Asp Asp Thr Gln Ile Ile 85 90 95 Thr Leu Gln Val Val Asn Met Glu Glu Gln Pro Ile Asn Ile Gly Glu 100 105 110 Leu Gln Leu Val Gln Val Pro Val Pro Val Thr Val Pro Val Ala Thr 115 120 125 Thr Ser Val Glu Glu Leu Gln Gly Ala Tyr Glu Asn Glu Val Ser Lys 130 135 140 Glu Gly Leu Ala Glu Ser Glu Pro Met Ile Cys His Thr Leu Pro Leu 145 150 155 160 Pro Glu Gly Phe Gln Val Val Lys Val Gly Ala Asn Gly Glu Val Glu 165 170 175 Thr Leu Glu Gln Gly Glu Leu Pro Pro Gln Glu Asp Pro Ser Trp Gln 180 185 190 Lys Asp Pro Asp Tyr Gln Pro Pro Ala Lys Lys Thr Lys Lys Thr Lys 195 200 205 Lys Ser Lys Leu Arg Tyr Thr Glu Glu Gly Lys Asp Val Asp Val Ser 210 215 220 Val Tyr Asp Phe Glu Glu Glu Gln Gln Glu Gly Leu Leu Ser Glu Val 225 230 235 240 Asn Ala Glu Lys Val Val Gly Asn Met Lys Pro Pro Lys Pro Thr Lys 245 250 255 Ile Lys Lys Lys Gly Val Lys Lys Thr Phe Gln Cys Glu Leu Cys Ser 260 265 270 Tyr Thr Cys Pro Arg Arg Ser Asn Leu Asp Arg His Met Lys Ser His 275 280 285 Thr Asp Glu Arg Pro His Lys Cys His Leu Cys Gly Arg Ala Phe Arg 290 295 300 Thr Val Thr Leu Leu Arg Asn His Leu Asn Thr His Thr Gly Thr Arg 305 310 315 320 Pro His Lys Cys Pro Asp Cys Asp Met Ala Phe Val Thr Ser Gly Glu 325 330 335 Leu Val Arg His Arg Arg Tyr Lys His Thr His Glu Lys Pro Phe Lys 340 345 350 Cys Ser Met Cys Asp Tyr Ala Ser Val Glu Val Ser Lys Leu Lys Arg 355 360 365 His Ile Arg Ser His Thr Gly Glu Arg Pro Phe Gln Cys Ser Leu Cys 370 375 380 Ser Tyr Ala Ser Arg Asp Thr Tyr Lys Leu Lys Arg His Met Arg Thr 385 390 395 400 His Ser Gly Glu Lys Pro Tyr Glu Cys Tyr Ile Cys His Ala Arg Phe 405 410 415 Thr Gln Ser Gly Thr Met Lys Met His Ile Leu Gln Lys His Thr Glu 420 425 430 Asn Val Ala Lys Phe His Cys Pro His Cys Asp Thr Val Ile Ala Arg 435 440 445 Lys Ser Asp Leu Gly Val His Leu Arg Lys Gln His Ser Tyr Ile Glu 450 455 460 Gln Gly Lys Lys Cys Arg Tyr Cys Asp Ala Val Phe His Glu Arg Tyr 465 470 475 480 Ala Leu Ile Gln His Gln Lys Ser His Lys Asn Glu Lys Arg Phe Lys 485 490 495 Cys Asp Gln Cys Asp Tyr Ala Cys Arg Gln Glu Arg His Met Ile Met 500 505 510 His Lys Arg Thr His Thr Gly Glu Lys Pro Tyr Ala Cys Ser His Cys 515 520 525 Asp Lys Thr Phe Arg Gln Lys Gln Leu Leu Asp Met His Phe Lys Arg 530 535 540 Tyr His Asp Pro Asn Phe Val Pro Ala Ala Phe Val Cys Ser Lys Cys 545 550 555 560 Gly Lys Thr Phe Thr Arg Arg Asn Thr Met Ala Arg His Ala Asp Asn 565 570 575 Cys Ala Gly Pro Asp Gly Val Glu Gly Glu Asn Gly Gly Glu Thr Lys 580 585 590 Lys Ser Lys Arg Gly Arg Lys Arg Lys Met Arg Ser Lys Lys Glu Asp 595 600 605 Ser Ser Asp Ser Glu Asn Ala Glu Pro Asp Leu Asp Asp Asn Glu Asp 610 615 620 Glu Glu Glu Pro Ala Val Glu Ile Glu Pro Glu Pro Glu Pro Gln Pro 625 630 635 640 Val Thr Pro Ala Pro Pro Pro Ala Lys Lys Arg Arg Gly Arg Pro Pro 645 650 655 Gly Arg Thr Asn Gln Pro Lys Gln Asn Gln Pro Ile Ile Gln Val Glu 660 665 670 Asp Gln Asn Thr Gly Ala Ile Glu Asn Ile Ile Val Glu Val Lys Lys 675 680 685 Glu Pro Asp Ala Glu Pro Ala Glu Gly Glu Glu Glu Glu Ala Gln Pro 690 695 700 Ala Ala Thr Asp Ala Pro Asn Gly Asp Leu Thr Pro Glu Met Ile Leu 705 710 715 720 Ser Met Met Asp Arg 725 <210> 10 <211> 763 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 10 Met Asp His Leu Asn Glu Ala Thr Gln Gly Lys Glu His Ser Glu Met 1 5 10 15 Ser Asn Asn Val Ser Asp Pro Lys Gly Pro Pro Ala Lys Ile Ala Arg 20 25 30 Leu Glu Gln Asn Gly Ser Pro Leu Gly Arg Gly Arg Leu Gly Ser Thr 35 40 45 Gly Ala Lys Met Gln Gly Val Pro Leu Lys His Ser Gly His Leu Met 50 55 60 Lys Thr Asn Leu Arg Lys Gly Thr Met Leu Pro Val Phe Cys Val Val 65 70 75 80 Glu His Tyr Glu Asn Ala Ile Glu Tyr Asp Cys Lys Glu Glu His Ala 85 90 95 Glu Phe Val Leu Val Arg Lys Asp Met Leu Phe Asn Gln Leu Ile Glu 100 105 110 Met Ala Leu Leu Ser Leu Gly Tyr Ser His Ser Ser Ala Ala Gln Ala 115 120 125 Lys Gly Leu Ile Gln Val Gly Lys Trp Asn Pro Val Pro Leu Ser Tyr 130 135 140 Val Thr Asp Ala Pro Asp Ala Thr Val Ala Asp Met Leu Gln Asp Val 145 150 155 160 Tyr His Val Val Thr Leu Lys Ile Gln Leu His Ser Cys Pro Lys Leu 165 170 175 Glu Asp Leu Pro Pro Glu Gln Trp Ser His Thr Thr Val Arg Asn Ala 180 185 190 Leu Lys Asp Leu Leu Lys Asp Met Asn Gln Ser Ser Leu Ala Lys Glu 195 200 205 Cys Pro Leu Ser Gln Ser Met Ile Ser Ser Ile Val Asn Ser Thr Tyr 210 215 220 Tyr Ala Asn Val Ser Ala Ala Lys Cys Gln Glu Phe Gly Arg Trp Tyr 225 230 235 240 Lys His Phe Lys Lys Thr Lys Asp Met Met Val Glu Met Asp Ser Leu 245 250 255 Ser Glu Leu Ser Gln Gln Gly Ala Asn His Val Asn Phe Gly Gln Gln 260 265 270 Pro Val Pro Gly Asn Thr Ala Glu Gln Pro Pro Ser Pro Ala Gln Leu 275 280 285 Ser His Gly Ser Gln Pro Ser Val Arg Thr Pro Leu Pro Asn Leu His 290 295 300 Pro Gly Leu Val Ser Thr Pro Ile Ser Pro Gln Leu Val Asn Gln Gln 305 310 315 320 Leu Val Met Ala Gln Leu Leu Asn Gln Gln Tyr Ala Val Asn Arg Leu 325 330 335 Leu Ala Gln Gln Ser Leu Asn Gln Gln Tyr Leu Asn His Pro Pro Pro 340 345 350 Val Ser Arg Ser Met Asn Lys Pro Leu Glu Gln Gln Val Ser Thr Asn 355 360 365 Thr Glu Val Ser Ser Glu Ile Tyr Gln Trp Val Arg Asp Glu Leu Lys 370 375 380 Arg Ala Gly Ile Ser Gln Ala Val Phe Ala Arg Val Ala Phe Asn Arg 385 390 395 400 Thr Gln Gly Leu Leu Ser Glu Ile Leu Arg Lys Glu Glu Asp Pro Lys 405 410 415 Thr Ala Ser Gln Ser Leu Leu Val Asn Leu Arg Ala Met Gln Asn Phe 420 425 430 Leu Gln Leu Pro Glu Ala Glu Arg Asp Arg Ile Tyr Gln Asp Glu Arg 435 440 445 Glu Arg Ser Leu Asn Ala Ala Ser Ala Met Gly Pro Ala Pro Leu Ile 450 455 460 Ser Thr Pro Pro Ser Arg Pro Pro Gln Val Lys Thr Ala Thr Ile Ala 465 470 475 480 Thr Glu Arg Asn Gly Lys Pro Glu Asn Asn Thr Met Asn Ile Asn Ala 485 490 495 Ser Ile Tyr Asp Glu Ile Gln Gln Glu Met Lys Arg Ala Lys Val Ser 500 505 510 Gln Ala Leu Phe Ala Lys Val Ala Ala Thr Lys Ser Gln Gly Trp Leu 515 520 525 Cys Glu Leu Leu Arg Trp Lys Glu Asp Pro Ser Pro Glu Asn Arg Thr 530 535 540 Leu Trp Glu Asn Leu Ser Met Ile Arg Arg Phe Leu Ser Leu Pro Gln 545 550 555 560 Pro Glu Arg Asp Ala Ile Tyr Glu Gln Glu Ser Asn Ala Val His His 565 570 575 His Gly Asp Arg Pro Pro His Ile Ile His Val Pro Ala Glu Gln Ile 580 585 590 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala 595 600 605 Pro Pro Pro Pro Gln Pro Gln Gln Gln Pro Gln Thr Gly Pro Arg Leu 610 615 620 Pro Pro Arg Gln Pro Thr Val Ala Ser Pro Ala Glu Ser Asp Glu Glu 625 630 635 640 Asn Arg Gln Lys Thr Arg Pro Arg Thr Lys Ile Ser Val Glu Ala Leu 645 650 655 Gly Ile Leu Gln Ser Phe Ile Gln Asp Val Gly Leu Tyr Pro Asp Glu 660 665 670 Glu Ala Ile Gln Thr Leu Ser Ala Gln Leu Asp Leu Pro Lys Tyr Thr 675 680 685 Ile Ile Lys Phe Phe Gln Asn Gln Arg Tyr Tyr Leu Lys His His Gly 690 695 700 Lys Leu Lys Asp Asn Ser Gly Leu Glu Val Asp Val Ala Glu Tyr Lys 705 710 715 720 Glu Glu Glu Leu Leu Lys Asp Leu Glu Glu Ser Val Gln Asp Lys Asn 725 730 735 Thr Asn Thr Leu Phe Ser Val Lys Leu Glu Glu Glu Leu Ser Val Glu 740 745 750 Gly Asn Thr Asp Ile Asn Thr Asp Leu Lys Asp 755 760 <210> 11 <211> 396 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 11 Met Pro Asn Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu 1 5 10 15 Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala 20 25 30 Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg 35 40 45 Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met 50 55 60 Pro Pro Ser Gln Leu Gln Leu Ser Thr Val Asp Ala His Ala Arg Thr 65 70 75 80 Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met Ile Ser Leu 85 90 95 Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys Ala Arg Pro 100 105 110 Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp Val Ser Arg 115 120 125 Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala Pro Arg Lys 130 135 140 Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro Leu Leu Ala 145 150 155 160 Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe Glu Glu Pro 165 170 175 Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu Asp Glu Lys 180 185 190 Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln Ser Leu Glu 195 200 205 Gln Gln Leu Val Leu Glu Lys Glu Lys Leu Ser Ala Met Gln Ala His 210 215 220 Leu Ala Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val Ala Ser Ser 225 230 235 240 Asp Lys Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln Gly Pro Val 245 250 255 Val Pro Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser Leu Phe Ala 260 265 270 Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Thr Phe Pro Glu 275 280 285 Phe Leu His Asn Met Asp Tyr Phe Lys Phe His Asn Met Arg Pro Pro 290 295 300 Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu Ala Pro Glu 305 310 315 320 Lys Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr Arg Met Phe 325 330 335 Ala Phe Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala Ile Arg His 340 345 350 Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser Glu Lys Gly 355 360 365 Ala Val Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys Arg Ser Gln 370 375 380 Arg Pro Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro 385 390 395 <210> 12 <211> 525 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 12 Met Val Ser Arg Pro Glu Pro Glu Gly Glu Ala Met Asp Ala Glu Leu 1 5 10 15 Ala Val Ala Pro Pro Gly Cys Ser His Leu Gly Ser Phe Lys Val Asp 20 25 30 Asn Trp Lys Gln Asn Leu Arg Ala Ile Tyr Gln Cys Phe Val Trp Ser 35 40 45 Gly Thr Ala Glu Ala Arg Lys Arg Lys Ala Lys Ser Cys Ile Cys His 50 55 60 Val Cys Gly Val His Leu Asn Arg Leu His Ser Cys Leu Tyr Cys Val 65 70 75 80 Phe Phe Gly Cys Phe Thr Lys Lys His Ile His Glu His Ala Lys Ala 85 90 95 Lys Arg His Asn Leu Ala Ile Asp Leu Met Tyr Gly Gly Ile Tyr Cys 100 105 110 Phe Leu Cys Gln Asp Tyr Ile Tyr Asp Lys Asp Met Glu Ile Ile Ala 115 120 125 Lys Glu Glu Gln Arg Lys Ala Trp Lys Met Gln Gly Val Gly Glu Lys 130 135 140 Phe Ser Thr Trp Glu Pro Thr Lys Arg Glu Leu Glu Leu Leu Lys His 145 150 155 160 Asn Pro Lys Arg Arg Lys Ile Thr Ser Asn Cys Thr Ile Gly Leu Arg 165 170 175 Gly Leu Ile Asn Leu Gly Asn Thr Cys Phe Met Asn Cys Ile Val Gln 180 185 190 Ala Leu Thr His Thr Pro Leu Leu Arg Asp Phe Phe Leu Ser Asp Arg 195 200 205 His Arg Cys Glu Met Gln Ser Pro Ser Ser Cys Leu Val Cys Glu Met 210 215 220 Ser Ser Leu Phe Gln Glu Phe Tyr Ser Gly His Arg Ser Pro His Ile 225 230 235 240 Pro Tyr Lys Leu Leu His Leu Val Trp Thr His Ala Arg His Leu Ala 245 250 255 Gly Tyr Glu Gln Gln Asp Ala His Glu Phe Leu Ile Ala Ala Leu Asp 260 265 270 Val Leu His Arg His Cys Lys Gly Asp Asp Asn Gly Lys Lys Ala Asn 275 280 285 Asn Pro Asn His Cys Asn Cys Ile Ile Asp Gln Ile Phe Thr Gly Gly 290 295 300 Leu Gln Ser Asp Val Thr Cys Gln Val Cys His Gly Val Ser Thr Thr 305 310 315 320 Ile Asp Pro Phe Trp Asp Ile Ser Leu Asp Leu Pro Gly Ser Ser Thr 325 330 335 Pro Phe Trp Pro Leu Ser Pro Gly Ser Glu Gly Asn Val Val Asn Gly 340 345 350 Glu Ser His Val Ser Gly Thr Thr Thr Leu Thr Asp Cys Leu Arg Arg 355 360 365 Phe Thr Arg Pro Glu His Leu Gly Ser Ser Ala Lys Ile Lys Cys Ser 370 375 380 Gly Cys His Ser Tyr Gln Glu Ser Thr Lys Gln Leu Thr Met Lys Lys 385 390 395 400 Leu Pro Ile Val Ala Cys Phe His Leu Lys Arg Phe Glu His Ser Ala 405 410 415 Lys Leu Arg Arg Lys Ile Thr Thr Tyr Val Ser Phe Pro Leu Glu Leu 420 425 430 Asp Met Thr Pro Phe Met Ala Ser Ser Lys Glu Ser Arg Met Asn Gly 435 440 445 Gln Tyr Gln Gln Pro Thr Asp Ser Leu Asn Asn Asp Asn Lys Tyr Ser 450 455 460 Leu Phe Ala Val Val Asn His Gln Gly Thr Leu Glu Ser Gly His Tyr 465 470 475 480 Thr Ser Phe Ile Arg Gln His Lys Asp Gln Trp Phe Lys Cys Asp Asp 485 490 495 Ala Ile Ile Thr Lys Ala Ser Ile Lys Asp Val Leu Asp Ser Glu Gly 500 505 510 Tyr Leu Leu Phe Tyr His Lys Gln Phe Leu Glu Tyr Glu 515 520 525 <210> 13 <211> 187 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 13 Met Pro Arg Val Val Pro Asp Gln Arg Ser Lys Phe Glu Asn Glu Glu 1 5 10 15 Phe Phe Arg Lys Leu Ser Arg Glu Cys Glu Ile Lys Tyr Thr Gly Phe 20 25 30 Arg Asp Arg Pro His Glu Glu Arg Gln Ala Arg Phe Gln Asn Ala Cys 35 40 45 Arg Asp Gly Arg Ser Glu Ile Ala Phe Val Ala Thr Gly Thr Asn Leu 50 55 60 Ser Leu Gln Phe Phe Pro Ala Ser Trp Gln Gly Glu Gln Arg Gln Thr 65 70 75 80 Pro Ser Arg Glu Tyr Val Asp Leu Glu Arg Glu Ala Gly Lys Val Tyr 85 90 95 Leu Lys Ala Pro Met Ile Leu Asn Gly Val Cys Val Ile Trp Lys Gly 100 105 110 Trp Ile Asp Leu Gln Arg Leu Asp Gly Met