Human enzyme-mediated homocysteine depletion for the treatment of hyperhomocysteinemia and homocystinuria patients

Abstract

Methods and compositions relating to the engineering of modified proteins having homocysteine ​​activity are described. For example, a modified cystathionine-γ-lyase (CGL) comprising one or more amino acid substitutions and capable of degrading homocysteine ​​is disclosed. Furthermore, compositions and methods for treating homocysteine ​​depletion or hyperhomocysteinemia using the disclosed enzymes or nucleic acids are provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 505,493, filed May 12, 2017, the entire contents of which are incorporated herein by reference.

[0003] Statement regarding federally sponsored research

[0004] This invention was made with the support of government grant number R01 CA139059 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0005] Reference to sequence list

[0006] This application contains a sequence list, which has been submitted via EFS-Web in ASCII format, and whose entire contents are incorporated herein by reference. The ASCII copy was created on April 24, 2018, named UTFBP1138WO_ST25.txt, and is 230KB in size. Technical Field

[0007] Recombinant engineered primate enzyme variants with homocysteine-degrading activity and stability suitable for human treatment are disclosed. Compositions and methods for treating homocysteine ​​and hyperhomocysteinemia using enzymes that degrade homocysteine ​​and homocysteine ​​are also provided.

[0008] Related technical specifications

[0009] Homocystinuria is a rare disease caused by a congenital metabolic defect involving sulfur amino acids. Typical homocystinuria, most commonly in its form, is caused by a deficiency of cystathionine β-synthase (CBS) (Mudd et al., 1964), while atypical homocystinuria is often associated with deficiencies in multiple enzymes involved in folate metabolism (e.g., MTHFR, MTRR, or MTR) (Kang et al., 1987). Patients with homocystinuria frequently present with thromboembolism, cognitive impairment, osteoporosis, and lens dislocation (Kruger et al., 2003). Patients also have hyperhomocysteinemia, with serum homocysteine ​​(Hcy) concentrations exceeding 15 μM. Patients with typical homocystinuria, as well as some atypical forms, often exhibit elevated serum methionine (Met) levels, while cystathionine (Cth) and cysteine ​​(Cys) levels are lower (Kruger et al., 2003). It is estimated that one in 344,000 people worldwide is affected, but in some countries the incidence is much higher. The most common medical condition associated with typical homocystinuria is cardiovascular complications, including an increased risk of blood clots. Other symptoms include skeletal deformities, intraocular lens dislocation, and developmental and learning disabilities.

[0010] Current treatments for typical homocystinuria include methionine restriction diets and high doses of vitamin B6 and betaine (N,N,N-trimethylglycine), the latter of which lowers homocysteine ​​levels. These treatments focus on preventing Hcy accumulation and have shown moderate to limited efficacy (Walter et al., 1998). While these therapies may be effective for some patients, responses are markedly volatile due to changes in the genetic mutations driving the disease. Consequently, there is an opportunity to develop a therapy that meets the needs of all patients by lowering blood homocysteine ​​levels back to normal. Therefore, new methods and compositions are needed to treat these patients. Summary of the Invention

[0011] A method for efficiently degrading homocysteine ​​using engineered human cystathionine-γ-lyase (CGL) is disclosed, making it a suitable therapy for treating patients with homocysteineuria and hyperhomocysteinemia by degrading excess serum homocysteine ​​and providing a reservoir of intracellular homocysteine. This method can reduce serum homocysteine ​​levels (tHcy) to less than 25 μM.

[0012] A treatment method is provided, comprising administering a modified CGL enzyme, a nucleic acid encoding a modified CGL enzyme, or a preparation containing a modified CGL enzyme in a gene therapy vector, particularly for treating subjects with homocystinuria or hyperhomocysteinemia. The subject can be any animal, such as a mouse. For example, the subject can be a mammal, rodent, primate, or human patient. The method may include selecting patients with homocystinuria or hyperhomocysteinemia. Treatment with the described composition may be combined with maintaining the subject or patient on a methionine-restricted diet or a normal diet.

[0013] This article provides a human cystathionine-γ-lyase (hCGL) mutant with catalytic activity against homocysteine ​​for therapeutic purposes. For example, the enzyme variant may have an amino acid sequence selected from the group consisting of SEQ ID NO: 2-6 and 37-39. In particular, the variant may be derived from human enzymes, such as human cystathionine-γ-lyase (CGL). A polypeptide comprising a modified human CGL capable of degrading homocysteine ​​is provided. This polypeptide may be capable of degrading homocysteine ​​under physiological conditions. For example, the polypeptide may exhibit catalytic activity against L-homocysteine ​​up to kJ. cat / K M Reaching 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001mM -1 s -1 Or any range derived from it.

[0014] These enzymes were modified by introducing amino acid substitutions into the human enzyme cystathionine-γ-lyase (CGL). Variants with amino acid substitutions include SEQ ID NO:2, hCGL-E59N-R119L-E339V (hCGL-NLV); SEQ ID NO:3, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8mut-1); SEQ ID NO:4, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-E339V-I353S (hCGL-8mut-2); SEQ ID NO:5, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8mut-3); SEQ ID NO: 6, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8mut-4); SEQ ID NO: 27, hCGL-E59N-R119L-T336D-E339V (hCGL-NLDV); SEQ ID NO: 28, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-1); SEQ ID NO: 29, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-2); SEQ ID NO: 30, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-3); SEQ IDNO: 31, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-4); SEQ ID NO: 32, hCGL-E59N-R119L-T336E-E339V (hCGL-NLEV); SEQ ID NO: 33, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-1); SEQ ID NO: 34, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-2);SEQ ID NO: 35, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-3); SEQ ID NO: 36, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-4); SEQ ID NO: 37, hCGL-E59I-S63L-L91M-R119D-K268R-T311G-E339V-I353S (mutant 3); SEQ ID SEQ ID NO:38, hCGL-E59I-S63L-L91M-R119H-K268R-T311G-E339V-I353S (mutant 4); and SEQ ID NO:39, hCGL-E59I-S63L-L91M-R119G-K268R-T311G-E339V-I353S (mutant 5).

[0015] The modified CGL enzymes discussed herein are characterized by a certain percentage of identity with unmodified CGL enzymes (e.g., native CGL enzymes). For example, unmodified CGL enzymes may be native primate cystathionine enzymes (i.e., cystathionine-γ-lyases). The unmodified portion of the modified CGL enzyme (i.e., the sequence of the modified CGL enzyme, excluding amino acids at positions 59, 63, 91, 119, 268, 311, 336, 339, and / or 353 of SEQ ID NO:1 and any substitutions at homologous positions of SEQ ID No:7-10, see [link to relevant documentation]). Figure 6 The percentage of identity between a modified human CGL enzyme and a natural CGL enzyme can be at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derived therefrom). It is also considered that the percentages of identity discussed above, compared to the unmodified regions of the corresponding natural enzyme, may relate to specific modified regions of the enzyme. For example, a modified CGL enzyme may contain a modified or mutated substrate recognition site, which can be characterized based on the identity of the amino acid sequence of the modified or mutated substrate recognition site with the amino acid sequence of an unmodified or natural CGL enzyme from the same species or across species. For example, a modified human CGL enzyme characterized by at least 90% identity with an unmodified human CGL enzyme means that at least 90% of the amino acids in the modified human CGL enzyme are the same as those in the unmodified human CGL enzyme.