Gly Cys Leu Glu Phe Asp 115 120 125 Glu Glu Arg Ala Gln Gln Glu Asp Ala Leu Ala Gln Gln Ala Phe Glu 130 135 140 Glu Ala Arg Arg Arg Thr Arg Glu Phe Glu Asp Arg Asp Arg Ser His 145 150 155 160 Arg Glu Glu Met Glu Ala Arg Arg Gln Gln Asp Pro Ser Pro Gly Ser 165 170 175 Asn Leu Gly Gly Gly Asp Asp Leu Lys Leu Arg 180 185 <210> 14 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 14 Met Arg Ile Pro Val Asp Ala Ser Thr Ser Arg Arg Phe Thr Pro Pro 1 5 10 15 Ser Thr Ala Leu Ser Pro Gly Lys Met Ser Glu Ala Leu Pro Leu Gly 20 25 30 Ala Pro Asp Ala Gly Ala Ala Leu Ala Gly Lys Leu Arg Ser Gly Asp 35 40 45 Arg Ser Met Val Glu Val Leu Ala Asp His Pro Gly Glu Leu Val Arg 50 55 60 Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu Pro Thr His Trp Arg 65 70 75 80 Cys Asn Lys Thr Leu Pro Ile Ala Phe Lys Val Val Ala Leu Gly Asp 85 90 95 Val Pro Asp Gly Thr Leu Val Thr Val Met Ala Gly Asn Asp Glu Asn 100 105 110 Tyr Ser Ala Glu Leu Arg Asn Ala Thr Ala Ala Met Lys Asn Gln Val 115 120 125 Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg Ser Gly Arg Gly Lys 130 135 140 Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn Pro Pro Gln Val Ala 145 150 155 160 Thr Tyr His Arg Ala Ile Lys Ile Thr Val Asp Gly Pro Arg Glu Pro 165 170 175 Arg Arg His Arg Gln Lys Leu Asp Asp Gln Thr Lys Pro Gly Ser Leu 180 185 190 Ser Phe Ser Glu Arg Leu Ser Glu Leu Glu Gln Leu Arg Arg Thr Ala 195 200 205 Met Arg Val Ser Pro His His Pro Ala Pro Thr Pro Asn Pro Arg Ala 210 215 220 Ser Leu Asn His Ser Thr Ala Phe Asn Pro Gln Pro Gln Ser Gln Met 225 230 235 240 Gln Asp Thr Arg Gln Ile Gln Pro Ser Pro Pro Trp Ser Tyr Asp Gln 245 250 255 Ser Tyr Gln Tyr Leu Gly Ser Ile Ala Ser Pro Ser Val His Pro Ala 260 265 270 Thr Pro Ile Ser Pro Gly Arg Ala Ser Gly Met Thr Thr Leu Ser Ala 275 280 285 Glu Leu Ser Ser Arg Leu Ser Thr Ala Pro Asp Leu Thr Ala Phe Ser 290 295 300 Asp Pro Arg Gln Phe Pro Ala Leu Pro Ser Ile Ser Asp Pro Arg Met 305 310 315 320 His Tyr Pro Gly Ala Phe Thr Tyr Ser Pro Thr Pro Val Thr Ser Gly 325 330 335 Ile Gly Ile Gly Met Ser Ala Met Gly Ser Ala Thr Arg Tyr His Thr 340 345 350 Tyr Leu Pro Pro Pro Tyr Pro Gly Ser Ser Gln Ala Gln Gly Gly Pro 355 360 365 Phe Gln Ala Ser Ser Pro Ser Tyr His Leu Tyr Tyr Gly Ala Ser Ala 370 375 380 Gly Ser Tyr Gln Phe Ser Met Val Gly Gly Glu Arg Ser Pro Pro Arg 385 390 395 400 Ile Leu Pro Pro Cys Thr Asn Ala Ser Thr Gly Ser Ala Leu Leu Asn 405 410 415 Pro Ser Leu Pro Asn Gln Ser Asp Val Val Glu Ala Glu Gly Ser His 420 425 430 Ser Asn Ser Pro Thr Asn Met Ala Pro Ser Ala Arg Leu Glu Glu Ala 435 440 445 Val Trp Arg Pro Tyr 450 <210> 15 <211> 453 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 15 Met Asp Phe Phe Arg Val Val Glu Asn Gln Pro Pro Ala Thr Met Pro 1 5 10 15 Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp Ser 20 25 30 Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr Gln Gln 35 40 45 Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp Ile Trp 50 55 60 Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser Arg Arg 65 70 75 80 Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe Ser Leu 85 90 95 Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala Asp Gln 100 105 110 Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn Gln Ser 115 120 125 Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile Ile 130 135 140 Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val Ser 145 150 155 160 Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser Pro 165 170 175 Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu Tyr Leu 180 185 190 Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser Val Val 195 200 205 Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys Ala Ser 210 215 220 Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu Ser Ser 225 230 235 240 Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His Glu 245 250 255 Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu Asp 260 265 270 Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro Gly 275 280 285 Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser Lys Pro 290 295 300 Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His Gln 305 310 315 320 His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala Ala 325 330 335 Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile Ser Asn 340 345 350 Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn Val 355 360 365 Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu Leu 370 375 380 Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu Asn 385 390 395 400 Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala Tyr 405 410 415 Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp 420 425 430 Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln Leu 435 440 445 Arg Asn Ser Cys Ala 450 <210> 16 <211> 395 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 16 Met Leu Asp Asp Asn Asn His Leu Ile Gln Cys Ile Met Asp Ser Gln 1 5 10 15 Asn Lys Gly Lys Thr Ser Glu Cys Ser Gln Tyr Gln Gln Met Leu His 20 25 30 Thr Asn Leu Val Tyr Leu Ala Thr Ile Ala Asp Ser Asn Gln Asn Met 35 40 45 Gln Ser Leu Leu Pro Ala Pro Pro Thr Gln Asn Met Pro Met Gly Pro 50 55 60 Gly Gly Met Asn Gln Ser Gly Pro Pro Pro Pro Pro Arg Ser His Asn 65 70 75 80 Met Pro Ser Asp Gly Met Val Gly Gly Gly Pro Pro Ala Pro His Met 85 90 95 Gln Asn Gln Met Asn Gly Gln Met Pro Gly Pro Asn His Met Pro Met 100 105 110 Gln Gly Pro Gly Pro Asn Gln Leu Asn Met Thr Asn Ser Ser Met Asn 115 120 125 Met Pro Ser Ser Ser His Gly Ser Met Gly Gly Tyr Asn His Ser Val 130 135 140 Pro Ser Ser Gln Ser Met Pro Val Gln Asn Gln Met Thr Met Ser Gln 145 150 155 160 Gly Gln Pro Met Gly Asn Tyr Gly Pro Arg Pro Asn Met Ser Met Gln 165 170 175 Pro Asn Gln Gly Pro Met Met His Gln Gln Pro Pro Ser Gln Gln Tyr 180 185 190 Asn Met Pro Gln Gly Gly Gly Gln His Tyr Gln Gly Gln Gln Pro Pro 195 200 205 Met Gly Met Met Gly Gln Val Asn Gln Gly Asn His Met Met Gly Gln 210 215 220 Arg Gln Ile Pro Pro Tyr Arg Pro Pro Gln Gln Gly Pro Pro Gln Gln 225 230 235 240 Tyr Ser Gly Gln Glu Asp Tyr Tyr Gly Asp Gln Tyr Ser His Gly Gly 245 250 255 Gln Gly Pro Pro Glu Gly Met Asn Gln Gln Tyr Tyr Pro Asp Gly His 260 265 270 Asn Asp Tyr Gly Tyr Gln Gln Pro Ser Tyr Pro Glu Gln Gly Tyr Asp 275 280 285 Arg Pro Tyr Glu Asp Ser Ser Gln His Tyr Tyr Glu Gly Gly Asn Ser 290 295 300 Gln Tyr Gly Gln Gln Gln Asp Ala Tyr Gln Gly Pro Pro Pro Gln Gln 305 310 315 320 Gly Tyr Pro Pro Gln Gln Gln Gln Tyr Pro Gly Gln Gln Gly Tyr Pro 325 330 335 Gly Gln Gln Gln Gly Tyr Gly Pro Ser Gln Gly Gly Pro Gly Pro Gln 340 345 350 Tyr Pro Asn Tyr Pro Gln Gly Gln Gly Gln Gln Tyr Gly Gly Tyr Arg 355 360 365 Pro Thr Gln Pro Gly Pro Pro Gln Pro Pro Gln Gln Arg Pro Tyr Gly 370 375 380 Tyr Asp Gln Gly Gln Tyr Gly Asn Tyr Gln Gln 385 390 395 <210> 17 <211> 157 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 17 Met Ser Thr Pro Pro Leu Ala Ala Ser Gly Met Ala Pro Gly Pro Phe 1 5 10 15 Ala Gly Pro Gln Ala Gln Gln Ala Ala Arg Glu Val Asn Thr Ala Ser 20 25 30 Leu Cys Arg Ile Gly Gln Glu Thr Val Gln Asp Ile Val Tyr Arg Thr 35 40 45 Met Glu Ile Phe Gln Leu Leu Arg Asn Met Gln Leu Pro Asn Gly Val 50 55 60 Thr Tyr His Thr Gly Thr Tyr Gln Asp Arg Leu Thr Lys Leu Gln Asp 65 70 75 80 Asn Leu Arg Gln Leu Ser Val Leu Phe Arg Lys Leu Arg Leu Val Tyr 85 90 95 Asp Lys Cys Asn Glu Asn Cys Gly Gly Met Asp Pro Ile Pro Val Glu 100 105 110 Gln Leu Ile Pro Tyr Val Glu Glu Asp Gly Ser Lys Asn Asp Asp Arg 115 120 125 Ala Gly Pro Pro Arg Phe Ala Ser Glu Glu Arg Arg Glu Ile Ala Glu 130 135 140 Val Asn Lys Ala Leu Ser Ser Val Pro Glu Phe Leu Pro 145 150 155 <210> 18 <211> 354 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 18 Met Lys Met Glu Glu Met Ser Leu Ser Gly Leu Asp Asn Ser Lys Leu 1 5 10 15 Glu Ala Ile Ala Gln Glu Ile Tyr Ala Asp Leu Val Glu Asp Ser Cys 20 25 30 Leu Gly Phe Cys Phe Glu Val His Arg Ala Val Lys Cys Gly Tyr Phe 35 40 45 Phe Leu Asp Asp Thr Asp Pro Asp Ser Met Lys Asp Phe Glu Ile Val 50 55 60 Asp Gln Pro Gly Leu Asp Ile Phe Gly Gln Val Phe Asn Gln Trp Lys 65 70 75 80 Ser Lys Glu Cys Val Cys Pro Asn Cys Ser Arg Ser Ile Ala Ala Ser 85 90 95 Arg Phe Ala Pro His Leu Glu Lys Cys Leu Gly Met Gly Arg Asn Ser 100 105 110 Ser Arg Ile Ala Asn Arg Arg Ile Ala Asn Ser Asn Asn Met Asn Lys 115 120 125 Ser Glu Ser Asp Gln Glu Asp Asn Asp Asp Ile Asn Asp Asn Asp Trp 130 135 140 Ser Tyr Gly Ser Glu Lys Lys Ala Lys Lys Arg Lys Ser Asp Lys Leu 145 150 155 160 Trp Tyr Leu Pro Phe Gln Asn Pro Asn Ser Pro Arg Arg Ser Lys Ser 165 170 175 Leu Lys His Lys Asn Gly Glu Leu Ser Asn Ser Asp Pro Phe Lys Tyr 180 185 190 Asn Asn Ser Thr Gly Ile Ser Tyr Glu Thr Leu Gly Pro Glu Glu Leu 195 200 205 Arg Ser Leu Leu Thr Thr Gln Cys Gly Val Ile Ser Glu His Thr Lys 210 215 220 Lys Met Cys Thr Arg Ser Leu Arg Cys Pro Gln His Thr Asp Glu Gln 225 230 235 240 Arg Arg Thr Val Arg Ile Tyr Phe Leu Gly Pro Ser Ala Val Leu Pro 245 250 255 Glu Val Glu Ser Ser Leu Asp Asn Asp Ser Phe Asp Met Thr Asp Ser 260 265 270 Gln Ala Leu Ile Ser Arg Leu Gln Trp Asp Gly Ser Ser Asp Leu Ser 275 280 285 Pro Ser Asp Ser Gly Ser Ser Lys Thr Ser Glu Asn Gln Gly Trp Gly 290 295 300 Leu Gly Thr Asn Ser Ser Glu Ser Arg Lys Thr Lys Lys Lys Lys Ser 305 310 315 320 His Leu Ser Leu Val Gly Thr Ala Ser Gly Leu Gly Ser Asn Lys Lys 325 330 335 Lys Lys Pro Lys Pro Pro Ala Pro Pro Thr Pro Ser Ile Tyr Asp Asp 340 345 350 Ile Asn <210> 19 <211> 2177 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 19 Met Ala Ala Phe Gly Ile Leu Ser Tyr Glu His Arg Pro Leu Lys Arg 1 5 10 15 Pro Arg Leu Gly Pro Pro Asp Val Tyr Pro Gln Asp Pro Lys Gln Lys 20 25 30 Glu Asp Glu Leu Thr Ala Leu Asn Val Lys Gln Gly Phe Asn Asn Gln 35 40 45 Pro Ala Val Ser Gly Asp Glu His Gly Ser Ala Lys Asn Val Ser Phe 50 55 60 Asn Pro Ala Lys Ile Ser Ser Asn Phe Ser Ser Ile Ile Ala Glu Lys 65 70 75 80 Leu Arg Cys Asn Thr Leu Pro Asp Thr Gly Arg Arg Lys Pro Gln Val 85 90 95 Asn Gln Lys Asp Asn Phe Trp Leu Val Thr Ala Arg Ser Gln Ser Ala 100 105 110 Ile Asn Thr Trp Phe Thr Asp Leu Ala Gly Thr Lys Pro Leu Thr Gln 115 120 125 Leu Ala Lys Lys Val Pro Ile Phe Ser Lys Lys Glu Glu Val Phe Gly 130 135 140 Tyr Leu Ala Lys Tyr Thr Val Pro Val Met Arg Ala Ala Trp Leu Ile 145 150 155 160 Lys Met Thr Cys Ala Tyr Tyr Ala Ala Ile Ser Glu Thr Lys Val Lys 165 170 175 Lys Arg His Val Asp Pro Phe Met Glu Trp Thr Gln Ile Ile Thr Lys 180 185 190 Tyr Leu Trp Glu Gln Leu Gln Lys Met Ala Glu Tyr Tyr Arg Pro Gly 195 200 205 Pro Ala Gly Ser Gly Gly Cys Gly Ser Thr Ile Gly Pro Leu Pro His 210 215 220 Asp Val Glu Val Ala Ile Arg Gln Trp Asp Tyr Thr Glu Lys Leu Ala 225 230 235 240 Met Phe Met Phe Gln Asp Gly Met Leu Asp Arg His Glu Phe Leu Thr 245 250 255 Trp Val Leu Glu Cys Phe Glu Lys Ile Arg Pro Gly Glu Asp Glu Leu 260 265 270 Leu Lys Leu Leu Leu Pro Leu Leu Leu Arg Tyr Ser Gly Glu Phe Val 275 280 285 Gln Ser Ala Tyr Leu Ser Arg Arg Leu Ala Tyr Phe Cys Thr Arg Arg 290 295 300 Leu Ala Leu Gln Leu Asp Gly Val Ser Ser His Ser Ser His Val Ile 305 310 315 320 Ser Ala Gln Ser Thr Ser Thr Leu Pro Thr Thr Pro Ala Pro Gln Pro 325 330 335 Pro Thr Ser Ser Thr Pro Ser Thr Pro Phe Ser Asp Leu Leu Met Cys 340 345 350 Pro Gln His Arg Pro Leu Val Phe Gly Leu Ser Cys Ile Leu Gln Thr 355 360 365 Ile Leu Leu Cys Cys Pro Ser Ala Leu Val Trp His Tyr Ser Leu Thr 370 375 380 Asp Ser Arg Ile Lys Thr Gly Ser Pro Leu Asp His Leu Pro Ile Ala 385 390 395 400 Pro Ser Asn Leu Pro Met Pro Glu Gly Asn Ser Ala Phe Thr Gln Gln 405 410 415 Val Arg Ala Lys Leu Arg Glu Ile Glu Gln Gln Ile Lys Glu Arg Gly 420 425 430 Gln Ala Val Glu Val Arg Trp Ser Phe Asp Lys Cys Gln Glu Ala Thr 435 440 445 Ala Gly Phe Thr Ile Gly Arg Val Leu His Thr Leu Glu Val Leu Asp 450 455 460 Ser His Ser Phe Glu Arg Ser Asp Phe Ser Asn Ser Leu Asp Ser Leu 465 470 475 480 Cys Asn Arg Ile Phe Gly Leu Gly Pro Ser Lys Asp Gly His Glu Ile 485 490 495 Ser Ser Asp Asp Asp Ala Val Val Ser Leu Leu Cys Glu Trp Ala Val 500 505 510 Ser Cys Lys Arg Ser Gly Arg His Arg Ala Met Val Val Ala Lys Leu 515 520 525 Leu Glu Lys Arg Gln Ala Glu Ile Glu Ala Glu Arg Cys Gly Glu Ser 530 535 540 Glu Ala Ala Asp Glu Lys Gly Ser Ile Ala Ser Gly Ser Leu Ser Ala 545 550 555 560 Pro Ser Ala Pro Ile Phe Gln Asp Val Leu Leu Gln Phe Leu Asp Thr 565 570 575 Gln Ala Pro Met Leu Thr Asp Pro Arg Ser Glu Ser Glu Arg Val Glu 580 585 590 Phe Phe Asn Leu Val Leu Leu Phe Cys Glu Leu Ile Arg His Asp Val 595 600 605 Phe Ser His Asn Met Tyr Thr Cys Thr Leu Ile Ser Arg Gly Asp Leu 610 615 620 Ala Phe Gly Ala Pro Gly Pro Arg Pro Pro Ser Pro Phe Asp Asp Pro 625 630 635 640 Ala Asp Asp Pro Glu His Lys Glu Ala Glu Gly Ser Ser Ser Ser Lys 645 650 655 Leu Glu Asp Pro Gly Leu Ser Glu Ser Met Asp Ile Asp Pro Ser Ser 660 665 670 Ser Val Leu Phe Glu Asp Met Glu Lys Pro Asp Phe Ser Leu Phe Ser 675 680 685 Pro Thr Met Pro Cys Glu Gly Lys Gly Ser Pro Ser Pro Glu Lys Pro 690 695 700 Asp Val Glu Lys Glu Val Lys Pro Pro Pro Lys Glu Lys Ile Glu Gly 705 710 715 720 Thr Leu Gly Val Leu Tyr Asp Gln Pro Arg His Val Gln Tyr Ala Thr 725 730 735 His Phe Pro Ile Pro Gln Glu Glu Ser Cys Ser His Glu Cys Asn Gln 740 745 750 Arg Leu Val Val Leu Phe Gly Val Gly Lys Gln Arg Asp Asp Ala Arg 755 760 765 His Ala Ile Lys Lys Ile Thr Lys Asp Ile Leu Lys Val Leu Asn Arg 770 775 780 Lys Gly Thr Ala Glu Thr Asp Gln Leu Ala Pro Ile Val Pro Leu Asn 785 790 795 800 Pro Gly Asp Leu Thr Phe Leu Gly Gly Glu Asp Gly Gln Lys Arg Arg 805 810 815 Arg Asn Arg Pro Glu Ala Phe Pro Thr Ala Glu Asp Ile Phe Ala Lys 820 825 830 Phe Gln His Leu Ser His Tyr Asp Gln His Gln Val Thr Ala Gln Val 835 840 845 Ser Arg Asn Val Leu Glu Gln Ile Thr Ser Phe Ala Leu Gly Met Ser 850 855 860 Tyr His Leu Pro Leu Val Gln His Val Gln Phe Ile Phe Asp Leu Met 865 870 875 880 Glu Tyr Ser Leu Ser Ile Ser Gly Leu Ile Asp Phe Ala Ile Gln Leu 885 890 895 Leu Asn Glu Leu Ser Val Val Glu Ala Glu Leu Leu Leu Lys Ser Ser 900 905 910 Asp Leu Val Gly Ser Tyr Thr Thr Ser Leu Cys Leu Cys Ile Val Ala 915 920 925 Val Leu Arg His Tyr His Ala Cys Leu Ile Leu Asn Gln Asp Gln Met 930 935 940 Ala Gln Val Phe Glu Gly Leu Cys Gly Val Val Lys His Gly Met Asn 945 950 955 960 Arg Ser Asp Gly Ser Ser Ala Glu Arg Cys Ile Leu Ala Tyr Leu Tyr 965 970 975 Asp Leu Tyr Thr Ser Cys Ser His Leu Lys Asn Lys Phe Gly Glu Leu 980 985 990 Phe Ser Asp Phe Cys Ser Lys Val Lys Asn Thr Ile Tyr Cys Asn Val 995 1000 1005 Glu Pro Ser Glu Ser Asn Met Arg Trp Ala Pro Glu Phe Met Ile 1010 1015 1020 Asp Thr Leu Glu Asn Pro Ala Ala His Thr Phe Thr Tyr Thr Gly 1025 1030 1035 Leu Gly Lys Ser Leu Ser Glu Asn Pro Ala Asn Arg Tyr Ser Phe 1040 1045 1050 Val Cys Asn Ala Leu Met His Val Cys Val Gly His His Asp Pro 1055 1060 1065 Asp Arg Val Asn Asp Ile Ala Ile Leu Cys Ala Glu Leu Thr Gly 1070 1075 1080 Tyr Cys Lys Ser Leu Ser Ala Glu Trp Leu Gly Val Leu Lys Ala 1085 1090 1095 Leu Cys Cys Ser Ser Asn Asn Gly Thr Cys Gly Phe Asn Asp Leu 1100 1105 1110 Leu Cys Asn Val Asp Val Ser Asp Leu Ser Phe His Asp Ser Leu 1115 1120 1125 Ala Thr Phe Val Ala Ile Leu Ile Ala Arg Gln Cys Leu Leu Leu 1130 1135 1140 Glu Asp Leu Ile Arg Cys Ala Ala Ile Pro Ser Leu Leu Asn Ala 1145 1150 1155 Ala Cys Ser Glu Gln Asp Ser Glu Pro Gly Ala Arg Leu Thr Cys 1160 1165 1170 Arg Ile Leu Leu His Leu Phe Lys Thr Pro Gln Leu Asn Pro Cys 1175 1180 1185 Gln Ser Asp Gly Asn Lys Pro Thr Val Gly Ile Arg Ser Ser Cys 1190 1195 1200 Asp Arg His Leu Leu Ala Ala Ser Gln Asn Arg Ile Val Asp Gly 1205 1210 1215 Ala Val Phe Ala Val Leu Lys Ala Val Phe Val Leu Gly Asp Ala 1220 1225 1230 Glu Leu Lys Gly Ser Gly Phe Thr Val Thr Gly Gly Thr Glu Glu 1235 1240 1245 Leu Pro Glu Glu Glu Gly Gly Gly Gly Ser Gly Gly Arg Arg Gln 1250 1255 1260 Gly Gly Arg Asn Ile Ser Val Glu Thr Ala Ser Leu Asp Val Tyr 1265 1270 1275 Ala Lys Tyr Val Leu Arg Ser Ile Cys Gln Gln Glu Trp Val Gly 1280 1285 1290 Glu Arg Cys Leu Lys Ser Leu Cys Glu Asp Ser Asn Asp Leu Gln 1295 1300 1305 Asp Pro Val Leu Ser Ser Ala Gln Ala Gln Arg Leu Met Gln Leu 1310 1315 1320 Ile Cys Tyr Pro His Arg Leu Leu Asp Asn Glu Asp Gly Glu Asn 1325 1330 1335 Pro Gln Arg Gln Arg Ile Lys Arg Ile Leu Gln Asn Leu Asp Gln 1340 1345 1350 Trp Thr Met Arg Gln Ser Ser Leu Glu Leu Gln Leu Met Ile Lys 1355 1360 1365 Gln Thr Pro Asn Asn Glu Met Asn Ser Leu Leu Glu Asn Ile Ala 1370 1375 1380 Lys Ala Thr Ile Glu Val Phe Gln Gln Ser Ala Glu Thr Gly Ser 1385 1390 1395 Ser Ser Gly Ser Thr Ala Ser Asn Met Pro Ser Ser Ser Lys Thr 1400 1405 1410 Lys Pro Val Leu Ser Ser Leu Glu Arg Ser Gly Val Trp Leu Val 1415 1420 1425 Ala Pro Leu Ile Ala Lys Leu Pro Thr Ser Val Gln Gly His Val 1430 1435 1440 Leu Lys Ala Ala Gly Glu Glu Leu Glu Lys Gly Gln His Leu Gly 1445 1450 1455 Ser Ser Ser Arg Lys Glu Arg Asp Arg Gln Lys Gln Lys Ser Met 1460 1465 1470 Ser Leu Leu Ser Gln Gln Pro Phe Leu Ser Leu Val Leu Thr Cys 1475 1480 1485 Leu Lys Gly Gln Asp Glu Gln Arg Glu Gly Leu Leu Thr Ser Leu 1490 1495 1500 Tyr Ser Gln Val His Gln Ile Val Asn Asn Trp Arg Asp Asp Gln 1505 1510 1515 Tyr Leu Asp Asp Cys Lys Pro Lys Gln Leu Met His Glu Ala Leu 1520 1525 1530 Lys Leu Arg Leu Asn Leu Val Gly Gly Met Phe Asp Thr Val Gln 1535 1540 1545 Arg Ser Thr Gln Gln Thr Thr Glu Trp Ala Met Leu Leu Leu Glu 1550 1555 1560 Ile Ile Ile Ser Gly Thr Val Asp Met Gln Ser Asn Asn Glu Leu 1565 1570 1575 Phe Thr Thr Val Leu Asp Met Leu Ser Val Leu Ile Asn Gly Thr 1580 1585 1590 Leu Ala Ala Asp Met Ser Ser Ile Ser Gln Gly Ser Met Glu Glu 1595 1600 1605 Asn Lys Arg Ala Tyr Met Asn Leu Ala Lys Lys Leu Gln Lys Glu 1610 1615 1620 Leu Gly Glu Arg Gln Ser Asp Ser Leu Glu Lys Val Arg Gln Leu 1625 1630 1635 Leu Pro Leu Pro Lys Gln Thr Arg Asp Val Ile Thr Cys Glu Pro 1640 1645 1650 Gln Gly Ser Leu Ile Asp Thr Lys Gly Asn Lys Ile Ala Gly Phe 1655 1660 1665 Asp Ser Ile Phe Lys Lys Glu Gly Leu Gln Val Ser Thr Lys Gln 1670 1675 1680 Lys Ile Ser Pro Trp Asp Leu Phe Glu Gly Leu Lys Pro Ser Ala 1685 1690 1695 Pro Leu Ser Trp Gly Trp Phe Gly Thr Val Arg Val Asp Arg Arg 1700 1705 1710 Val Ala Arg Gly Glu Glu Gln Gln Arg Leu Leu Leu Tyr His Thr 1715 1720 1725 His Leu Arg Pro Arg Pro Arg Ala Tyr Tyr Leu Glu Pro Leu Pro 1730 1735 1740 Leu Pro Pro Glu Asp Glu Glu Pro Pro Ala Pro Thr Leu Leu Glu 1745 1750 1755 Pro Glu Lys Lys Ala Pro Glu Pro Pro Lys Thr Asp Lys Pro Gly 1760 1765 1770 Ala Ala Pro Pro Ser Thr Glu Glu Arg Lys Lys Lys Ser Thr Lys 1775 1780 1785 Gly Lys Lys Arg Ser Gln Pro Ala Thr Lys Thr Glu Asp Tyr Gly 1790 1795 1800 Met Gly Pro Gly Arg Ser Gly Pro Tyr Gly Val Thr Val Pro Pro 1805 1810 1815 Asp Leu Leu His His Pro Asn Pro Gly Ser Ile Thr His Leu Asn 1820 1825 1830 Tyr Arg Gln Gly Ser Ile Gly Leu Tyr Thr Gln Asn Gln Pro Leu 1835 1840 1845 Pro Ala Gly Gly Pro Arg Val Asp Pro Tyr Arg Pro Val Arg Leu 1850 1855 1860 Pro Met Gln Lys Leu Pro Thr Arg Pro Thr Tyr Pro Gly Val Leu 1865 1870 1875 Pro Thr Thr Met Thr Gly Val Met Gly Leu Glu Pro Ser Ser Tyr 1880 1885 1890 Lys Thr Ser Val Tyr Arg Gln Gln Gln Pro Ala Val Pro Gln Gly 1895 1900 1905 Gln Arg Leu Arg Gln Gln Leu Gln Gln Ser Gln Gly Met Leu Gly 1910 1915 1920 Gln Ser Ser Val His Gln Met Thr Pro Ser Ser Ser Tyr Gly Leu 1925 1930 1935 Gln Thr Ser Gln Gly Tyr Thr Pro Tyr Val Ser His Val Gly Leu 1940 1945 1950 Gln Gln His Thr Gly Pro Ala Gly Thr Met Val Pro Pro Ser Tyr 1955 1960 1965 Ser Ser Gln Pro Tyr Gln Ser Thr His Pro Ser Thr Asn Pro Thr 1970 1975 1980 Leu Val Asp Pro Thr Arg His Leu Gln Gln Arg Pro Ser Gly Tyr 1985 1990 1995 Val His Gln Gln Ala Pro Thr Tyr Gly His Gly Leu Thr Ser Thr 2000 2005 2010 Gln Arg Phe Ser His Gln Thr Leu Gln Gln Thr Pro Met Ile Ser 2015 2020 2025 Thr Met Thr Pro Met Ser Ala Gln Gly Val Gln Ala Gly Val Arg 2030 2035 2040 Ser Thr Ala Ile Leu Pro Glu Gln Gln Gln Gln Gln Gln Gln Gln 2045 2050 2055 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 2060 2065 2070 Gln Gln Gln Tyr His Ile Arg Gln Gln Gln Gln Gln Gln Ile Leu 2075 2080 2085 Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 2090 2095 2100 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Gln Gln 2105 2110 2115 Gln Gln Gln Gln Gln Ala Ala Pro Pro Gln Pro Gln Pro Gln Ser 2120 2125 2130 Gln Pro Gln Phe Gln Arg Gln Gly Leu Gln Gln Thr Gln Gln Gln 2135 2140 2145 Gln Gln Thr Ala Ala Leu Val Arg Gln Leu Gln Gln Gln Leu Ser 2150 2155 2160 Asn Thr Gln Pro Gln Pro Ser Thr Asn Ile Phe Gly Arg Tyr 2165 2170 2175 <210> 20 <211> 440 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 20 Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn 1 5 10 15 Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala 20 25 30 Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45 Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60 Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp 65 70 75 80 Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95 Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Tyr Gln 100 105 110 His Tyr Lys Gly Ser Asp Phe Asp Cys Glu Leu Arg Leu Leu Ile His 115 120 125 Gln Ser Leu Ala Gly Gly Ile Ile Gly Val Lys Gly Ala Lys Ile Lys 130 135 140 Glu Leu Arg Glu Asn Thr Gln Thr Thr Ile Lys Leu Phe Gln Glu Cys 145 150 155 160 Cys Pro His Ser Thr Asp Arg Val Val Leu Ile Gly Gly Lys Pro Asp 165 170 175 Arg Val Val Glu Cys Ile Lys Ile Ile Leu Asp Leu Ile Ser Glu Ser 180 185 190 Pro Ile Lys Gly Arg Ala Gln Pro Tyr Asp Pro Asn Phe Tyr Asp Glu 195 200 205 Thr Tyr Asp Tyr Gly Gly Phe Thr Met Met Phe Asp Asp Arg Arg Gly 210 215 220 Arg Pro Val Gly Phe Pro Met Arg Gly Arg Gly Gly Phe Asp Arg Met 225 230 235 240 Pro Pro Gly Arg Gly Gly Arg Pro Met Pro Pro Ser Arg Arg Asp Tyr 245 250 255 Asp Asp Met Ser Pro Arg Arg Gly Pro Pro Pro Pro Pro Pro Gly Arg 260 265 270 Gly Gly Arg Gly Gly Ser Arg Ala Arg Asn Leu Pro Leu Pro Pro Pro 275 280 285 Pro Pro Pro Arg Gly Gly Asp Leu Met Ala Tyr Asp Arg Arg Gly Arg 290 295 300 Pro Gly Asp Arg Tyr Asp Gly Met Val Gly Phe Ser Ala Asp Glu Thr 305 310 315 320 Trp Asp Ser Ala Ile Asp Thr Trp Ser Pro Ser Glu Trp Gln Met Ala 325 330 335 Tyr Glu Pro Gln Gly Gly Ser Gly Tyr Asp Tyr Ser Tyr Ala Gly Gly 340 345 350 Arg Gly Ser Tyr Gly Asp Leu Gly Gly Pro Ile Ile Thr Thr Gln Val 355 360 365 Thr Ile Pro Lys Asp Leu Ala Gly Ser Ile Ile Gly Lys Gly Gly Gln 370 375 380 Arg Ile Lys Gln Ile Arg His Glu Ser Gly Ala Ser Ile Lys Ile Asp 385 390 395 400 Glu Pro Leu Glu Gly Ser Glu Asp Arg Ile Ile Thr Ile Thr Gly Thr 405 410 415 Gln Asp Gln Ile Gln Asn Ala Gln Tyr Leu Leu Gln Asn Ser Val Lys 420 425 430 Gln Tyr Ala Asp Val Glu Gly Phe 435 440 <210> 21 <211> 859 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 21 Met Lys Ile Gly Ser Gly Phe Leu Ser Gly Gly Gly Gly Thr Gly Ser 1 5 10 15 Ser Gly Gly Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly 20 25 30 Gly Gly Ser Ser Gly Arg Arg Ala Glu Met Glu Pro Thr Phe Pro Gln 35 40 45 Ala Pro Ala Ala Glu Pro Pro Pro Pro Pro Ala Pro Asp Met Thr Phe 50 55 60 Lys Lys Glu Pro Ala Ala Ser Ala Ala Ala Phe Pro Ser Gln Arg Thr 65 70 75 80 Ser Trp Gly Phe Leu Gln Ser Leu Val Ser Ile Lys Gln Glu Lys Pro 85 90 95 Ala Asp Pro Glu Glu Gln Gln Ser His His His His His His His His 100 105 110 Tyr Gly Gly Leu Phe Ala Gly Ala Glu Glu Arg Ser Pro Gly Leu Gly 115 120 125 Gly Gly Glu Gly Gly Ser His Gly Val Ile Gln Asp Leu Ser Ile Leu 130 135 140 His Gln His Val Gln Gln Gln Pro Ala Gln His His Arg Asp Val Leu 145 150 155 160 Leu Ser Ser Ser Ser Arg Thr Asp Asp His His Gly Thr Glu Glu Pro 165 170 175 Lys Gln Asp Thr Asn Val Lys Lys Ala Lys Arg Pro Lys Pro Glu Ser 180 185 190 Gln Gly Ile Lys Ala Lys Arg Lys Pro Ser Ala Ser Ser Lys Pro Ser 195 200 205 Leu Val Gly Asp Gly Glu Gly Ala Ile Leu Ser Pro Ser Gln Lys Pro 210 215 220 His Ile Cys Asp His Cys Ser Ala Ala Phe Arg Ser Ser Tyr His Leu 225 230 235 240 Arg Arg His Val Leu Ile His Thr Gly Glu Arg Pro Phe Gln Cys Ser 245 250 255 Gln Cys Ser Met Gly Phe Ile Gln Lys Tyr Leu Leu Gln Arg His Glu 260 265 270 Lys Ile His Ser Arg Glu Lys Pro Phe Gly Cys Asp Gln Cys Ser Met 275 280 285 Lys Phe Ile Gln Lys Tyr His Met Glu Arg His Lys Arg Thr His Ser 290 295 300 Gly Glu Lys Pro Tyr Lys Cys Asp Thr Cys Gln Gln Tyr Phe Ser Arg 305 310 315 320 Thr Asp Arg Leu Leu Lys His Arg Arg Thr Cys Gly Glu Val Ile Val 325 330 335 Lys Gly Ala Thr Ser Ala Glu Pro Gly Ser Ser Asn His Thr Asn Met 340 345 350 Gly Asn Leu Ala Val Leu Ser Gln Gly Asn Thr Ser Ser Ser Arg Arg 355 360 365 Lys Thr Lys Ser Lys Ser Ile Ala Ile Glu Asn Lys Glu Gln Lys Thr 370 375 380 Gly Lys Thr Asn Glu Ser Gln Ile Ser Asn Asn Ile Asn Met Gln Ser 385 390 395 400 Tyr Ser Val