[0016] Unmodified CGL enzymes can be natural CGL enzymes, particularly human or other primate isotypes. For example, a natural human CGL enzyme may have SEQ ID NO:1. Non-limiting examples of other natural primate CGL enzymes include Sumatran orangutan CGL (gene bank ID: NP_001124635.1; SEQ ID NO:7), cynomolgus monkey CGL (gene bank ID: AAW71993.1; SEQ ID NO:8), chimpanzee CGL (gene bank ID: XP_513486.2; SEQ ID NO:9), and bonobo CGL (gene bank ID: XP_003830652.1; SEQ ID NO:10). Exemplary natural CGL enzymes comprise sequences having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity (or any range derived therefrom) with SEQ ID NOs:1 or 7-10.

[0017] Natural CGLs can be modified by one or more other modifications, such as chemical modification, substitution, insertion, deletion, and / or truncation. Modifications can occur at the substrate recognition site of the natural enzyme. Natural CGLs can be modified by substitution. For example, the number of substitutions can be four, five, six, seven, or more. Natural CGLs can be modified at the substrate recognition site or any position that may affect substrate specificity. The modified polypeptide can have at least one amino acid substitution at the amino acid positions corresponding to E59, S63, L91, R119, K268, T311, T336, E339, and / or I353 of SEQ ID NO:1 or at the amino acid positions 59, 63, 91, 119, 268, 311, 336, 339, and / or 353 of primate CGLs. For example, primates can be humans, Sumatran orangutans, cynomolgus monkeys, chimpanzees, or bonobos. For example, the equivalent substitution E59 for SEQ ID NO:7 in SEQ ID NO:1 would modify valine instead of glutamic acid in SEQ ID NO:1. Another anticipated substitution for SEQ ID NO:7 is V353, which is as follows: Figure 6 The isoleucine at the equivalent position of SEQ ID NO:1 shown.

[0018] The substitutions in the modified CGL enzyme can occur at amino acid positions 59, 63, 91, 119, 268, 311, 339, and / or 353, and can be aspartic (N), valine (V), leucine (L), methionine (M), arginine (R), glycine (G), alanine (A), or serine (S). The modification can be selected from the group consisting of: E / V59N, E59I, S63L, L91M, R119L, R119A, R119D, R119H, R119G, K268R, T311G, T336D, T336E, E339V, and I / V353S. Substitutions can include S63L, L91M, K268R, T311G, E339V, and I / V353S. The alternatives may include other alternatives such as E / V59N or E / V59I; any one of R119L, R119A, R119D, R119H and R119G; and / or T336D or T336E.

[0019] The substituents can be combinations of E59N, S63L, L91M, R119L, K268R, T311G, I353S, and E339V of human CGL (e.g., modified polypeptides, fragments, or homologues having the amino acid sequence of SEQ ID NO:3), combinations of E59I, S63L, L91M, R119L, K268R, T311G, E339V, and I353S of human CGL (e.g., modified polypeptides, fragments, or homologues having the amino acid sequence of SEQ ID NO:4), combinations of E59N, S63L, L91M, R119A, K268R, T311G, E339V, and I353S of human CGL (e.g., modified polypeptides, fragments, or homologues having the amino acid sequence of SEQ ID NO:4), or combinations of E59N, S63L, L91M, R119A, K268R, T311G, E339V, and I353S of human CGL (e.g., modified polypeptides, fragments, or homologues having the amino acid sequence of SEQ ID NO:3). Modified polypeptides, fragments or homologs of the amino acid sequence NO: 5, combinations of human CGL's E59I, S63L, L91M, R119A, K268R, T311G, E339V and I353S (e.g., modified polypeptides, fragments or homologs of the amino acid sequence SEQ ID NO: 6), combinations of E59I, S63L, L91M, R119D, K268R, T311G, E339V and I353S (e.g., modified polypeptides, fragments or homologs of the amino acid sequence SEQ ID NO: 37), combinations of E59I, S63L, L91M, R119H, K268R, T311G, E339V and I353S (e.g., modified polypeptides, fragments or homologs of the amino acid sequence SEQ ID NO: 6), combinations of E59I, S63L, L91M, R119A, K268R, T311G, E339V and I353S (e.g., modified polypeptides, fragments or homologs of the amino acid sequence SEQ ID NO: 6), combinations of E59I, S63L, L91M, R119A, K268R, T311G, E339V and I353S (e.g., modified polypeptides, fragments or homologs of the amino acid sequence SEQ ID NO: 6), combinations of human CGL's ... Modified polypeptides of the amino acid sequence NO: 38, fragments thereof or homologs thereof, combinations of E59I, S63L, L91M, R119G, K268R, T311G, E339V and I353S (e.g., modified polypeptides of the amino acid sequence having SEQ ID NO: 39, fragments thereof or homologs thereof), or any of these modifications of SEQ ID NO: 2-6 and 37-39 combined with T336D or T336E. The modified CGL enzyme can be a Sumatran orangutan CGL-NLMLRGVS mutant (SEQ ID NO: 11), a Sumatran orangutan CGL-ILMLRGVS mutant (SEQ ID NO: 12), a Sumatran orangutan CGL-NLMARGVS mutant (SEQ ID NO: 13), a Sumatran orangutan CGL-ILMARGVS mutant (SEQ ID NO: 14), a Sumatran orangutan CGL-ILMARGVS mutant (SEQ ID NO: 40), a Sumatran orangutan CGL-ILMARGVS mutant (SEQ ID NO: 41), a Sumatran orangutan CGL-ILMARGVS mutant (SEQ ID NO: 42), or a Sumatran orangutan CGL-NLMARGVS mutant (SEQ ID NO: 15).NO:52), Sumatran orangutan CGL-NLMARGVS mutant (SEQ ID NO:53), Sumatran orangutan CGL-NLMARGVS mutant (SEQ ID NO:54), cynomolgus monkey CGL-NLMLRGVS mutant (SEQ ID NO:15), cynomolgus monkey CGL-ILMARGVS mutant (SEQ ID NO:16), cynomolgus monkey CGL-NLMLRGVS mutant (SEQ ID NO:17), cynomolgus monkey CGL-ILMARGVS mutant (SEQ ID NO:18), cynomolgus monkey CGL-ILMARGVS mutant (SEQ ID NO:43), cynomolgus monkey CGL-ILMARGVS mutant (SEQ ID NO:44), cynomolgus monkey CGL-ILMARGVS mutant (SEQ ID NO:45), cynomolgus monkey CGL-NLMLRGVS mutant (SEQ ID NO:55), cynomolgus monkey CGL-NLMLRGVS mutant (SEQ ID NO:56), cynomolgus monkey CGL-NLMLRGVS mutant (SEQ ID NO:56). (SEQ ID NO:57), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:19), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:20), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:21), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:22), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:46), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:47), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:48), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:58), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:59), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:60), Bonobo CGL-NLMLRGVS mutant (SEQ ID NO:23), Bonobo CGL-ILMARGVS mutant (SEQ ID NO:57), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:20), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:21), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:22), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:46), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:47), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:48), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:58), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:59), Chimpanzee CGL-NLMLRGVS mutant (SEQ ID NO:60), Bonobo CGL-NLMLRGVS mutant (SEQ ID NO:23), Bonobo CGL-ILMARGVS mutant (SEQ ID NO:20), Chimpanzee CGL-ILMARGVS mutant (SEQ ID NO:20), Chimpanzee CGL NO:24), bonobo CGL-NLMLRGVS mutant (SEQ ID NO:25), bonobo CGL-ILMARGVS mutant (SEQ ID NO:26), bonobo CGL-ILMARGVS mutant (SEQ ID NO:49), bonobo CGL-ILMARGVS mutant (SEQ ID NO:50), or bonobo CGL-ILMGRGVS mutant (SEQ ID NO:51), bonobo CGL-NLMDRGVS mutant (SEQ ID NO:24), bonobo CGL-NLMDRGVS mutant (SEQ ID NO:25), or bonobo CGL-ILMGRGVS mutant (SEQ ID NO:26), or bonobo CGL-NLMDRGVS mutant (SEQ ID NO:27).NO:61), bonobo CGL-NLMDRGVS mutant (SEQ ID NO:62), bonobo CGL-NLMDRGVS mutant (SEQ ID NO:63).