Glu Met Pro Thr Val Ser Ser Ser Gly Gly Ile Ile Gly 405 410 415 Thr Gly Ile Asp Glu Leu Gln Lys Arg Val Pro Lys Leu Ile Phe Lys 420 425 430 Lys Gly Ser Arg Lys Asn Thr Asp Lys Asn Tyr Leu Asn Phe Val Ser 435 440 445 Pro Leu Pro Asp Ile Val Gly Gln Lys Ser Leu Ser Gly Lys Pro Ser 450 455 460 Gly Ser Leu Gly Ile Val Ser Asn Asn Ser Val Glu Thr Ile Gly Leu 465 470 475 480 Leu Gln Ser Thr Ser Gly Lys Gln Gly Gln Ile Ser Ser Asn Tyr Asp 485 490 495 Asp Ala Met Gln Phe Ser Lys Lys Arg Arg Tyr Leu Pro Thr Ala Ser 500 505 510 Ser Asn Ser Ala Phe Ser Ile Asn Val Gly His Met Val Ser Gln Gln 515 520 525 Ser Val Ile Gln Ser Ala Gly Val Ser Val Leu Asp Asn Glu Ala Pro 530 535 540 Leu Ser Leu Ile Asp Ser Ser Ala Leu Asn Ala Glu Ile Lys Ser Cys 545 550 555 560 His Asp Lys Ser Gly Ile Pro Asp Glu Val Leu Gln Ser Ile Leu Asp 565 570 575 Gln Tyr Ser Asn Lys Ser Glu Ser Gln Lys Glu Asp Pro Phe Asn Ile 580 585 590 Ala Glu Pro Arg Val Asp Leu His Thr Ser Gly Glu His Ser Glu Leu 595 600 605 Val Gln Glu Glu Asn Leu Ser Pro Gly Thr Gln Thr Pro Ser Asn Asp 610 615 620 Lys Ala Ser Met Leu Gln Glu Tyr Ser Lys Tyr Leu Gln Gln Ala Phe 625 630 635 640 Glu Lys Ser Thr Asn Ala Ser Phe Thr Leu Gly His Gly Phe Gln Phe 645 650 655 Val Ser Leu Ser Ser Pro Leu His Asn His Thr Leu Phe Pro Glu Lys 660 665 670 Gln Ile Tyr Thr Thr Ser Pro Leu Glu Cys Gly Phe Gly Gln Ser Val 675 680 685 Thr Ser Val Leu Pro Ser Ser Leu Pro Lys Pro Pro Phe Gly Met Leu 690 695 700 Phe Gly Ser Gln Pro Gly Leu Tyr Leu Ser Ala Leu Asp Ala Thr His 705 710 715 720 Gln Gln Leu Thr Pro Ser Gln Glu Leu Asp Asp Leu Ile Asp Ser Gln 725 730 735 Lys Asn Leu Glu Thr Ser Ser Ala Phe Gln Ser Ser Ser Gln Lys Leu 740 745 750 Thr Ser Gln Lys Glu Gln Lys Asn Leu Glu Ser Ser Thr Gly Phe Gln 755 760 765 Ile Pro Ser Gln Glu Leu Ala Ser Gln Ile Asp Pro Gln Lys Asp Ile 770 775 780 Glu Pro Arg Thr Thr Tyr Gln Ile Glu Asn Phe Ala Gln Ala Phe Gly 785 790 795 800 Ser Gln Phe Lys Ser Gly Ser Arg Val Pro Met Thr Phe Ile Thr Asn 805 810 815 Ser Asn Gly Glu Val Asp His Arg Val Arg Thr Ser Val Ser Asp Phe 820 825 830 Ser Gly Tyr Thr Asn Met Met Ser Asp Val Ser Glu Pro Cys Ser Thr 835 840 845 Arg Val Lys Thr Pro Thr Ser Gln Ser Tyr Arg 850 855 <210> 22 <211> 589 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 22 Met Lys Arg Val Arg Thr Glu Gln Ile Gln Met Ala Val Ser Cys Tyr 1 5 10 15 Leu Lys Arg Arg Gln Tyr Val Asp Ser Asp Gly Pro Leu Lys Gln Gly 20 25 30 Leu Arg Leu Ser Gln Thr Ala Glu Glu Met Ala Ala Asn Leu Thr Val 35 40 45 Gln Ser Glu Ser Gly Cys Ala Asn Ile Val Ser Ala Ala Pro Cys Gln 50 55 60 Ala Glu Pro Gln Gln Tyr Glu Val Gln Phe Gly Arg Leu Arg Asn Phe 65 70 75 80 Leu Thr Asp Ser Asp Ser Gln His Ser His Glu Val Met Pro Leu Leu 85 90 95 Tyr Pro Leu Phe Val Tyr Leu His Leu Asn Leu Val Gln Asn Ser Pro 100 105 110 Lys Ser Thr Val Glu Ser Phe Tyr Ser Arg Phe His Gly Met Phe Leu 115 120 125 Gln Asn Ala Ser Gln Lys Asp Val Ile Glu Gln Leu Gln Thr Thr Gln 130 135 140 Thr Ile Gln Asp Ile Leu Ser Asn Phe Lys Leu Arg Ala Phe Leu Asp 145 150 155 160 Asn Lys Tyr Val Val Arg Leu Gln Glu Asp Ser Tyr Asn Tyr Leu Ile 165 170 175 Arg Tyr Leu Gln Ser Asp Asn Asn Thr Ala Leu Cys Lys Val Leu Thr 180 185 190 Leu His Ile His Leu Asp Val Gln Pro Ala Lys Arg Thr Asp Tyr Gln 195 200 205 Leu Tyr Ala Ser Gly Ser Ser Ser Arg Ser Glu Asn Asn Gly Leu Glu 210 215 220 Pro Pro Asp Met Pro Ser Pro Ile Leu Gln Asn Glu Ala Ala Leu Glu 225 230 235 240 Val Leu Gln Glu Ser Ile Lys Arg Val Lys Asp Gly Pro Pro Ser Leu 245 250 255 Thr Thr Ile Cys Phe Tyr Ala Phe Tyr Asn Thr Glu Gln Leu Leu Asn 260 265 270 Thr Ala Glu Ile Ser Pro Asp Ser Lys Leu Leu Ala Ala Gly Phe Asp 275 280 285 Asn Ser Cys Ile Lys Leu Trp Ser Leu Arg Ser Lys Lys Leu Lys Ser 290 295 300 Glu Pro His Gln Val Asp Val Ser Arg Ile His Leu Ala Cys Asp Ile 305 310 315 320 Leu Glu Glu Glu Asp Asp Glu Asp Asp Asn Ala Gly Thr Glu Met Lys 325 330 335 Ile Leu Arg Gly His Cys Gly Pro Val Tyr Ser Thr Arg Phe Leu Ala 340 345 350 Asp Ser Ser Gly Leu Leu Ser Cys Ser Glu Asp Met Ser Ile Arg Tyr 355 360 365 Trp Asp Leu Gly Ser Phe Thr Asn Thr Val Leu Tyr Gln Gly His Ala 370 375 380 Tyr Pro Val Trp Asp Leu Asp Ile Ser Pro Tyr Ser Leu Tyr Phe Ala 385 390 395 400 Ser Gly Ser His Asp Arg Thr Ala Arg Leu Trp Ser Phe Asp Arg Thr 405 410 415 Tyr Pro Leu Arg Ile Tyr Ala Gly His Leu Ala Asp Val Asp Cys Val 420 425 430 Lys Phe His Pro Asn Ser Asn Tyr Leu Ala Thr Gly Ser Thr Asp Lys 435 440 445 Thr Val Arg Leu Trp Ser Ala Gln Gln Gly Asn Ser Val Arg Leu Phe 450 455 460 Thr Gly His Arg Gly Pro Val Leu Ser Leu Ala Phe Ser Pro Asn Gly 465 470 475 480 Lys Tyr Leu Ala Ser Ala Gly Glu Asp Gln Arg Leu Lys Leu Trp Asp 485 490 495 Leu Ala Ser Gly Thr Leu Tyr Lys Glu Leu Arg Gly His Thr Asp Asn 500 505 510 Ile Thr Ser Leu Thr Phe Ser Pro Asp Ser Gly Leu Ile Ala Ser Ala 515 520 525 Ser Met Asp Asn Ser Val Arg Val Trp Asp Ile Arg Asn Thr Tyr Cys 530 535 540 Ser Ala Pro Ala Asp Gly Ser Ser Ser Glu Leu Val Gly Val Tyr Thr 545 550 555 560 Gly Gln Met Ser Asn Val Leu Ser Val Gln Phe Met Ala Cys Asn Leu 565 570 575 Leu Leu Val Thr Gly Ile Thr Gln Glu Asn Gln Glu His 580 585 <210> 23 <211> 169 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 23 Met Ala Ala Glu Ser Leu Pro Phe Ser Phe Gly Thr Leu Ser Ser Trp 1 5 10 15 Glu Leu Glu Ala Trp Tyr Glu Asp Leu Gln Glu Val Leu Ser Ser Asp 20 25 30 Glu Asn Gly Gly Thr Tyr Val Ser Pro Pro Gly Asn Glu Glu Glu Glu 35 40 45 Ser Lys Ile Phe Thr Thr Leu Asp Pro Ala Ser Leu Ala Trp Leu Thr 50 55 60 Glu Glu Glu Pro Glu Pro Ala Glu Val Thr Ser Thr Ser Gln Ser Pro 65 70 75 80 His Ser Pro Asp Ser Ser Gln Ser Ser Leu Ala Gln Glu Glu Glu Glu 85 90 95 Glu Asp Gln Gly Arg Thr Arg Lys Arg Lys Gln Ser Gly His Ser Pro 100 105 110 Ala Arg Ala Gly Lys Gln Arg Met Lys Glu Lys Glu Gln Glu Asn Glu 115 120 125 Arg Lys Val Ala Gln Leu Ala Glu Glu Asn Glu Arg Leu Lys Gln Glu 130 135 140 Ile Glu Arg Leu Thr Arg Glu Val Glu Ala Thr Arg Arg Ala Leu Ile 145 150 155 160 Asp Arg Met Val Asn Leu His Gln Ala 165 <210> 24 <211> 1186 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 24 Met Asp Ile Ser Thr Arg Ser Lys Asp Pro Gly Ser Ala Glu Arg Thr 1 5 10 15 Ala Gln Lys Arg Lys Phe Pro Ser Pro Pro His Ser Ser Asn Gly His 20 25 30 Ser Pro Gln Asp Thr Ser Thr Ser Pro Ile Lys Lys Lys Lys Lys Pro 35 40 45 Gly Leu Leu Asn Ser Asn Asn Lys Glu Gln Ser Glu Leu Arg His Gly 50 55 60 Pro Phe Tyr Tyr Met Lys Gln Pro Leu Thr Thr Asp Pro Val Asp Val 65 70 75 80 Val Pro Gln Asp Gly Arg Asn Asp Phe Tyr Cys Trp Val Cys His Arg 85 90 95 Glu Gly Gln Val Leu Cys Cys Glu Leu Cys Pro Arg Val Tyr His Ala 100 105 110 Lys Cys Leu Arg Leu Thr Ser Glu Pro Glu Gly Asp Trp Phe Cys Pro 115 120 125 Glu Cys Glu Lys Ile Thr Val Ala Glu Cys Ile Glu Thr Gln Ser Lys 130 135 140 Ala Met Thr Met Leu Thr Ile Glu Gln Leu Ser Tyr Leu Leu Lys Phe 145 150 155 160 Ala Ile Gln Lys Met Lys Gln Pro Gly Thr Asp Ala Phe Gln Lys Pro 165 170 175 Val Pro Leu Glu Gln His Pro Asp Tyr Ala Glu Tyr Ile Phe His Pro 180 185 190 Met Asp Leu Cys Thr Leu Glu Lys Asn Ala Lys Lys Lys Met Tyr Gly 195 200 205 Cys Thr Glu Ala Phe Leu Ala Asp Ala Lys Trp Ile Leu His Asn Cys 210 215 220 Ile Ile Tyr Asn Gly Gly Asn His Lys Leu Thr Gln Ile Ala Lys Val 225 230 235 240 Val Ile Lys Ile Cys Glu His Glu Met Asn Glu Ile Glu Val Cys Pro 245 250 255 Glu Cys Tyr Leu Ala Ala Cys Gln Lys Arg Asp Asn Trp Phe Cys Glu 260 265 270 Pro Cys Ser Asn Pro His Pro Leu Val Trp Ala Lys Leu Lys Gly Phe 275 280 285 Pro Phe Trp Pro Ala Lys Ala Leu Arg Asp Lys Asp Gly Gln Val Asp 290 295 300 Ala Arg Phe Phe Gly Gln His Asp Arg Ala Trp Val Pro Ile Asn Asn 305 310 315 320 Cys Tyr Leu Met Ser Lys Glu Ile Pro Phe Ser Val Lys Lys Thr Lys 325 330 335 Ser Ile Phe Asn Ser Ala Met Gln Glu Met Glu Val Tyr Val Glu Asn 340 345 350 Ile Arg Arg Lys Phe Gly Val Phe Asn Tyr Ser Pro Phe Arg Thr Pro 355 360 365 Tyr Thr Pro Asn Ser Gln Tyr Gln Met Leu Leu Asp Pro Thr Asn Pro 370 375 380 Ser Ala Gly Thr Ala Lys Ile Asp Lys Gln Glu Lys Val Lys Leu Asn 385 390 395 400 Phe Asp Met Thr Ala Ser Pro Lys Ile Leu Met Ser Lys Pro Val Leu 405 410 415 Ser Gly Gly Thr Gly Arg Arg Ile Ser Leu Ser Asp Met Pro Arg Ser 420 425 430 Pro Met Ser Thr Asn Ser Ser Val His Thr Gly Ser Asp Val Glu Gln 435 440 445 Asp Ala Glu Lys Lys Ala Thr Ser Ser His Phe Ser Ala Ser Glu Glu 450 455 460 Ser Met Asp Phe Leu Asp Lys Ser Thr Ala Ser Pro Ala Ser Thr Lys 465 470 475 480 Thr Gly Gln Ala Gly Ser Leu Ser Gly Ser Pro Lys Pro Phe Ser Pro 485 490 495 Gln Leu Ser Ala Pro Ile Thr Thr Lys Thr Asp Lys Thr Ser Thr Thr 500 505 510 Gly Ser Ile Leu Asn Leu Asn Leu Asp Arg Ser Lys Ala Glu Met Asp 515 520 525 Leu Lys Glu Leu Ser Glu Ser Val Gln Gln Gln Ser Thr Pro Val Pro 530 535 540 Leu Ile Ser Pro Lys Arg Gln Ile Arg Ser Arg Phe Gln Leu Asn Leu 545 550 555 560 Asp Lys Thr Ile Glu Ser Cys Lys Ala Gln Leu Gly Ile Asn Glu Ile 565 570 575 Ser Glu Asp Val Tyr Thr Ala Val Glu His Ser Asp Ser Glu Asp Ser 580 585 590 Glu Lys Ser Asp Ser Ser Asp Ser Glu Tyr Ile Ser Asp Asp Glu Gln 595 600 605 Lys Ser Lys Asn Glu Pro Glu Asp Thr Glu Asp Lys Glu Gly Cys Gln 610 615 620 Met Asp Lys Glu Pro Ser Ala Val Lys Lys Lys Pro Lys Pro Thr Asn 625 630 635 640 Pro Val Glu Ile Lys Glu Glu Leu Lys Ser Thr Ser Pro Ala Ser Glu 645 650 655 Lys Ala Asp Pro Gly Ala Val Lys Asp Lys Ala Ser Pro Glu Pro Glu 660 665 670 Lys Asp Phe Ser Glu Lys Ala Lys Pro Ser Pro His Pro Ile Lys Asp 675 680 685 Lys Leu Lys Gly Lys Asp Glu Thr Asp Ser Pro Thr Val His Leu Gly 690 695 700 Leu Asp Ser Asp Ser Glu Ser Glu Leu Val Ile Asp Leu Gly Glu Asp 705 710 715 720 His Ser Gly Arg Glu Gly Arg Lys Asn Lys Lys Glu Pro Lys Glu Pro 725 730 735 Ser Pro Lys Gln Asp Val Val Gly Lys Thr Pro Pro Ser Thr Thr Val 740 745 750 Gly Ser His Ser Pro Pro Glu Thr Pro Val Leu Thr Arg Ser Ser Ala 755 760 765 Gln Thr Ser Ala Ala Gly Ala Thr Ala Thr Thr Ser Thr Ser Ser Thr 770 775 780 Val Thr Val Thr Ala Pro Ala Pro Ala Ala Thr Gly Ser Pro Val Lys 785 790 795 800 Lys Gln Arg Pro Leu Leu Pro Lys Glu Thr Ala Pro Ala Val Gln Arg 805 810 815 Val Val Trp Asn Ser Ser Ser Lys Phe Gln Thr Ser Ser Gln Lys Trp 820 825 830 His Met Gln Lys Met Gln Arg Gln Gln Gln Gln Gln Gln Gln Gln Asn 835 840 845 Gln Gln Gln Gln Pro Gln Ser Ser Gln Gly Thr Arg Tyr Gln Thr Arg 850 855 860 Gln Ala Val Lys Ala Val Gln Gln Lys Glu Ile Thr Gln Ser Pro Ser 865 870 875 880 Thr Ser Thr Ile Thr Leu Val Thr Ser Thr Gln Ser Ser Pro Leu Val 885 890 895 Thr Ser Ser Gly Ser Met Ser Thr Leu Val Ser Ser Val Asn Ala Asp 900 905 910 Leu Pro Ile Ala Thr Ala Ser Ala Asp Val Ala Ala Asp Ile Ala Lys 915 920 925 Tyr Thr Ser Lys Met Met Asp Ala Ile Lys Gly Thr Met Thr Glu Ile 930 935 940 Tyr Asn Asp Leu Ser Lys Asn Thr Thr Gly Ser Thr Ile Ala Glu Ile 945 950 955 960 Arg Arg Leu Arg Ile Glu Ile Glu Lys Leu Gln Trp Leu His Gln Gln 965 970 975 Glu Leu Ser Glu Met Lys His Asn Leu Glu Leu Thr Met Ala Glu Met 980 985 990 Arg Gln Ser Leu Glu Gln Glu Arg Asp Arg Leu Ile Ala Glu Val Lys 995 1000 1005 Lys Gln Leu Glu Leu Glu Lys Gln Gln Ala Val Asp Glu Thr Lys 1010 1015 1020 Lys Lys Gln Trp Cys Ala Asn Cys Lys Lys Glu Ala Ile Phe Tyr 1025 1030 1035 Cys Cys Trp Asn Thr Ser Tyr Cys Asp Tyr Pro Cys Gln Gln Ala 1040 1045 1050 His Trp Pro Glu His Met Lys Ser Cys Thr Gln Ser Ala Thr Ala 1055 1060 1065 Pro Gln Gln Glu Ala Asp Ala Glu Val Asn Thr Glu Thr Leu Asn 1070 1075 1080 Lys Ser Ser Gln Gly Ser Ser Ser Ser Thr Gln Ser Ala Pro Ser 1085 1090 1095 Glu Thr Ala Ser Ala Ser Lys Glu Lys Glu Thr Ser Ala Glu Lys 1100 1105 1110 Ser Lys Glu Ser Gly Ser Thr Leu Asp Leu Ser Gly Ser Arg Glu 1115 1120 1125 Thr Pro Ser Ser Ile Leu Leu Gly Ser Asn Gln Gly Ser Asp His 1130 1135 1140 Ser Arg Ser Asn Lys Ser Ser Trp Ser Ser Ser Asp Glu Lys Arg 1145 1150 1155 Gly Ser Thr Arg Ser Asp His Asn Thr Ser Thr Ser Thr Lys Ser 1160 1165 1170 Leu Leu Pro Lys Glu Ser Arg Leu Asp Thr Phe Trp Asp 1175 1180 1185 <210> 25 <211> 1454 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 25 Met Ala Pro Val Gln Leu Glu Asn His Gln Leu Val Pro Pro Gly Gly 1 5 10 15 Gly Gly Gly Gly Ser Gly Gly Pro Ser Ala Pro Ala Pro Pro Pro 20 25 30 Pro Gly Ala Ala Val Ala Ala Ala Ala Ala Ala Ala Ala Ser Pro Gly 35 40 45 Tyr Arg Leu Ser Thr