[0020] Modified CGL enzymes can be linked to heterologous amino acid sequences. For example, modified CGL enzymes can be linked to heterologous amino acid sequences as fusion proteins. Modified CGL enzymes can be linked to amino acid sequences such as IgG Fc, albumin, albumin-binding peptides, polysialic acid time-extended peptides, or XTEN peptides to increase their in vivo half-life.

[0021] To increase serum stability, the modified CGL enzyme can be linked to one or more polyether molecules. The polyether can be polyethylene glycol (PEG). The modified CGL enzyme can be linked to PEG via specific amino acid residues such as lysine or cysteine. For therapeutic administration, this polypeptide containing the modified CGL enzyme can be dispersed in a pharmaceutically acceptable carrier.

[0022] Nucleic acids encoding modified CGL enzymes were considered. The nucleic acids can be codon-optimized for expression in bacteria, such as *Escherichia coli* (E. coli). The codon-optimized nucleic acids for expression of the modified CGL enzymes provided in SEQ ID NO:37-39 in *E. coli* are provided in SEQ ID NO:64-66, respectively. The sequences provided in SEQ ID NO:64-66 encode an N-terminal 6x-histidine tag. Therefore, the twelfth and subsequent amino acids encoded by SEQ ID NO:64-66 correspond to the second and subsequent amino acids of SEQ ID NO:37-39, respectively. Alternatively, the nucleic acids can be codon-optimized for expression in fungi (e.g., yeast), insects, or mammals. Vectors containing such nucleic acids, such as expression vectors, are also considered. The nucleic acids encoding the modified CGL enzymes can be operatively linked to promoters, including but not limited to heterologous promoters. The modified CGL enzymes can be delivered to target cells via vectors (e.g., gene therapy vectors). Such vectors can be modified using recombinant DNA technology to achieve the expression of modified CGL-encoded nucleic acids in target cells. These vectors can be derived from non-viral (e.g., plasmids) or viral (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, herpesvirus, or vaccinia virus) sources. Non-viral vectors can be complexed with reagents to facilitate DNA transmembrane entry. Examples of such non-viral vector complexes include formulations containing polycationic agents that promote DNA condensation, and lipid-based delivery systems. Lipid-based delivery systems include liposome-based nucleic acid delivery.

[0023] A host cell containing such a vector is provided. The host cell can be a bacterium (e.g., Escherichia coli), a fungal cell (e.g., yeast), an insect cell, or a mammalian cell.

[0024] The vector can be introduced into host cells to express the modified CGL enzyme. The protein can be expressed in any suitable manner. The protein can be expressed in the host cell, resulting in glycosylation. Alternatively, the protein can be expressed in the host cell, resulting in non-glycosylation.

[0025] Therapeutic agents containing modified CGL enzymes can be administered intravenously, intradermally, intraarterially, intraperitoneally, intramuscularly, subcutaneously, by infusion, continuous infusion, or via catheter in a lipid composition (e.g., liposomes). The subject may have previously received treatment for homocystinuria or hyperhomocysteinemia. Administration of the enzyme is intended to treat or improve relapses of homocystinuria or hyperhomocysteinemia; administration of the compounds or compositions described herein may improve one or more conditions and / or symptoms associated with homocystinuria and hyperhomocysteinemia. Known symptoms of homocystinuria include intraocular lens dislocation, myopia, abnormal blood clots, osteoporosis, learning disabilities, developmental problems, chest deformities, scoliosis, megaloblastic anemia, and seizures. Known symptoms of hyperhomocysteinemia include blood clots, damaged vascular lining, dementia (e.g., Alzheimer's disease), and fractures.

[0026] The method may also include administering at least a second therapy to the subject, such as a second therapy for homocystinuria or hyperhomocysteinemia. The second therapy for homocystinuria or hyperhomocysteinemia may be high-dose vitamin B6 or betaine (N,N,N-trimethylglycine) therapy.

[0027] Compositions comprising a modified CGL enzyme or a nucleic acid encoding a modified CGL enzyme are provided for treating homocystinuria or hyperhomocysteinemia in a subject. Use of the modified CGL enzyme or a nucleic acid encoding a modified CGL enzyme in the preparation of a medicament for treating homocystinuria or hyperhomocysteinemia is also provided. The modified CGL enzyme can be any modified CGL enzyme disclosed herein.

[0028] This article also provides:

[0029] 1. An isolated and modified primate cystathionine-γ-lyase (CGL) comprising at least the following substitutions relative to the amino acid sequence of natural human CGL (SEQ ID NO: 1), wherein the modified enzyme has both homocystinase activity and homocysteine ​​activity, and the substitutions are selected from the group consisting of:

[0030] (a) Isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0031] (b) Asparagine at position 59, leucine at position 119, aspartic acid at position 336, and valine at position 339.

[0032] (c) Asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353.

[0033] (d) Isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353.

[0034] (e) Asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353.

[0035] (f) Isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, aspartic acid at position 336, valine at position 339, and serine at position 353.