Leu Ile Glu Phe Leu Leu His Arg Ala Tyr Ser 50 55 60 Glu Leu Met Val Leu Thr Asp Leu Leu Pro Arg Lys Ser Asp Val Glu 65 70 75 80 Arg Lys Ile Glu Ile Val Gln Phe Ala Ser Arg Thr Arg Gln Leu Phe 85 90 95 Val Arg Leu Leu Ala Leu Val Lys Trp Ala Asn Asn Ala Gly Lys Val 100 105 110 Glu Lys Cys Ala Met Ile Ser Ser Phe Leu Asp Gln Gln Ala Ile Leu 115 120 125 Phe Val Asp Thr Ala Asp Arg Leu Ala Ser Leu Ala Arg Asp Ala Leu 130 135 140 Val His Ala Arg Leu Pro Ser Phe Ala Ile Pro Tyr Ala Ile Asp Val 145 150 155 160 Leu Thr Thr Gly Ser Tyr Pro Arg Leu Pro Thr Cys Ile Arg Asp Lys 165 170 175 Ile Ile Pro Pro Asp Pro Ile Thr Lys Ile Glu Lys Gln Ala Thr Leu 180 185 190 His Gln Leu Asn Gln Ile Leu Arg His Arg Leu Val Thr Thr Asp Leu 195 200 205 Pro Pro Gln Leu Ala Asn Leu Thr Val Ala Asn Gly Arg Val Lys Phe 210 215 220 Arg Val Glu Gly Glu Phe Glu Ala Thr Leu Thr Val Met Gly Asp Asp 225 230 235 240 Pro Asp Val Pro Trp Arg Leu Leu Lys Leu Glu Ile Leu Val Glu Asp 245 250 255 Lys Glu Thr Gly Asp Gly Arg Ala Leu Val His Ser Met Gln Ile Ser 260 265 270 Phe Ile His Gln Leu Val Gln Ser Arg Leu Phe Ala Asp Glu Lys Pro 275 280 285 Leu Gln Asp Met Tyr Asn Cys Leu His Ser Phe Cys Leu Ser Leu Gln 290 295 300 Leu Glu Val Leu His Ser Gln Thr Leu Met Leu Ile Arg Glu Arg Trp 305 310 315 320 Gly Asp Leu Val Gln Val Glu Arg Tyr His Ala Gly Lys Cys Leu Ser 325 330 335 Leu Ser Val Trp Asn Gln Gln Val Leu Gly Arg Lys Thr Gly Thr Ala 340 345 350 Ser Val His Lys Val Thr Ile Lys Ile Asp Glu Asn Asp Val Ser Lys 355 360 365 Pro Leu Gln Ile Phe His Asp Pro Pro Leu Pro Ala Ser Asp Ser Lys 370 375 380 Leu Val Glu Arg Ala Met Lys Ile Asp His Leu Ser Ile Glu Lys Leu 385 390 395 400 Leu Ile Asp Ser Val His Ala Arg Ala His Gln Lys Leu Gln Glu Leu 405 410 415 Lys Ala Ile Leu Arg Gly Phe Asn Ala Asn Glu Asn Ser Ser Ile Glu 420 425 430 Thr Ala Leu Pro Ala Leu Val Val Pro Ile Leu Glu Pro Cys Gly Asn 435 440 445 Ser Glu Cys Leu His Ile Phe Val Asp Leu His Ser Gly Met Phe Gln 450 455 460 Leu Met Leu Tyr Gly Leu Asp Gln Ala Thr Leu Asp Asp Met Glu Lys 465 470 475 480 Ser Val Asn Asp Asp Met Lys Arg Ile Ile Pro Trp Ile Gln Gln Leu 485 490 495 Lys Phe Trp Leu Gly Gln Gln Arg Cys Lys Gln Ser Ile Lys His Leu 500 505 510 Pro Thr Ile Ser Ser Glu Thr Leu Gln Leu Ser Asn Tyr Ser Thr His 515 520 525 Pro Ile Gly Asn Leu Ser Lys Asn Lys Leu Phe Ile Lys Leu Thr Arg 530 535 540 Leu Pro Gln Tyr Tyr Ile Val Val Glu Met Leu Glu Val Pro Asn Lys 545 550 555 560 Pro Thr Gln Leu Ser Tyr Lys Tyr Tyr Phe Met Ser Val Asn Ala Ala 565 570 575 Asp Arg Glu Asp Ser Pro Ala Met Ala Leu Leu Leu Gln Gln Phe Lys 580 585 590 Glu Asn Ile Gln Asp Leu Val Phe Arg Thr Lys Thr Gly Lys Gln Thr 595 600 605 Arg Thr Asn Ala Lys Arg Lys Leu Ser Asp Asp Pro Cys Pro Val Glu 610 615 620 Ser Lys Lys Thr Lys Arg Ala Gly Glu Met Cys Ala Phe Asn Lys Val 625 630 635 640 Leu Ala His Phe Val Ala Met Cys Asp Thr Asn Met Pro Phe Val Gly 645 650 655 Leu Arg Leu Glu Leu Ser Asn Leu Glu Ile Pro His Gln Gly Val Gln 660 665 670 Val Glu Gly Asp Gly Phe Ser His Ala Ile Arg Leu Leu Lys Ile Pro 675 680 685 Pro Cys Lys Gly Ile Thr Glu Glu Thr Gln Lys Ala Leu Asp Arg Ser 690 695 700 Leu Leu Asp Cys Thr Phe Arg Leu Gln Gly Arg Asn Asn Arg Thr Trp 705 710 715 720 Val Ala Glu Leu Val Phe Ala Asn Cys Pro Leu Asn Gly Thr Ser Thr 725 730 735 Arg Glu Gln Gly Pro Ser Arg His Val Tyr Leu Thr Tyr Glu Asn Leu 740 745 750 Leu Ser Glu Pro Val Gly Gly Arg Lys Val Val Glu Met Phe Leu Asn 755 760 765 Asp Trp Asn Ser Ile Ala Arg Leu Tyr Glu Cys Val Leu Glu Phe Ala 770 775 780 Arg Ser Leu Pro Asp Ile Pro Ala His Leu Asn Ile Phe Ser Glu Val 785 790 795 800 Arg Val Tyr Asn Tyr Arg Lys Leu Ile Leu Cys Tyr Gly Thr Thr Lys 805 810 815 Gly Ser Ser Ile Ser Ile Gln Trp Asn Ser Ile His Gln Lys Phe His 820 825 830 Ile Ser Leu Gly Thr Val Gly Pro Asn Ser Gly Cys Ser Asn Cys His 835 840 845 Asn Thr Ile Leu His Gln Leu Gln Glu Met Phe Asn Lys Thr Pro Asn 850 855 860 Val Val Gln Leu Leu Gln Val Leu Phe Asp Thr Gln Ala Pro Leu Asn 865 870 875 880 Ala Ile Asn Lys Leu Pro Thr Val Pro Met Leu Gly Leu Thr Gln Arg 885 890 895 Thr Asn Thr Ala Tyr Gln Cys Phe Ser Ile Leu Pro Gln Ser Ser Thr 900 905 910 His Ile Arg Leu Ala Phe Arg Asn Met Tyr Cys Ile Asp Ile Tyr Cys 915 920 925 Arg Ser Arg Gly Val Val Ala Ile Arg Asp Gly Ala Tyr Ser Leu Phe 930 935 940 Asp Asn Ser Lys Leu Val Glu Gly Phe Tyr Pro Ala Pro Gly Leu Lys 945 950 955 960 Thr Phe Leu Asn Met Phe Val Asp Ser Asn Gln Asp Ala Arg Arg Arg 965 970 975 Ser Val Asn Glu Asp Asp Asn Pro Pro Ser Pro Ile Gly Gly Asp Met 980 985 990 Met Asp Ser Leu Ile Ser Gln Leu Gln Pro Pro Pro Gln Gln Gln Pro 995 1000 1005 Phe Pro Lys Gln Pro Gly Thr Ser Gly Ala Tyr Pro Leu Thr Ser 1010 1015 1020 Pro Pro Thr Ser Tyr His Ser Thr Val Asn Gln Ser Pro Ser Met 1025 1030 1035 Met His Thr Gln Ser Pro Gly Asn Leu His Ala Ala Ser Ser Pro 1040 1045 1050 Ser Gly Ala Leu Arg Ala Pro Ser Pro Ala Ser Phe Val Pro Thr 1055 1060 1065 Pro Pro Pro Ser Ser His Gly Ile Ser Ile Gly Pro Gly Ala Ser 1070 1075 1080 Phe Ala Ser Pro His Gly Thr Leu Asp Pro Ser Ser Pro Tyr Thr 1085 1090 1095 Met Val Ser Pro Ser Gly Arg Ala Gly Asn Trp Pro Gly Ser Pro 1100 1105 1110 Gln Val Ser Gly Pro Ser Pro Ala Ala Arg Met Pro Gly Met Ser 1115 1120 1125 Pro Ala Asn Pro Ser Leu His Ser Pro Val Pro Asp Ala Ser His 1130 1135 1140 Ser Pro Arg Ala Gly Thr Ser Ser Gln Thr Met Pro Thr Asn Met 1145 1150 1155 Pro Pro Pro Arg Lys Leu Pro Gln Arg Ser Trp Ala Ala Ser Ile 1160 1165 1170 Pro Thr Ile Leu Thr His Ser Ala Leu Asn Ile Leu Leu Leu Pro 1175 1180 1185 Ser Pro Thr Pro Gly Leu Val Pro Gly Leu Ala Gly Ser Tyr Leu 1190 1195 1200 Cys Ser Pro Leu Glu Arg Phe Leu Gly Ser Val Ile Met Arg Arg 1205 1210 1215 His Leu Gln Arg Ile Ile Gln Gln Glu Thr Leu Gln Leu Ile Asn 1220 1225 1230 Ser Asn Glu Pro Gly Val Ile Met Phe Lys Thr Asp Ala Leu Lys 1235 1240 1245 Cys Arg Val Ala Leu Ser Pro Lys Thr Asn Gln Thr Leu Gln Leu 1250 1255 1260 Lys Val Thr Pro Glu Asn Ala Gly Gln Trp Lys Pro Asp Glu Leu 1265 1270 1275 Gln Val Leu Glu Lys Phe Phe Glu Thr Arg Val Ala Gly Pro Pro 1280 1285 1290 Phe Lys Ala Asn Thr Leu Ile Ala Phe Thr Lys Leu Leu Gly Ala 1295 1300 1305 Pro Thr His Ile Leu Arg Asp Cys Val His Ile Met Lys Leu Glu 1310 1315 1320 Leu Phe Pro Asp Gln Ala Thr Gln Leu Lys Trp Asn Val Gln Phe 1325 1330 1335 Cys Leu Thr Ile Pro Ser Ala Pro Pro Ile Ala Pro Pro Gly 1340 1345 1350 Thr Pro Ala Val Val Leu Lys Ser Lys Met Leu Phe Phe Leu Gln 1355 1360 1365 Leu Thr Gln Lys Thr Ser Val Pro Pro Gln Glu Pro Val Ser Ile 1370 1375 1380 Ile Val Pro Ile Ile Tyr Asp Met Ala Ser Gly Thr Thr Gln Gln 1385 1390 1395 Ala Asp Ile Pro Arg Gln Gln Asn Ser Ser Val Ala Ala Pro Met 1400 1405 1410 Met Val Ser Asn Ile Leu Lys Arg Phe Ala Glu Met Asn Pro Pro 1415 1420 1425 Arg Gln Gly Glu Cys Thr Ile Phe Ala Ala Val Arg Asp Leu Met 1430 1435 1440 Ala Asn Leu Thr Leu Pro Pro Gly Gly Arg Pro 1445 1450 <210> 26 <211> 631 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 26 Met Phe Tyr Ala His Phe Val Leu Ser Lys Arg Gly Pro Leu Ala Lys 1 5 10 15 Ile Trp Leu Ala Ala His Trp Asp Lys Lys Leu Thr Lys Ala His Val 20 25 30 Phe Glu Cys Asn Leu Glu Ser Ser Val Glu Ser Ile Ile Ser Pro Lys 35 40 45 Val Lys Met Ala Leu Arg Thr Ser Gly His Leu Leu Leu Gly Val Val 50 55 60 Arg Ile Tyr His Arg Lys Ala Lys Tyr Leu Leu Ala Asp Cys Asn Glu 65 70 75 80 Ala Phe Ile Lys Ile Lys Met Ala Phe Arg Pro Gly Val Val Asp Leu 85 90 95 Pro Glu Glu Asn Arg Glu Ala Ala Tyr Asn Ala Ile Thr Leu Pro Glu 100 105 110 Glu Phe His Asp Phe Asp Gln Pro Leu Pro Asp Leu Asp Asp Ile Asp 115 120 125 Val Ala Gln Gln Phe Ser Leu Asn Gln Ser Arg Val Glu Glu Ile Thr 130 135 140 Met Arg Glu Glu Val Gly Asn Ile Ser Ile Leu Gln Glu Asn Asp Phe 145 150 155 160 Gly Asp Phe Gly Met Asp Asp Arg Glu Ile Met Arg Glu Gly Ser Ala 165 170 175 Phe Glu Asp Asp Asp Met Leu Val Ser Thr Thr Thr Ser Asn Leu Leu 180 185 190 Leu Glu Ser Glu Gln Ser Thr Ser Asn Leu Asn Glu Lys Ile Asn His 195 200 205 Leu Glu Tyr Glu Asp Gln Tyr Lys Asp Asp Asn Phe Gly Glu Gly Asn 210 215 220 Asp Gly Gly Ile Leu Asp Asp Lys Leu Ile Ser Asn Asn Asp Gly Gly 225 230 235 240 Ile Phe Asp Asp Pro Pro Ala Leu Ser Glu Ala Gly Val Met Leu Pro 245 250 255 Glu Gln Pro Ala His Asp Asp Met Asp Glu Asp Asp Asn Val Ser Met 260 265 270 Gly Gly Pro Asp Ser Pro Asp Ser Val Asp Pro Val Glu Pro Met Pro 275 280 285 Thr Met Thr Asp Gln Thr Thr Leu Val Pro Asn Glu Glu Glu Ala Phe 290 295 300 Ala Leu Glu Pro Ile Asp Ile Thr Val Lys Glu Thr Lys Ala Lys Arg 305 310 315 320 Lys Arg Lys Leu Ile Val Asp Ser Val Lys Glu Leu Asp Ser Lys Thr 325 330 335 Ile Arg Ala Gln Leu Ser Asp Tyr Ser Asp Ile Val Thr Thr Leu Asp 340 345 350 Leu Ala Pro Pro Thr Lys Lys Leu Met Met Trp Lys Glu Thr Gly Gly 355 360 365 Val Glu Lys Leu Phe Ser Leu Pro Ala Gln Pro Leu Trp Asn Asn Arg 370 375 380 Leu Leu Lys Leu Phe Thr Arg Cys Leu Thr Pro Leu Val Pro Glu Asp 385 390 395 400 Leu Arg Lys Arg Arg Lys Gly Gly Glu Ala Asp Asn Leu Asp Glu Phe 405 410 415 Leu Lys Glu Phe Glu Asn Pro Glu Val Pro Arg Glu Asp Gln Gln Gln 420 425 430 Gln His Gln Gln Arg Asp Val Ile Asp Glu Pro Ile Ile Glu Glu Pro 435 440 445 Ser Arg Leu Gln Glu Ser Val Met Glu Ala Ser Arg Thr Asn Ile Asp 450 455 460 Glu Ser Ala Met Pro Pro Pro Pro Pro Gln Gly Val Lys Arg Lys Ala 465 470 475 480 Gly Gln Ile Asp Pro Glu Pro Val Met Pro Pro Gln Gln Val Glu Gln 485 490 495 Met Glu Ile Pro Pro Val Glu Leu Pro Pro Glu Glu Pro Pro Asn Ile 500 505 510 Cys Gln Leu Ile Pro Glu Leu Glu Leu Leu Pro Glu Lys Glu Lys Glu 515 520 525 Lys Glu Lys Glu Lys Glu Asp Asp Glu Glu Glu Glu Asp Glu Asp Ala 530 535 540 Ser Gly Gly Asp Gln Asp Gln Glu Glu Arg Arg Trp Asn Lys Arg Thr 545 550 555 560 Gln Gln Met Leu His Gly Leu Gln Arg Ala Leu Ala Lys Thr Gly Ala 565 570 575 Glu Ser Ile Ser Leu Leu Glu Leu Cys Arg Asn Thr Asn Arg Lys Gln 580 585 590 Ala Ala Ala Lys Phe Tyr Ser Phe Leu Val Leu Lys Lys Gln Gln Ala 595 600 605 Ile Glu Leu Thr Gln Glu Glu Pro Tyr Ser Asp Ile Ile Ala Thr Pro 610 615 620 Gly Pro Arg Phe His Ile Ile 625 630 <210> 27 <211> 467 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 27 Met Ala Thr Gly Ala Asp Val Arg Asp Ile Leu Glu Leu Gly Gly Pro 1 5 10 15 Glu Gly Asp Ala Ala Ser Gly Thr Ile Ser Lys Lys Asp Ile Ile Asn 20 25 30 Pro Asp Lys Lys Lys Ser Lys Lys Ser Ser Glu Thr Leu Thr Phe Lys 35 40 45 Arg Pro Glu Gly Met His Arg Glu Val Tyr Ala Leu Leu Tyr Ser Asp 50 55 60 Light Light Asp Ala Pro Pro Leu Leu Pro Ser Asp Thr Gly Gln Gly Tyr 65 70 75 80 Arg Thr Val Lys Ala Lys Leu Gly Ser Lys Lys Val Arg Pro Trp Lys 85 90 95 Trp Met Pro Phe Thr Asn Pro Ala Arg Lys Asp Gly Ala Met Phe Phe 100 105 110 His Trp Arg Arg Ala Ala Glu Glu Gly Lys Asp Tyr Pro Phe Ala Arg 115 120 125 Phe Asn Lys Thr Val Gln Val Pro Val Tyr Ser Glu Gln Glu Tyr Gln 130 135 140 Leu Tyr Leu His Asp Asp Ala Trp Thr Lys Ala Glu Thr Asp His Leu 145 150 155 160 Phe Asp Leu Ser Arg Arg Phe Asp Leu Arg Phe Val Val Ile His Asp 165 170 175 Arg Tyr Asp His Gln Gln Phe Lys Lys Arg Ser Val Glu Asp Leu Lys 180 185 190 Glu Arg Tyr Tyr His Ile Cys Ala Lys Leu Ala Asn Val Arg Ala Val 195 200 205 Pro Gly Thr Asp Leu Lys Ile Pro Val Phe Asp Ala Gly His Glu Arg 210 215 220 Arg Arg Lys Glu Gln Leu Glu Arg Leu Tyr Asn Arg Thr Pro Glu Gln 225 230 235 240 Val Ala Glu Glu Glu Tyr Leu Leu Gln Glu Leu Arg Lys Ile Glu Ala 245 250 255 Arg Lys Lys Glu Arg Glu Lys Arg Ser Gln Asp Leu Gln Lys Leu Ile 260 265 270 Thr Ala Ala Asp Thr Thr Ala Glu Gln Arg Arg Thr Glu Arg Lys Ala 275 280 285 Pro Lys Lys Lys Leu Pro Gln Lys Lys Glu Ala Glu Lys Pro Ala Val 290 295 300 Pro Glu Thr Ala Gly Ile Lys Phe Pro Asp Phe Lys Ser Ala Gly Val 305 310 315 320 Thr Leu Arg Ser Gln Arg Met Lys Leu Pro Ser Ser Val Gly Gln Lys 325 330 335 Lys Ile Lys Ala Leu Glu Gln Met Leu Leu Glu Leu Gly Val Glu Leu 340 345 350 Ser Pro Thr Pro Thr Glu Glu Leu Val His Met Phe Asn Glu Leu Arg 355 360 365 Ser Asp Leu Val Leu Leu Tyr Glu Leu Lys Gln Ala Cys Ala Asn Cys 370 375 380 Glu Tyr Glu Leu Gln Met Leu Arg His Arg His Glu Ala Leu Ala Arg 385 390 395 400 Ala Gly Val Leu Gly Gly Pro Ala Thr Pro Ala Ser Gly Pro Gly Pro 405 410 415 Ala Ser Ala Glu Pro Ala Val Thr Glu Pro Gly Leu Gly Pro Asp Pro 420 425 430 Lys Asp Thr Ile Ile Asp Val Val Gly Ala Pro Leu Thr Pro Asn Ser 435 440 445 Arg Lys Arg Arg Glu Ser Ala Ser Ser Ser Ser Ser Val Lys Lys Ala 450 455 460 Lys Lys Pro 465 <210> 28 <211> 85 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 28 Met Ala Thr Tyr Ser Leu Ala Asn Glu Arg Leu Arg Ala Leu Glu Asp 1 5 10 15 Ile Glu Arg Glu Ile Gly Ala Ile Leu Gln Asn Ala Gly Thr Val Ile 20 25 30 Leu Glu Leu Ser Lys Glu Lys Thr Asn Glu Arg Leu Leu Asp Arg Gln 35 40 45 Ala Ala Ala Phe Thr Ala Ser Val Gln His Val Glu Ala Glu Leu Ser 50 55 60 Ala Gln Ile Arg Tyr Leu Thr Gln Leu Pro Asp Gly Leu Thr Asn Ser 65 70 75 80 Asn Ser Gly Lys Lys 85 <210> 29 <211> 390 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 29 Met Ala Leu Leu Thr Pro Ala Pro Gly Ser Gln Ser Ser Gln Phe Gln 1 5 10 15 Leu Met Lys Ala Leu Leu Lys His Glu Ser Val Gly Ser Gln Pro Leu 20 25 30 Gln Asp Arg Val Leu Gln Val Pro Val Leu Ala His Gly Gly Cys Cys 35 40 45 Arg Glu Asp Lys Val Val Ala Ser Arg Leu Thr Pro Glu Ser Gln Gly 50 55 60 Leu Leu Lys Val Glu Asp Val Ala Leu Thr Leu Thr Pro Glu Trp Thr 65 70 75 80 Gln Gln Asp Ser Ser Gln Gly Asn Leu Cys Arg Asp Glu Lys Gln Glu 85 90 95 Asn His Gly Ser Leu Val Ser Leu Gly Asp Glu Lys Gln Thr Lys Ser 100 105 110 Arg Asp Leu Pro Pro Ala Glu Glu Leu Pro Glu Lys Glu His Gly Lys 115 120 125 Ile Ser Cys His Leu Arg Glu Asp Ile Ala Gln Ile Pro Thr Cys Ala 130 135 140 Glu Ala Gly Glu Gln Glu Gly Arg Leu Gln Arg Lys Gln Lys Asn Ala 145 150 155 160 Thr Gly Gly Arg Arg His Ile Cys His Glu Cys Gly Lys Ser Phe Ala 165 170 175 Gln Ser Ser Gly Leu Ser Lys His Arg Arg Ile His Thr Gly Glu Lys 180 185 190 Pro Tyr Glu Cys Glu Glu Cys Gly Lys Ala Phe Ile Gly Ser Ser Ala 195 200 205 Leu Val Ile His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys 210 215 220 Glu Glu Cys Gly Lys Ala Phe Ser His Ser Ser Asp Leu Ile Lys His 225 230 235 240 Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asp Asp Cys Gly 245 250 255 Lys Thr Phe Ser Gln Ser Cys Ser Leu Leu Glu His His Arg Ile His 260 265 270 Thr Gly Glu Lys Pro Tyr Gln Cys Ser Met Cys Gly Lys Ala Phe Arg 275 280 285 Arg Ser Ser His Leu Leu Arg His Gln Arg Ile His Thr Gly Asp Lys 290 295 300 Asn Val Gln Glu Pro Glu Gln Gly Glu Ala Trp Lys Ser Arg Met Glu 305 310 315 320 Ser Gln Leu Glu Asn Val Glu Thr Pro Met Ser Tyr Lys Cys Asn Glu 325 330 335 Cys Glu Arg Ser Phe Thr Gln Asn Thr Gly Leu Ile Glu His Gln Lys 340 345 350 Ile His Thr Gly Glu Lys Pro Tyr Gln Cys Asn Ala Cys Gly Lys Gly 355 360 365 Phe Thr Arg Ile Ser Tyr Leu Val Gln His Gln Arg Ser His Val Gly 370 375 380 Lys Asn Ile Leu Ser Gln 385 390 <210> 30 <211> 677 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 30 Met Met Gln Glu Ser Gly Thr Glu Thr Lys Ser Asn Gly Ser Ala Ile 1 5 10 15 Gln Asn Gly Ser Gly Gly Ser Asn His Leu Leu Glu Cys Gly Gly Leu 20 25 30 Arg Glu Gly Arg Ser Asn Gly Glu Thr Pro Ala Val Asp Ile Gly Ala 35 40 45 Ala Asp Leu Ala His Ala Gln Gln Gln Gln Gln Gln Ala Leu Gln Val 50 55 60 Ala Arg Gln Leu Leu Leu Gln Gln Gln Gln Gln Gln Gln Val Ser Gly 65 70 75 80 Leu Lys Ser Pro Lys Arg Asn Asp Lys Gln Pro Ala Leu Gln Val Pro 85 90 95 Val Ser Val Ala Met Met Thr Pro Gln Val Ile Thr Pro Gln Gln Met 100 105 110 Gln Gln Ile Leu Gln Gln Gln Val Leu Ser Pro Gln Gln Leu Gln Val 115 120 125 Leu Leu Gln Gln Gln Gln Ala Leu Met Leu Gln Gln Gln Gln Leu Gln 130 135 140 Glu Phe Tyr Lys Lys Gln Gln Glu Gln Leu Gln Leu Gln Leu Leu Gln 145 150 155 160 Gln Gln His Ala Gly Lys Gln Pro Lys Glu Gln Gln Gln Val Ala Thr 165 170 175 Gln Gln Leu Ala Phe Gln Gln Gln Leu Leu Gln Met Gln Gln Leu Gln 180 185 190 Gln Gln His Leu Leu Ser Leu Gln Arg Gln Gly Leu Leu Thr Ile Gln 195 200 205 Pro Gly Gln Pro Ala Leu Pro Leu Gln Pro Leu Ala Gln Gly Met Ile 210 215 220 Pro Thr Glu Leu Gln Gln Leu Trp Lys Glu Val Thr Ser Ala His Thr 225 230 235 240 Ala Glu Glu Thr Thr Gly Asn Asn His Ser Ser Leu Asp Leu Thr Thr 245 250 255 Thr Cys Val Ser Ser Ser Ala Pro Ser Lys Thr Ser Leu Ile Met Asn 260 265 270 Pro His Ala Ser Thr Asn Gly Gln Leu Ser Val His Thr Pro Lys Arg 275 280 285 Glu Ser Leu Ser His Glu Glu His Pro His Ser His Pro Leu Tyr Gly 290 295 300 His Gly Val Cys Lys Trp Pro Gly Cys Glu Ala Val Cys Glu Asp Phe 305 310 315 320 Gln Ser Phe Leu Lys His Leu Asn Ser Glu His Ala Leu Asp Asp Arg 325 330 335 Ser Thr Ala Gln Cys Arg Val Gln Met Gln Val Val Gln Gln Leu Glu 340 345 350 Leu Gln Leu Ala Lys Asp Lys Glu Arg Leu Gln Ala Met Met Thr His 355 360 365 Leu His Val Lys Ser Thr Glu Pro Lys Ala Ala Pro Gln Pro Leu Asn 370 375 380 Leu Val Ser Ser Val Thr Leu Ser Lys Ser Ala Ser Glu Ala Ser Pro 385 390 395 400 Gln Ser Leu Pro His Thr Pro Thr Thr Pro Thr Ala Pro Leu Thr Pro 405 410 415 Val Thr Gln Gly Pro Ser Val Ile Thr Thr Thr Ser Met His Thr Val 420 425 430 Gly Pro Ile Arg Arg Arg Tyr Ser Asp Lys Tyr Asn Val Pro Ile Ser 435 440 445 Ser Ala Asp Ile Ala Gln Asn Gln Glu Phe Tyr Lys Asn Ala Glu Val 450 455 460 Arg Pro Pro Phe Thr Tyr Ala Ser Leu Ile Arg Gln Ala Ile Leu Glu 465 470 475 480 Ser Pro Glu Lys Gln Leu Thr Leu Asn Glu Ile Tyr Asn Trp Phe Thr 485 490 495 Arg Met Phe Ala Tyr Phe Arg Arg Asn Ala Ala Thr Trp Lys Asn Ala 500 505 510 Val Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Asn 515 520 525 Val Lys Gly Ala Val Trp Thr Val Asp Glu Val Glu Phe Gln Lys Arg 530 535 540 Arg Pro Gln Lys Ile Ser Gly Asn Pro Ser Leu Ile Lys Asn Met Gln 545 550 555 560 Ser Ser His Ala Tyr Cys Thr Pro Leu Asn Ala Ala Leu Gln Ala Ser 565 570 575 Met Ala Glu Asn Ser Ile Pro Leu Tyr Thr Thr Ala Ser Met Gly Asn 580 585 590 Pro Thr Leu Gly Asn Leu Ala Ser Ala Ile Arg Glu Glu Leu Asn Gly 595 600 605 Ala Met Glu His Thr Asn Ser Asn Glu Ser Asp Ser Ser Pro Gly Arg 610 615 620 Ser Pro Met Gln Ala Val His Pro Val His Val Lys Glu Glu Pro Leu 625 630 635 640 Asp Pro Glu Glu Ala Glu Gly Pro Leu Ser Leu Val Thr Thr Ala Asn 645 650 655 His Ser Pro Asp Phe Asp His Asp Arg Asp Tyr Glu Asp Glu Pro Val 660 665 670 Asn Glu Asp Met Glu 675 <210> 31 <211> 787 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 31 Met Ala Val Trp Ile Gln Ala Gln Gln Leu Gln Gly Glu Ala Leu His 1 5 10 15 Gln Met Gln Ala Leu Tyr Gly Gln His Phe Pro Ile Glu Val Arg His 20 25 30 Tyr Leu Ser Gln Trp Ile Glu Ser Gln Ala Trp Asp Ser Val Asp Leu 35 40 45 Asp Asn Pro Gln Glu Asn Ile Lys Ala Thr Gln Leu Leu Glu Gly Leu 50 55 60 Val Gln Glu Leu Gln Lys Lys Ala Glu His Gln Val Gly Glu Asp Gly 65 70 75 80 Phe Leu Leu Lys Ile Lys Leu Gly His Tyr Ala Thr Gln Leu Gln Asn 85 90 95 Thr Tyr Asp Arg Cys Pro Met Glu Leu Val Arg Cys Ile Arg His Ile 100 105 110 Leu Tyr Asn Glu Gln Arg Leu Val Arg Glu Ala Asn Asn Gly Ser Ser 115 120 125 Pro Ala Gly Ser Leu Ala Asp Ala Met Ser Gln Lys His Leu Gln Ile 130 135 140 Asn Gln Thr Phe Glu Glu Leu Arg Leu Val Thr Gln Asp Thr Glu Asn 145 150 155 160 Glu Leu Lys Lys Leu Gln Gln Thr Gln Glu Tyr Phe Ile Ile Gln Tyr 165 170 175 Gln Glu Ser Leu Arg Ile Gln Ala Gln Phe Gly Pro Leu Ala Gln Leu 180 185 190 Ser Pro Gln Glu Arg Leu Ser Arg Glu Thr Ala Leu Gln Gln Lys Gln 195 200 205 Val Ser Leu Glu Ala Trp Leu Gln Arg Glu Ala Gln Thr Leu Gln Gln 210 215 220 Tyr Arg Val Glu Leu Ala Glu Lys His Gln Lys Thr Leu Gln Leu Leu 225 230 235 240 Arg Lys Gln Gln Thr Ile Ile Leu Asp Asp Glu Leu Ile Gln Trp Lys 245 250 255 Arg Arg Gln Gln Leu Ala Gly Asn Gly Gly Pro Pro Glu Gly Ser Leu 260 265 270 Asp Val Leu Gln Ser Trp Cys Glu Lys Leu Ala Glu Ile Ile Trp Gln 275 280 285 Asn Arg Gln Gln Ile Arg Arg Ala Glu His Leu Cys Gln Gln Leu Pro 290 295 300 Ile Pro Gly Pro Val Glu Glu Met Leu Ala Glu Val Asn Ala Thr Ile 305 310 315 320 Thr Asp Ile Ile Ser Ala Leu Val Thr Ser Thr Phe Ile Ile Glu Lys 325 330 335 Gln Pro Pro Gln Val Leu Lys Thr Gln Thr Lys Phe Ala Ala Thr Val 340 345 350 Arg Leu Leu Val Gly Gly Lys Leu Asn Val His Met Asn Pro Pro Gln 355 360 365 Val Lys Ala Thr Ile Ile Ser Glu Gln Gln Ala Lys Ser Leu Leu Lys 370 375 380 Asn Glu Asn Thr Arg Asn Asp Tyr Ser Gly Glu Ile Leu Asn Asn Cys 385 390 395 400 Cys Val Met Glu Tyr His Gln Ala Thr Gly Thr Leu Ser Ala His Phe 405 410 415 Arg Asn Met Ser Leu Lys Arg Ile Lys Arg Ser Asp Arg Arg Gly Ala 420 425 430 Glu Ser Val Thr Glu Glu Lys Phe Thr Ile Leu Phe Glu Ser Gln Phe 435 440 445 Ser Val Gly Gly Asn Glu Leu Val Phe Gln Val Lys Thr Leu Ser Leu 450 455 460 Pro Val Val Val Ile Val His Gly Ser Gln Asp Asn Asn Ala Thr Ala 465 470 475 480 Thr Val Leu Trp Asp Asn Ala Phe Ala Glu Pro Gly Arg Val Pro Phe 485 490 495 Ala Val Pro Asp Lys Val Leu Trp Pro Gln Leu Cys Glu Ala Leu Asn 500 505 510 Met Lys Phe Lys Ala Glu Val Gln Ser Asn Arg Gly Leu Thr Lys Glu 515 520 525 Asn Leu Val Phe Leu Ala Gln Lys Leu Phe Asn Asn Ser Ser Ser His 530 535 540 Leu Glu Asp Tyr Ser Gly Leu Ser Val Ser Trp Ser Gln Phe Asn Arg 545 550 555 560 Glu Asn Leu Pro Gly Arg Asn Tyr Thr Phe Trp Gln Trp Phe Asp Gly 565 570 575 Val Met Glu Val Leu Lys Lys His Leu Lys Pro His Trp Asn Asp Gly 580 585 590 Ala Ile Leu Gly Phe Val Asn Lys Gln Gln Ala His Asp Leu Leu Ile 595 600 605 Asn Lys Pro Asp Gly Thr Phe Leu Leu Arg Phe Ser Asp Ser Glu Ile 610 615 620 Gly Gly Ile Thr Ile Ala Trp Lys Phe Asp Ser Gln Glu Arg Met Phe 625 630 635 640 Trp Asn Leu Met Pro Phe Thr Thr Arg Asp Phe Ser Ile Arg Ser Leu 645 650 655 Ala Asp Arg Leu Gly Asp Leu Asn Tyr Leu Ile Tyr Val Phe Pro Asp 660 665 670 Arg Pro Lys Asp Glu Val Tyr Ser Lys Tyr Tyr Thr Pro Val Pro Cys 675 680 685 Glu Ser Ala Thr Ala Lys Ala Val Asp Gly Tyr Val Lys Pro Gln Ile 690 695 700 Lys Gln Val Val Pro Glu Phe Val Asn Ala Ser Ala Asp Ala Gly Gly 705 710 715 720 Gly Ser Ala Thr Tyr Met Asp Gln Ala Pro Ser Pro Ala Val Cys Pro 725 730 735 Gln Ala His Tyr Asn Met Tyr Pro Gln Asn Pro Asp Ser Val Leu Asp 740 745 750 Thr Asp Gly Asp Phe Asp Leu Glu Asp Thr Met Asp Val Ala Arg Arg 755 760 765 Val Glu Glu Leu Leu Gly Arg Pro Met Asp Ser Gln Trp Ile Pro His 770 775 780 Ala Gln Ser 785 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 32 caacagatta tcacaaatcg 20 <210> 33 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 33 catcatccgg acaccaacag 20 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 34 gtatgtgacc aatgtaccag 20 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 35 ttactaccag tggatcatca 20 <210> 36 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 36 acttcggcaa gactttgagg 20 <210> 37 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 37 gcatcgcacc atgtctcagg 20 <210> 38 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 38 ggagggcact accactacgc 20 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 39 tcggttgaca atcaatagtg 20 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 40 acaacgatac caataggttg 20 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 41 agctgaatca ctgataacaa 20 <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 42 ctggattcgg aataccctag 20 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 43 gaagcgggct aattccaaga 20 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 44 aggttggtga ctgtgaacgc 20 <210> 45 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 45 agttcatcga caacaagctg 20 <210> 46 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 46 gggctgacac tagcagacac 20 <210> 47 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 47 tctgttgtgg ggtctgaacg 20 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 48 aatttcatgc caagtcacct 20 <210> 49 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 49 ccagtaccaa tattagcatg 20 <210> 50 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 50 gttattgtac aggttcgagt 20 <210> 51 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 51 tgataatcaa gtgattcact 20 <210> 52 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 52 aagcacataa agatgaacgg 20 <210> 53 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 53 gaattgctag ttaaaacgcc 20 <210> 54 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 54 gccctatcgg cagtactacg 20 <210> 55 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 55 tatgcatgat agtaagacga 20 <210> 56 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 56 gagaacctcg gaacatacgg 20 <210> 57 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 57 gcagatcgag tcctacccca 20 <210> 58 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 