[0036] (g) Asparagine at position 59, leucine at position 119, glutamic acid at position 336 and valine at position 339;

[0037] (h) Asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353.

[0038] (i) Isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353.

[0039] (j) Asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353.

[0040] (i) Asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353.

[0041] (k) isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, glutamic acid at position 336, valine at position 339, and serine at position 353.

[0042] (l) isoleucine at position 59, leucine at position 63, methionine at position 91, histidine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353; and

[0043] (m) isoleucine at position 59, leucine at position 63, methionine at position 91, glycine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0044] 2. The enzyme of claim 1, wherein the substitutions include asparagine at position 59, leucine at position 119, aspartic acid at position 336, and valine at position 339.

[0045] 3. The enzyme of claim 2, wherein the substitutions further comprise leucine at position 63, methionine at position 91, arginine at position 268, glycine at position 311, and serine at position 353.

[0046] 4. The enzyme of claim 1, wherein the substitutions include asparagine at position 59, leucine at position 119, glutamic acid at position 336, and valine at position 339.

[0047] 5. The enzyme of claim 4, wherein the substitution further comprises leucine at position 63, methionine at position 91, arginine at position 268, glycine at position 311, and serine at position 353.

[0048] 6. The enzyme of claim 1, wherein the substitutions include isoleucine at position 59, leucine at position 63, methionine at position 91, aspartic acid at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0049] 7. The enzyme of claim 1, wherein the substitutions include isoleucine at position 59, leucine at position 63, methionine at position 91, histidine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0050] 8. The enzyme of claim 1, wherein the substitutions include isoleucine at position 59, leucine at position 63, methionine at position 91, glycine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0051] 9. The enzyme of claim 1, wherein the modified CGL enzyme is a modified Sumatran orangutan CGL enzyme.

[0052] 10. The enzyme of claim 9, wherein the modified Sumatran orangutan CGL enzyme comprises substitutions selected from the group consisting of:

[0053] (a) V59I, S63L, L91M, R119D, K268R, T311G, E339V and V353S;

[0054] (b) V59N, R119L, T336D and E339V;

[0055] (c) V59N, S63L, L91M, R119L, K268R, T311G, T336D, E339V and V353S;

[0056] (d) V59I, S63L, L91M, R119L, K268R, T311G, T336D, E339V and V353S;

[0057] (e)V59N, S63L, L91M, R119A, K268R, T311G, T336D, E339V and V353S;

[0058] (f) V59I, S63L, L91M, R119A, K268R, T311G, T336D, E339V and V353S;

[0059] (g)V59N, R119L, T336E and E339V;

[0060] (h)V59N, S63L, L91M, R119L, K268R, T311G, T336E, E339V and V353S;

[0061] (i) V59I, S63L, L91M, R119L, K268R, T311G, T336E, E339V and V353S;

[0062] (j)V59N, S63L, L91M, R119A, K268R, T311G, T336E, E339V and V353S;

[0063] (k)V59I,S63L,L91M,R119A,K268R,T311G,T336E,E339V andV353S;

[0064] (l) V59I, S63L, L91M, R119H, K268R, T311G, E339V and V353S; and

[0065] (m)V59I,S63L,L91M,R119G,K268R,T311G,E339V andV353S.

[0066] 11. The enzyme of claim 1, wherein the modified CGL enzyme is a modified human CGL enzyme, a modified cynomolgus monkey CGL enzyme, a modified chimpanzee CGL enzyme, or a modified bonobo CGL enzyme.

[0067] 12. The enzyme of claim 11, wherein the modified human CGL enzyme, modified cynomolgus monkey CGL enzyme, modified chimpanzee CGL enzyme, or modified bonobo CGL enzyme comprises substitutions selected from the group consisting of:

[0068] (a) E59I, S63L, L91M, R119D, K268R, T311G, E339V and I353S;

[0069] (b) E59N, R119L, T336D and E339V;

[0070] (c) E59N, S63L, L91M, R119L, K268R, T311G, T336D, E339V and I353S;

[0071] (d) E59I, S63L, L91M, R119L, K268R, T311G, T336D, E339V and I353S;

[0072] (e) E59N, S63L, L91M, R119A, K268R, T311G, T336D, E339V and I353S;

[0073] (f) E59I, S63L, L91M, R119A, K268R, T311G, T336D, E339V and I353S;

[0074] (g)E59N, R119L, T336E and E339V;

[0075] (h)E59N, S63L, L91M, R119L, K268R, T311G, T336E, E339V and I353S;

[0076] (i) E59I, S63L, L91M, R119L, K268R, T311G, T336E, E339V and I353S;

[0077] (j)E59N, S63L, L91M, R119A, K268R, T311G, T336E, E339V and I353S;

[0078] (k)E59I, S63L, L91M, R119A, K268R, T311G, T336E, E339V and I353S;

[0079] (l) E59I, S63L, L91M, R119H, K268R, T311G, E339V and I353S; and

[0080] (m)E59I, S63L, L91M, R119G, K268R, T311G, E339V and I353S.

[0081] 13. The enzyme according to any one of claims 1 to 8, further comprising a heteropeptide fragment or a polysaccharide.

[0082] 14. The enzyme of claim 13, wherein the heteropeptide fragment is an XTEN polypeptide, IgGFc, albumin, or albumin-binding peptide.

[0083] 15. The enzyme of claim 13, wherein the polysaccharide comprises a polysialic acid polymer.

[0084] 16. The enzyme according to any one of claims 1-15, wherein the enzyme is coupled with polyethylene glycol (PEG).

[0085] 17. The enzyme of claim 16, wherein the enzyme is coupled to PEG via one or more lysine residues.

[0086] 18. A nucleic acid comprising a nucleotide sequence encoding an enzyme as described in any one of claims 1-14.

[0087] 19. The nucleic acid of claim 18, wherein the nucleic acid is codon-optimized for expression in bacteria, fungi, insects, or mammals.

[0088] 20. The nucleic acid of claim 19, wherein the bacteria is Escherichia coli.

[0089] 21. The nucleic acid of claim 20, wherein the nucleic acid comprises one of SEQ ID NO:64 to SEQ ID NO:66.

[0090] 22. An expression vector comprising the nucleic acid as described in claim 18 or 19.

[0091] 23. A host cell comprising the nucleic acid as described in claim 18 or 19.

[0092] 24. The host cell of claim 23, wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

[0093] 25. A therapeutic preparation comprising an enzyme as described in any one of claims 1-17 or a nucleic acid as described in any one of claims 18 or 19 in a pharmaceutically acceptable carrier.

[0094] 26. A method of treating a subject suffering from homocystinuria or hyperhomocysteinemia or at risk of developing homocystinuria or hyperhomocysteinemia, comprising administering to the subject a therapeutically effective amount of the formulation of claim 25.

[0095] 27. A method for treating a subject suffering from homocystinuria or hyperhomocysteinemia or at risk of developing homocystinuria or hyperhomocysteinemia, the method comprising:

[0096] The subject is administered a therapeutically effective amount of the formulation of claim 25, or a formulation comprising isolated, modified primate cystathionine-γ-lyase (CGL), said enzyme having a substitution relative to the amino acid sequence (SEQ ID NO: 1) of natural human CGL, or comprising a nucleic acid encoding a nucleotide sequence of said modified enzyme, said substitution being selected from the group consisting of:

[0097] (a) Asparagine at position 59, leucine at position 119, and valine at position 339;

[0098] (b) Valine at position 59, leucine at position 119, and valine at position 339;

[0099] (c) Asparagine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0100] (d) Isoleucine at position 59, leucine at position 63, methionine at position 91, leucine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0101] (e) Asparagine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353; and

[0102] (f) Isoleucine at position 59, leucine at position 63, methionine at position 91, alanine at position 119, arginine at position 268, glycine at position 311, valine at position 339, and serine at position 353.

[0103] 28. The method of claim 27, wherein the modified CGL enzyme is a modified Sumatran orangutan CGL enzyme.

[0104] 29. The method of claim 28, wherein the modified Sumatran orangutan CGL enzyme comprises a substitution selected from the group consisting of:

[0105] (a) V59N, R119L and E339V;

[0106] (b) R119L and E339V;

[0107] (c) V59N, S63L, L91M, R119L, K268R, T311G, E339V and V353S;

[0108] (d) V59I, S63L, L91M, R119L, K268R, T311G, E339V and V353S;

[0109] (e) V59N, S63L, L91M, R119A, K268R, T311G, E339V, and V353S; and

[0110] (f) V59I, S63L, L91M, R119A, K268R, T311G, E339V and V353S.

[0111] 30. The method of claim 27, wherein the modified CGL enzyme is a modified human CGL enzyme, a modified cynomolgus monkey CGL enzyme, a modified chimpanzee CGL enzyme, or a modified bonobo CGL enzyme.

[0112] 31. The method of claim 30, wherein the modified human CGL enzyme, modified cynomolgus monkey CGL enzyme, modified chimpanzee CGL enzyme, or modified bonobo CGL enzyme comprises substitutions selected from the group consisting of:

[0113] (a) E59N, R119L and E339V;

[0114] (b) E59V, R119L and E339V;

[0115] (c)E59N, S63L, L91M, R119L, K268R, T311G, E339V and I353S;

[0116] (d) E59I, S63L, L91M, R119L, K268R, T311G, E339V and I353S;

[0117] (e) E59N, S63L, L91M, R119A, K268R, T311G, E339V, and I353S; and

[0118] (f) E59I, S63L, L91M, R119A, K268R, T311G, E339V and I353S.

[0119] 32. The method of any one of claims 27-31, wherein the enzyme further comprises a heteropeptide fragment.

[0120] 33. The method of claim 32, wherein the heteropeptide fragment is an XTEN polypeptide, IgG Fc, albumin, albumin-binding peptide, or polysialic acid with extended time.

[0121] 34. The method of any one of claims 27-31, wherein the enzyme is coupled with polyethylene glycol (PEG).

[0122] 35. The method of claim 34, wherein the enzyme is coupled to PEG via one or more lysine or cysteine ​​residues.

[0123] 36. The method of claim 27, wherein the subject maintains a methionine-restricted diet.

[0124] 37. The method of claim 27, wherein the subject maintains a normal diet.

[0125] 38. The method of claim 27, wherein the subject is a human patient.

[0126] 39. The method of claim 27, wherein the formulation is administered via injection, infusion, continuous infusion, direct local perfusion of target cells, or via a catheter into a vein, artery, peritoneum, lesion, joint, prostate, pleura, trachea, vitreous body, muscle, vesicle, or umbilical cord.

[0127] 40. The method of claim 27, wherein the subject has previously been treated for homocystinuria or hyperhomocysteinemia, and the enzyme is administered to prevent recurrence of the homocystinuria or hyperhomocysteinemia.

[0128] 41. The method of claim 27, further comprising administering at least a second homocystinuria or hyperhomocysteinemia therapy to the subject.

[0129] 42. The method of claim 41, wherein the treatment for second homocystinuria or hyperhomocysteinemia is high-dose vitamin B6 or betaine (N,N,N-trimethylglycine) therapy.

[0130] 43. The therapeutic formulation of claim 25, used as a medicine for treating a subject suffering from homocystinuria or hyperhomocysteinemia.

[0131] 44. The enzyme according to any one of claims 1-14, used to treat homocystinuria or hyperhomocysteinemia in a subject.

[0132] As used herein, the term "encode" or "encoding" in relation to nucleic acids is used to facilitate understanding of the invention by those skilled in the art. However, these terms may be used interchangeably with "comprise" or "comprising," respectively.

[0133] As used herein, "substantially free" with respect to a particular component is used to mean that no particular component was intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the particular component cannot be detected using standard analytical methods.

[0134] As used in this specification, "a" or "an" may refer to one or more. As used in one or more claims, when used with the word "comprising," "a" or "an" may refer to one or more.

[0135] Although this disclosure supports the use of only substitutes and the definition of "and / or", the term "or" as used in the claims means "and / or" unless it is explicitly stated that only substitutes are mentioned or that substitutes are mutually exclusive. "Another" as used herein may mean at least a second or more.

[0136] As used herein, the term "about" is as understood by one of ordinary skill in the art and will vary to some extent in the context in which it is used. Typically, about includes a range of values ​​plus or minus 10% of a reference value.

[0137] Brief description of the attached figures

[0138] The following figures form part of and are included in this specification to further illustrate the methods, compounds, and compositions.

[0139] Figure 1 – Evaluation of modified CGL in vivo. The enzyme was administered as a single dose of 50 mg / kg (intraperitoneal). Mutant 1 corresponds to hCGL-NLV (SEQ ID NO: 2), and mutant 2 corresponds to hCGL-8mut-4 (SEQ ID NO: 6). Serum was collected for one week before and every 24 hours after injection.

[0140] Figure 2 –Multi-dose pharmacodynamics. The enzyme (hCGL-8mut-4; SEQ ID NO:6) was administered every 5 days at 50 mg / kg (intraperitoneal). Serum was collected before each injection and 24 hours after each injection.

[0141] Figure 3 - Therapeutic effects of modified CGL in vivo. The enzyme (hCGL-8mut-4; SEQ ID NO:6) was administered twice weekly, starting on day 10 after birth. The top line represents the modified CGL. The bottom line represents the inactive enzyme.

[0142] Figure 4 - Evaluation of in vivo mutant 3. The enzyme (Mutant 3; SEQ ID NO:37) was administered once at a dose of 50 mg / kg (intraperitoneal). Serum was collected before and every 24 hours after injection for one week. The top line represents the inactivated enzyme. The bottom line represents mutant 3 (SEQ ID NO:37).