58 tcttcttgtc tcggcccatg 20 <210> 59 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 59 tgagaacgca tctcagcccg 20 <210> 60 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 60 aaaccagggc caccgaaagg 20 <210> 61 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 61 ccctcgcgct tgaggccgcg 20 <210> 62 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 62 cttcggtctc ttcgacgacg 20 <210> 63 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 63 tcggggtaat agaacgcagg 20 <210> 64 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 64 cgatccaaat ttgaacgccg 20 <210> 65 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 65 gagcaaactg cgttatacag 20 <210> 66 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 66 ttaccccaga accagacgga 20 <210> 67 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 67 tttgtgcagt tatgccagca 20 <210> 68 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 68 atgctaagta cctgtgaaag 20 <210> 69 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 69 cattgaatat gattgcaagg 20 <210> 70 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 70 taggtgttga tacgagccca 20 <210> 71 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 71 tattcataga tctactgaca 20 <210> 72 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 72 acccaggcat catccgacaa 20 <210> 73 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 73 cccacccaca gggatcaacg 20 <210> 74 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 74 cctacttagg cactgccagg 20 <210> 75 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 75 gagggtgcca ccatgactag 20 <210> 76 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 76 acctggtgtg gacccacgcg 20 <210> 77 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 77 cctcgaactg caccataggt 20 <210> 78 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 78 gccattgatc tgatgtacgg 20 <210> 79 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 79 tggggctctg catctcacag 20 <210> 80 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 80 aagtcgacat actctcggct 20 <210> 81 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 81 cctgcctcac ctcacactcg 20 <210> 82 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 82 gccgacttac gatttccgag 20 <210> 83 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 83 ggagtctgtg ttatctggaa 20 <210> 84 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 84 cacttcgacc gacaaacctg 20 <210> 85 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 85 ctgatcgtag gaccacggtg 20 <210> 86 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 86 ggcagtggag tggttcaggg 20 <210> 87 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 87 tagatgatca gaccaagccc 20 <210> 88 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 88 agagtgcatc gacccctcgg 20 <210> 89 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 89 ctgcggggag gactccgtcg 20 <210> 90 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 90 cttcggggag acaacgacgg 20 <210> 91 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 91 gctgcaccga gtcgtagtcg 20 <210> 92 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 92 aatcagatga caatgagtca 20 <210> 93 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 93 caatacaata tgccacaggg 20 <210> 94 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 94 cctaaccata tgcctatgca 20 <210> 95 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 95 ggcatgttgt gagagcgtgg 20 <210> 96 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 96 acactggaac atatcaagac 20 <210> 97 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 97 gacaaatgca atgaaaactg 20 <210> 98 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 98 ggacatcgtg taccgcacca 20 <210> 99 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 99 ggccgcccgg gaagtcaaca 20 <210> 100 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 100 cacggaccct gatagcatga 20 <210> 101 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 101 catcgctcag gagatatacg 20 <210> 102 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 102 gcagccgaat cgccaaccgc 20 <210> 103 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 103 gcttcgcagc ctgctaacca 20 <210> 104 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 104 acatcgactg ctggacaatg 20 <210> 105 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 105 cagtgagtag tgccaaacca 20 <210> 106 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 106 gtggcgtact gcacgtgtcg 20 <210> 107 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 107 ttcacattat gaccaacacc 20 <210> 108 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 108 atgatgtttg atgaccgtcg 20 <210> 109 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 109 ctgttgggac ataccgctcg 20 <210> 110 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 110 gatgatatga gccctcgtcg 20 <210> 111 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 111 taaaatcaaa gaacttcgag 20 <210> 112 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 112 cctccactgg aagacacggt 20 <210> 113 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 113 cgaacagccc cccatagtgg 20 <210> 114 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 114 gaggataaca cgcattgcgg 20 <210> 115 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 115 tgctgagtaa tacgtcacgg 20 <210> 116 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 116 cgggacacgt ctacttggtg 20 <210> 117 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 117 gcagaacgag gctgccctag 20 <210> 118 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 118 gcggaccagt gtacagcacg 20 <210> 119 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 119 taaggtgagg actttgcaca 20 <210> 120 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 120 atttccagga ggtgaaacat 20 <210> 121 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 121 ctggtatgag gacctgcaag 20 <210> 122 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 122 gactggaatc tggagagtga 20 <210> 123 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 123 tcagccaagc cagagaagca 20 <210> 124 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 124 agatgtattc cgcatagtca 20 <210> 125 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 125 cacttagcgt gataaacccg 20 <210> 126 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 126 ctcttccgcc caaacttccg 20 <210> 127 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 127 ggagcgcggc atatccgaca 20 <210> 128 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 128 atcacacata gcgacgaagt 20 <210> 129 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 129 cagagcatct ctagctaacg 20 <210> 130 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 130 ctaactctgc tacccaagtg 20 <210> 131 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 131 taatgttaat ccgagaacgg 20 <210> 132 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 132 aagtgttgtt tgatcagtca 20 <210> 133 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 133 acatactcta agtcaggcag 20 <210> 134 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 134 gtgtaattta gagagcagcg 20 <210> 135 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 135 tctgttcaga ctctaatagg 20 <210> 136 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 136 atgctgggca cgaacgacgg 20 <210> 137 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 137 catggataac aacaaaacgc 20 <210> 138 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 138 gaagctaccc cagaaaaagg 20 <210> 139 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 139 ggacattatc aacccggaca 20 <210> 140 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 140 cacagcagga ttcatctcag 20 <210> 141 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 141 gccgactcag cgcctcgcgg 20 <210> 142 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 142 gctcaggcct gagtaaacac 20 <210> 143 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 143 tcaccagctt ctgcacatgt 20 <210> 144 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 144 agaggaggag acacatgtcg 20 <210> 145 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 145 catacaccat gtccatagag 20 <210> 146 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 146 gccttctgac aattcagccc 20 <210> 147 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 147 gttctgtaga cttcacatgc 20 <210> 148 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 148 cagccaggac aacaatgcga 20 <210> 149 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 149 gtggccttaa tgttctcctg 20 <210> 150 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 150 gttcattgta caatatatgg 20 <210> 151 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 151 taagaggtca gaccgtcgtg 20 <210> 152 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 152 aaaccaacag attatcacaa atcgaggaag 30 <210> 153 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 153 ccatcatcat ccggacacca acagtggggc 30 <210> 154 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 154 ctcagtatgt gaccaatgta ccagtggccc 30 <210> 155 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 155 aactttacta ccagtggatc atcagggacc 30 <210> 156 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 156 agaaacttcg gcaagacttt gaggaggtca 30 <210> 157 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 157 aaaagcatcg caccatgtct caggaggtac 30 <210> 158 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 158 tgcaggaggg cactaccact acgcaggcgt 30 <210> 159 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 159 agtatcggtt gacaatcaat agtgaggcat 30 <210> 160 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 160 tgccacaacg ataccaatag gttgaggaga 30 <210> 161 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 161 ccaaagctga atcactgata acaagggcag 30 <210> 162 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 162 tttcctggat tcggaatacc ctagaggaac 30 <210> 163 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 163 caaggaagcg ggctaattcc aagacggtgt 30 <210> 164 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 164 cggcaggttg gtgactgtga acgccggctt 30 <210> 165 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 165 atggagttca tcgacaacaa gctgcggcgc 30 <210> 166 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 166 actggggctg acactagcag acactggtgc 30 <210> 167 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 167 ctggtctgtt gtggggtctg aacggggtgg 30 <210> 168 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 168 ttgcaatttc atgccaagtc acctgggtaa 30 <210> 169 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 169 tcccccagta ccaatattag catgtggcaa 30 <210> 170 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 170 gtctgttatt gtacaggttc gagtaggtga 30 <210> 171 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 171 gatctgataa tcaagtgatt cactgggaaa 30 <210> 172 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 172 tttgaagcac ataaagatga acggagggga 30 <210> 173 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 173 tgaggaattg ctagttaaaa cgccaggtaa 30 <210> 174 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 174 ggaggcccta tcggcagtac tacgtggagg 30 <210> 175 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 175 aagatatgca tgatagtaag acgaaggagc 30 <210> 176 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 176 tacagagaac ctcggaacat acggaggtag 30 <210> 177 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 177 gacagcagat cgagtcctac cccacggaca 30 <210> 178 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 178 gttctcttct tgtctcggcc catgcggttc 30 <210> 179 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 179 tccatgagaa cgcatctcag cccgaggtgc 30 <210> 180 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 180 gccgaaacca gggccaccga aaggcggcgg 30 <210> 181 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 181 ggcgccctcg cgcttgaggc cgcgcggtcc 30 <210> 182 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 182 cagccttcgg tctcttcgac gacgcggccg 30 <210> 183 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 183 gggttcgggg taatagaacg caggcggcgg 30 <210> 184 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 184 gtgacgatcc aaatttgaac gccgtggaca 30 <210> 185 