[0143] Figure 5 - Therapeutic effects of mutant 3 in vivo. The enzyme (Mutant 3; SEQ ID NO:37) is administered twice weekly, starting on day 10 after birth.

[0144] Figure 6 – Sequence alignment of SEQ ID NO: 1 and 7-10. Asterisks indicate engineered locations in various modified CGL enzymes. Detailed Implementation

[0145] Methods for treating diseases such as homocystinuria and hyperhomocysteinemia using modified therapeutic enzymes that degrade homocysteine ​​and homocysteine ​​are provided. Therapeutic enzymes include hCGL-NLV mutants (E59N, R119L, E339V) and recombinant engineered human cystathionine-γ-lyase (hCGL) with modified homocysteine ​​(cysteine) enzyme activity relative to hCGL-NLV mutants. Mutants exhibiting higher catalytic activity require lower concentrations of therapeutic agents for patient administration.

[0146] I. Definition

[0147] As used herein, the terms “enzyme”, “protein”, and “polypeptide” refer to compounds containing amino acids linked by peptide bonds and are used interchangeably.

[0148] As used herein, the term "fusion protein" refers to a chimeric protein that comprises proteins or protein fragments that are operatively linked in a non-natural manner.

[0149] As used herein, the term "half-life" (1 / 2-lifetime) refers, for example, the time required for the concentration of a polypeptide to decrease by half, either in vitro (e.g., as measured in cell culture media) or in vivo (e.g., as measured in serum), following injection into a mammal. Methods for measuring "half-life" include using antibodies specific to CGL or PEG, as used in ELISA (enzyme-linked immunosorbent assay) formats, to measure the physical quantity of the protein over time. Other methods closely related to measuring half-life include determining changes in the catalytic activity of an enzyme drug over time by any detection method that detects the production of any substrate resulting from the conversion of homocysteine ​​to products (e.g., α-ketobutyrate, methanethiol, and / or ammonia).

[0150] The terms “operable combination,” “operable sequence,” and “operable linking” refer to a link in which the components are in a relationship that allows them to function in their intended manner, such as nucleic acid sequences linked in a way that enables the transcription of a given gene and / or the synthesis of a desired protein molecule, or amino acid sequences linked in a way that produces a fusion protein.

[0151] The term "connector" refers to a compound or part that acts as a molecular bridge to operatively connect two different molecules, wherein a portion of the connector is operatively connected to a first molecule and another portion of the connector is operatively connected to a second molecule.

[0152] The term "PEGylation" refers to the conjugation with polyethylene glycol (PEG), which is widely used as a drug carrier due to its high biocompatibility and ease of modification. PEG can be chemically coupled (e.g., covalently linked) to active agents via hydroxyl groups at the ends of the PEG chain; however, PEG itself is limited to a maximum of two active agents per molecule. Among different approaches, copolymers of PEG and amino acids have been investigated as novel biomaterials that retain the biocompatibility of PEG but offer the additional advantage of numerous attachment sites per molecule (thus providing greater drug loading capacity) and can be engineered to suit a variety of applications.

[0153] The term "gene" refers to a DNA sequence that contains both control and coding sequences necessary for the production of a polypeptide or its precursor. This polypeptide can be encoded by the full-length coding sequence or any portion thereof, in order to retain the desired enzymatic activity.

[0154] The term "natural" refers to the typical or wild-type form of a gene or gene product when isolated from a naturally occurring source, or a characteristic of that gene or gene product. Conversely, the terms "modified," "variant," "mutant protein," or "mutant" refer to a gene or gene product that exhibits modifications in sequence and functional characteristics (i.e., altered characteristics) compared to a natural gene or gene product, wherein the modified gene or gene product has been genetically engineered and is not naturally occurring.

[0155] The term "vector" is used to refer to a vector nucleic acid molecule into which a nucleic acid sequence can be inserted to be introduced into a cell for replication. The nucleic acid sequence can be "exogenous," meaning that the sequence is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell but located in the host cell nucleic acid where the nucleic acid sequence is not normally found. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well-equipped to construct vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0156] The term "expression vector" refers to any type of genetic structure, including nucleic acids encoding RNA that can be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In other cases, such as during the generation of antisense molecules or ribonucleases, these sequences are not translated. Expression vectors can contain a variety of "control sequences," which are nucleic acid sequences essential for the transcription and, possibly, translation of the coding sequences that are operablely linked in a particular host cell. In addition to control sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0157] As used herein, the term "therapeutic effective amount" refers to the amount of the therapeutic composition (i.e., the modified CGL enzyme or the nucleic acid encoding such enzyme) used in a method for achieving a therapeutic effect (i.e., depleting homocysteine ​​in the patient's bloodstream to a level at or below the normal reference value). The terms "therapeutic benefit" or "therapeutic effectiveness" as used throughout this application mean anything that promotes or enhances the medical well-being of the subject under this condition. This includes, but is not limited to, a reduction in the frequency or severity of disease symptoms or signs, such as an elevated (e.g., above 15 μmol / L) serum level of total homocysteine. The dosage range of the therapeutic composition should be sufficiently broad to produce the intended effect of reducing the symptoms of homocysteineuria. For example, a therapeutically effective amount of the therapeutic composition may be an amount sufficient, when administered in a physiologically permissible manner, to achieve an intravascular (plasma) concentration of the modified CGL enzyme from about 0.001 to about 100 U / mL, preferably above 0.1 U / mL, and more preferably above 1.0 U / mL. Typical dosages are administered based on body weight, ranging from approximately 1-100 U / kg / day, preferably approximately 2-25 U / kg / day, and more preferably approximately 2.0-8.0 U / kg / day. A typical dose may be 5.0 U / kg / day or 35 U / kg / week. Excessive dosage should be avoided to prevent adverse side effects such as hyperviscosity syndrome, pulmonary edema, and congestive heart failure. In general, those skilled in the art can determine the dosage based on the patient's age, symptoms, sex, and condition. The dosage may also be adjusted by a physician in case of complications. The dosage should cause a reduction of at least 50%, at least 60%, and at least 70% in the serum tHcy level of the subject within approximately 12 to 24 hours. The dosage should cause a reduction of at least 50% to 70% in the serum tHcy level of the subject within 6 hours. A statistically significant response should result in a reduction of the serum tHcy level of the subject to within two standard deviations from the reference level published by Nygard et al. (1998). Therefore, this dose can reduce the tHcy level in the subject's serum to below 20-25 μM.

[0158] As used in this article, the term "K" M "k" refers to the Michaelis-Menten constant of an enzyme, defined as the concentration of a specific substrate at which a given enzyme produces half of its maximum rate in a catalytic reaction. As used in this paper, "k" is a different term. cat "k" refers to the number of substrate molecules that are converted into product per unit time at each enzyme site, where the enzyme works at its highest efficiency. As used in this article, "k" is a term that... cat / K M "" refers to the specificity constant, which is a unit of measurement for the efficiency of an enzyme in converting a substrate into a product.

[0159] The term "cystathionine-γ-lyase" (CGL or cystathionase) refers to any enzyme that catalyzes the hydrolysis of cystathionine to cysteine. As used herein, the terminology also takes into account the primate forms of cystathionine-γ-lyase, including the human forms.

[0160] "Treatment" refers to the administration or application of a therapeutic agent to a subject to obtain therapeutic benefit from a disease or health-related condition, or the performance of surgery or physical therapy on a subject. For example, treatment may include the administration of a therapeutically effective amount of homocysteine ​​enzyme.

[0161] "Subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. Subjects can be human.

[0162] II. Cystathione-γ-lyase

[0163] Lyases are enzymes that catalyze the breaking of various chemical bonds, usually resulting in the formation of new double bonds or new ring structures. For example, the enzyme catalyzing this reaction would be a lyase: ATP → cAMP + PPi. Lyases differ from other enzymes in that they require only one substrate for a unidirectional reaction, while they require two substrates for a reverse reaction.

[0164] Many enzymes dependent on pyridoxal (PLP) 5'-phosphate are involved in the metabolism of cysteine, homocysteine, and methionine. These enzymes form an evolutionarily related family known as Cys / Met metabolic PLP-dependent enzymes. These enzymes are proteins composed of approximately 400 amino acids, with the PLP group linked to a lysine residue at the center of the polypeptide. Members of this family include cystathionine-γ-lyase (CGL), cystathionine-γ-synthetase (CGS), cystathionine-β-lyase (CBL), methionine-γ-lyase (MGL), and O-acetylhomoserine (OAH) / O-acetyl-serine (OAS) hydrogen sulfide hydrolase (OSHS). A common feature of all of them is the formation of a Michaelis-Menten complex, which results in the formation of an aldehyde imine as the exosubstrate. Further reaction processes are determined by the substrate specificity of the particular enzyme.

[0165] For example, the inventors introduced specific mutations into members of the PLP-dependent lyase family (such as human cystathionine-γ-lyase) to alter their substrate specificity. In this way, the inventors produced novel variants with de novo synthesis capabilities to degrade homocysteine ​​as a substrate with catalytic activity superior to hGGL-NLV. Modification of other PLP-dependent enzymes can also be considered to generate novel homocysteine ​​degradation activities.

[0166] CGL is a tetramer that catalyzes the final step of the transsulfurization pathway in mammals (Rao et al., 1990). CGL catalyzes the conversion of L-cystathionine to L-cysteine, α-ketobutyrate, and ammonia. Pyridoxal phosphate is the cofactor of this enzyme. Protein engineering was used to convert cystathionases, which exhibit only weak degradative activity towards homocysteine ​​and homocysteine, into enzymes capable of rapidly degrading homocysteine ​​and homocysteine ​​(as described in U.S. Patent No. 9,481,877, the entire contents of which are incorporated herein by reference).

[0167] III. Homocysteinase Engineering

[0168] Because humans do not produce homocysteine ​​enzymes, homocysteine ​​enzymes need to be engineered for human treatment to have high activity and specificity in degrading homocysteine ​​under physiological conditions, high stability in physiological fluids (such as serum), and generally non-immunogenic, since they are natural proteins that usually induce immune tolerance.

[0169] Because undesirable immunogenic effects were observed in animal studies using pMGL (MGL derived from *Pseudomonas putida*), there is a need to engineer the homocysteine ​​degradation activity of human enzymes. Immune tolerance to human proteins suggests that this enzyme may be non-immunogenic or minimally immunogenic, and therefore well tolerated.

[0170] Although mammals lack homocysteine ​​enzymes, they possess cystathionine-γ-lyase (CGL). CGL is a tetramer that catalyzes the final step in the mammalian transsulfurization pathway (Rao et al., 1990). CGL catalyzes the conversion of L-cystathionine to L-cysteine, α-ketobutyrate, and ammonia. Human CGL (hCGL) cDNA has previously been cloned and expressed, but in relatively low yields (approximately 5 mg / L culture) (Lu et al., 1992; Steegborn et al., 1999).

[0171] Therefore, methods and compositions relating to primate (particularly human) cystathionine-γ-lyases (CGL or cystathionases) that have been mutagenized to efficiently hydrolyze homocysteine ​​are provided.

[0172] Modified CGL enzymes are described, exhibiting at least one functional activity comparable to that of unmodified CGL enzymes. Modified CGL enzymes can be further modified to improve serum stability. Compared to unmodified CGL enzymes, modified CGL enzymes include proteins with additional advantages, such as homocysteine ​​activity. Unmodified proteins or peptides can be native cystathionine-γ-lyases, such as human cystathionine-γ-lyase.

[0173] Activity assays, particularly assays of enzyme activity, can be performed using detection methods familiar to those skilled in the art, and for comparative purposes may include, for example, the use of natural and / or recombinant forms of modified or unmodified enzymes. For example, homocysteine ​​enzyme activity can be determined by any assay that detects any substrates (such as α-ketobutyrate, methanethiol, and / or ammonia) produced by homocysteine ​​conversion.

[0174] Modified CGL enzymes can be identified based on their enhanced homocysteine ​​degradation activity. For example, they can recognize substrate recognition sites of unmodified peptides. This recognition can be based on structural analysis or homology analysis. A population of mutants may be generated, including modifications to such substrate recognition sites. Mutants with enhanced homocysteine ​​degradation activity can be selected from this population. The selection of desired mutants may include methods such as detecting byproducts or products derived from homocysteine ​​degradation.

[0175] Modified CGL enzymes may have amino acid deletions and / or substitutions; therefore, enzymes with deletions, enzymes with substitutions, and enzymes with both deletions and substitutions are all modified CGL enzymes. These modified CGL enzymes may further include inserted or added amino acids, such as fusion proteins or adaptor proteins. A “modified deletion CGL enzyme” lacks one or more residues of a native enzyme but may possess the specificity and / or activity of the native enzyme. Modified deletion CGL enzymes may also have reduced immunogenicity or antigenicity. An example of a modified deletion CGL enzyme is an enzyme having an amino acid residue missing from at least one antigenically active region, said antigenically active region being a region of an enzyme identified as antigenically active in a particular organism (such as the type of organism to which the modified CGL enzyme can be administered).

[0176] Substitution or alternative variants may involve the exchange of one amino acid for another at one or more sites within a protein, and can be programmed to modulate one or more properties of the polypeptide, particularly its effector function and / or bioavailability. Substitutions may be conserved or non-conserved, meaning that one amino acid may be replaced by an amino acid with a similar shape and charge. Conservative substitutions are well known in the art and include, for example: alanine replaced by serine; arginine replaced by lysine; asparagine replaced by glutamine or histidine; aspartic acid replaced by glutamic acid; cysteine ​​replaced by serine; glutamine replaced by asparagine; glutamic acid replaced by aspartic acid; glycine replaced by proline; histidine replaced by asparagine or glutamine; isoleucine replaced by leucine or valine; leucine replaced by valine or isoleucine; lysine replaced by arginine; methionine replaced by leucine or isoleucine; phenylalanine replaced by tyrosine, leucine, or methionine; serine replaced by threonine; threonine replaced by serine; tryptophan replaced by tyrosine; tyrosine replaced by tryptophan or phenylalanine; valine replaced by isoleucine or leucine.