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 185 aaaagagcaa actgcgttat acagaggagg 30 <210> 186 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 186 ccacttaccc cagaaccaga cggatggggg 30 <210> 187 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 187 gcagtttgtg cagttatgcc agcagggaca 30 <210> 188 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 188 ttctatgcta agtacctgtg aaagggggca 30 <210> 189 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 189 acgccattga atatgattgc aaggaggagc 30 <210> 190 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 190 ctgataggtg ttgatacgag cccagggtgc 30 <210> 191 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 191 ggcttattca tagatctact gacaggggga 30 <210> 192 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 192 cctcacccag gcatcatccg acaagggctc 30 <210> 193 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 193 tgtccccacc cacagggatc aacgtggcca 30 <210> 194 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 194 tctccctact taggcactgc caggcggacc 30 <210> 195 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 195 cccggagggt gccaccatga ctaggggcag 30 <210> 196 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 196 ctgcacctgg tgtggaccca cgcgaggcac 30 <210> 197 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 197 atcacctcga actgcaccat aggtgggtgg 30 <210> 198 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 198 ctcagccatt gatctgatgt acggaggcat 30 <210> 199 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 199 gagctggggc tctgcatctc acagcggtgc 30 <210> 200 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 200 ttctaagtcg acatactctc ggctaggtgt 30 <210> 201 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 201 cccgcctgcc tcacctcaca ctcgcggctc 30 <210> 202 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 202 gccagccgac ttacgatttc cgagcggccg 30 <210> 203 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 203 gaatggagtc tgtgttatct ggaaaggctg 30 <210> 204 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 204 cttccacttc gaccgacaaa cctgaggtca 30 <210> 205 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 205 aggactgatc gtaggaccac ggtggggatg 30 <210> 206 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 206 taaaggcagt ggagtggttc agggaggcac 30 <210> 207 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 207 aaactagatg atcagaccaa gcccgggagc 30 <210> 208 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 208 cctcagagtg catcgacccc tcggtggtct 30 <210> 209 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 209 tgccctgcgg ggaggactcc gtcgaggaga 30 <210> 210 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 210 ctcccttcgg ggagacaacg acggcggtgg 30 <210> 211 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 211 tacggctgca ccgagtcgta gtcgaggtca 30 <210> 212 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 212 acagaatcag atgacaatga gtcagggaca 30 <210> 213 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 213 tcagcaatac aatatgccac agggaggcgg 30 <210> 214 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 214 agggcctaac catatgccta tgcagggacc 30 <210> 215 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 215 tgaaggcatg ttgtgagagc gtggaggtgg 30 <210> 216 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 216 taccacactg gaacatatca agaccggtta 30 <210> 217 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 217 atatgacaaa tgcaatgaaa actgtggtgg 30 <210> 218 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 218 tgcaggacat cgtgtaccgc accatggaga 30 <210> 219 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 219 agcaggccgc ccgggaagtc aacacggcgt 30 <210> 220 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 220 acgacacgga ccctgatagc atgaaggatt 30 <210> 221 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 221 aggccatcgc tcaggagata tacgcggacc 30 <210> 222 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 222 aacagcagcc gaatcgccaa ccgccggtga 30 <210> 223 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 223 aggagcttcg cagcctgcta accacggtga 30 <210> 224 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 224 atccacatcg actgctggac aatgaggatg 30 <210> 225 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 225 cagtcagtga gtagtgccaa accaaggcac 30 <210> 226 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 226 atgggtggcg tactgcacgt gtcgtggctg 30 <210> 227 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 227 acctttcaca ttatgaccaa caccaggtca 30 <210> 228 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 228 tacaatgatg tttgatgacc gtcgcggacg 30 <210> 229 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 229 taaactgttg ggacataccg ctcggggcca 30 <210> 230 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 230 ttatgatgat atgagccctc gtcgaggacc 30 <210> 231 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 231 gtgctaaaat caaagaactt cgagaggtaa 30 <210> 232 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 232 tatgcctcca ctggaagaca cggtaggcat 30 <210> 233 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 233 ccagcgaaca gccccccata gtggtggtgg 30 <210> 234 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 234 agaggaggat aacacgcatt gcgggggagg 30 <210> 235 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 235 ctgctgctga gtaatacgtc acggtggtgc 30 <210> 236 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 236 gatgcgggac acgtctactt ggtggggctc 30 <210> 237 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 237 ttctgcagaa cgaggctgcc ctagaggtct 30 <210> 238 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 238 cactgcggac cagtgtacag cacgaggttc 30 <210> 239 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 239 tatgtaaggt gaggactttg cacagggcag 30 <210> 240 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 240 cttcatttcc aggaggtgaa acataggtac 30 <210> 241 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 241 aagcctggta tgaggacctg caagaggtcc 30 <210> 242 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 242 ctctgactgg aatctggaga gtgagggctc 30 <210> 243 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 243 tcagtcagcc aagccagaga agcagggtca 30 <210> 244 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 244 tggaagatgt attccgcata gtcagggtgc 30 <210> 245 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 245 cagacactta gcgtgataaa cccggggaca 30 <210> 246 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 246 cccgctcttc cgcccaaact tccgcggctg 30 <210> 247 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 247 tgggggagcg cggcatatcc gacaaggaaa 30 <210> 248 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 248 ttgtatcaca catagcgacg aagtgggcta 30 <210> 249 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 249 ggaccagagc atctctagct aacgaggcca 30 <210> 250 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 250 aacactaact ctgctaccca agtgcggtta 30 <210> 251 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 251 actctaatgt taatccgaga acggtgggga 30 <210> 252 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 252 gaacaagtgt tgtttgatca gtcatggttg 30 <210> 253 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 253 agacacatac tctaagtcag gcagtggctg 30 <210> 254 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 254 tcgagtgtaa tttagagagc agcgtggaga 30 <210> 255 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 255 gtgctctgtt cagactctaa taggaggtta 30 <210> 256 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 256 tttgatgctg ggcacgaacg acggcggaag 30 <210> 257 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 257 cggtcatgga taacaacaaa acgcaggtca 30 <210> 258 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 258 aaaagaagct accccagaaa aaggaggctg 30 <210> 259 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 259 agaaggacat tatcaacccg gacaaggtag 30 <210> 260 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 260 tggacacagc aggattcatc tcaggggaat 30 <210> 261 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 261 cggagccgac tcagcgcctc gcgggggcct 30 <210> 262 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 262 caaagctcag gcctgagtaa acacaggaga 30 <210> 263 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 263 ctgttcacca gcttctgcac atgtaggaat 30 <210> 264 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 264 gtgcagagga ggagacacat gtcgtggtca 30 <210> 265 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 265 cttgcataca ccatgtccat agagaggatg 30 <210> 266 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 266 caaggccttc tgacaattca gcccgggcag 30 <210> 267 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 267 ttgggttctg tagacttcac atgcaggtgg 30 <210> 268 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 268 atggcagcca ggacaacaat gcgacggcca 30 <210> 269 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 269 ctgggtggcc ttaatgttct cctgtggatt 30 <210> 270 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 270 ctctgttcat tgtacaatat atggcggatg 30 <210> 271 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 271 gaattaagag gtcagaccgt cgtggggcag 30

Claims

1. An ex vivo method for enhancing the stability of human regulatory T (Treg) cells, comprising: inhibiting expression of Rnf20, The inhibition step is By decreasing the expression of Rnf20, or Reducing the expression of a polynucleotide encoding Rnf20 Contains, or Mutating a polynucleotide encoding Rnf20, The method.

2. 2. The method of claim 1, wherein the inhibiting step comprises contacting a polynucleotide encoding Rnf20 with a targeting nuclease or a guide RNA (gRNA), or wherein the inhibiting step comprises contacting a polynucleotide encoding Rnf20 with at least one gRNA and a targeting nuclease, wherein at least one gRNA comprises a sequence set forth in any of SEQ ID NOs: 36-39.

3. the inhibiting step comprises contacting the polynucleotide with a targeted nuclease; or the inhibiting step comprises contacting the polynucleotide with a targeted nuclease, where the targeted nuclease introduces a double-stranded break at a target region within the polynucleotide; or the inhibiting step comprises contacting the polynucleotide with a targeted nuclease, the targeted nuclease being an RNA-guided nuclease; or or the inhibiting step comprises contacting the polynucleotide with a targeting nuclease, the targeting nuclease being a Cpf1 nuclease or a Cas9 nuclease, and the method further comprises introducing into the Treg cell a gRNA that specifically hybridizes to a target region within the polynucleotide; or the inhibiting step comprises contacting the polynucleotide with a targeting nuclease, the targeting nuclease being a Cpf1 nuclease or a Cas9 nuclease, and the method further comprises introducing into the Treg cell a gRNA that specifically hybridizes to a target region within the polynucleotide, the Cpf1 nuclease or the Cas9 nuclease and the gRNA being introduced into the Treg cell as a ribonucleoprotein (RNP) complex. The method of claim 1.

4. 4. The method of claim 3, wherein the inhibiting step comprises performing clustered regularly interspaced short palindromic repeats (CRISPR) / Cas genome editing.

5. (a) the Treg cells are administered to a human after the inhibiting step; and / or (b) the Treg cells are from a human, the Treg cells are treated to inhibit expression of Rnf20, and the treated Treg cells are reintroduced into the human, where inhibition of expression results in Treg cells with improved stability, or the human has an autoimmune disorder. The method according to any one of claims 1 to 4. (a) comprising a heterologous polynucleotide that inhibits expression of Rnf20; or (b) comprising at least one guide RNA (gRNA) comprising a sequence set forth in any one of SEQ ID NOs: 36-39, wherein expression of Rnf20 is reduced in the Treg cell compared to expression of Rnf20 in a Treg cell not comprising a gRNA; Treg cells.

7. A pharmaceutical composition for treating an autoimmune disorder in a subject, comprising the population of Treg cells described in claim 6.

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