[0177] Besides deletions or substitutions, modified CGL enzymes may have residue insertions, which typically involve adding at least one residue to the enzyme. This can include the insertion of a targeted peptide or polypeptide or a simple single residue. Terminal additions (also known as fusion proteins) will be discussed below.

[0178] The term "biological functional equivalent" is well understood in the art and is further defined in detail herein. Therefore, a CGL enzyme sequence comprising about 90% or more sequence identity with SEQ ID NO:1, or even about 91% to about 99% (including 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) of the amino acids identical or conservedly substituted with those of the modified CGL enzymes disclosed herein, such that, while maintaining the enzyme's biological activity, measurable biological activity parameters (e.g., homocysteine ​​conversion to α-ketobutyrate, methanethiol, and ammonia) are within about 20%, about 15%, about 10%, or about 5% of those of the modified CGL enzymes disclosed herein. Modified CGL enzymes may be biologically functionally identical to unmodified CGL enzymes.

[0179] It is also understood that amino acid and nucleic acid sequences may include additional residues (such as additional N- or C-terminal amino acids or 5′ and 3′ sequences); however, as long as the sequence conforms to the above-mentioned criteria, including maintaining the biological protein activity in terms of protein expression, these sequences are essentially still one of the sequences disclosed herein as previously described. The addition of terminal sequences is particularly applicable to nucleic acid sequences that, for example, may include various non-coding sequences flanking the 5′ or 3′ portions of coding regions, or may include various internal sequences known to exist in genes, i.e., introns.

[0180] Specifically, in addition to three mutation sites (i.e., E59N-R119L-E339V), five amino acid positions were found to be associated with hCGL-NLV (SEQ ID NO:2). These extra positions are located at residues 63, 91, 268, 311, and 353 of hCGL (SEQ ID NO:1) (see [link to relevant documentation]). Figure 6Mutations in one or more of these positions, such as S63L, L91M, K268R, T311G, and I353S, combined with residue mutations at positions 59, 119, and 339, result in increased activity compared to hCGL-NLV. Specifically, the variants include those corresponding to SEQ ID NO:3, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-E339V-I353S(hCGL-8mut-1); SEQ ID NO:4, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-E339V-I353S(hCGL-8mut-2); SEQ ID NO:5, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-E339V-I353S(hCGL-8mut-3); SEQ ID NO: 6, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-E339V-I353S (hCGL-8mut-4); SEQ ID NO: 27, hCGL-E59N-R119L-T336D-E339V (hCGL-NLDV); SEQ ID NO: 28, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-1); SEQ ID NO: 29, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-2); SEQ ID NO: 30, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-3); SEQ IDNO: 31, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336D-E339V-I353S (hCGL-9mutD-4); SEQ ID NO: 32, hCGL-E59N-R119L-T336E-E339V (hCGL-NLEV); SEQ ID NO: 33, hCGL-E59N-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-1); SEQ ID NO: 34, hCGL-E59I-S63L-L91M-R119L-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-2);SEQ ID NO: 35, hCGL-E59N-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-3); SEQ ID NO: 36, hCGL-E59I-S63L-L91M-R119A-K268R-T311G-T336E-E339V-I353S (hCGL-9mutE-4); SEQ ID NO: 37, hCGL-E59I-S63L-L91M-R119D-K268R-T311G-E339V-I353S (mutant 3); SEQ ID Amino acid substitutions in SEQ ID NO:38, hCGL-E59I-S63L-L91M-R119H-K268R-T311G-E339V-I353S (mutant 4); and SEQ ID NO:39, hCGL-E59I-S63L-L91M-R119G-K268R-T311G-E339V-I353S (mutant 5).

[0181] IV. Enzymatic homocysteine ​​depletion used for treatment

[0182] This polypeptide can be used to treat diseases such as homocysteine ​​and / or homocysteine ​​by utilizing novel enzymes that deplete homocysteine ​​and / or homocysteine. A therapeutic method using modified CGLs containing L-(cysteine)degradation activity is disclosed. Enzymes with homocysteine ​​degradation activity are provided to enhance therapeutic efficacy.

[0183] A modified CGL enzyme with homocysteine ​​degradation activity is provided for the treatment of diseases such as homocysteineuria. In particular, the modified polypeptide may have a human polypeptide sequence, thus preventing adverse immunogenic reactions when administered to human patients, allowing for repeated dosing, and improving therapeutic efficacy.

[0184] Homocysteine ​​depletion can be performed in the following situations: in the blood circulation of mammals, in vitro conditions where tissue culture or other biological culture media require homocysteine ​​deficiency, and in ex vivo surgeries where biological fluids, cells, or tissues are manipulated in vitro and subsequently returned to a mammalian patient. Homocysteine ​​depletion in blood circulation, culture media, biological fluids, or cells reduces the amount of homocysteine ​​available in the material being treated, and therefore includes contacting the material to be depleted with an engineered enzyme of homocysteine ​​depletion amount under homocysteine ​​depletion conditions to degrade environmental homocysteine ​​in the contacted material.

[0185] Homocysteine ​​depletion efficiency varies considerably depending on the application and generally depends on the amount of homocysteine ​​present in the material, the required depletion rate, and the material's tolerance to homocysteine ​​exposure. The level of homocysteine ​​in a material and the rate of homocysteine ​​depletion in that material can be readily monitored using various chemical and biochemical methods known in the art. Exemplary homocysteine ​​depletion amounts are further described herein, ranging from 0.001 to 100 units (U) of engineered enzyme per milliliter (mL) of the material to be treated, preferably between about 0.01 and 10 U, more preferably between about 0.1 and 5 U.

[0186] Homocysteine ​​depletion conditions are buffered and temperature conditions adapted to the biological activity of homocysteine ​​enzymes, including mild temperature, salinity, and pH conditions adapted to the enzyme, such as physiological conditions. Exemplary conditions include approximately 4–40°C, an ionic strength of approximately 0.05–0.2 M NaCl, and a pH of approximately 5–9, while also including physiological conditions.

[0187] In vivo contact can be achieved by administration, intravenous injection or intraperitoneal injection. Physiologically tolerable therapeutic amounts of the composition include administration of a modified CGL enzyme to the patient, thereby depleting circulating homocysteine ​​in the patient's blood.

[0188] The modified CGL enzyme can be administered extracorporeally via injection or gradual infusion. The modified CGL enzyme can be administered intravenously, intraperitoneally, or intramuscularly; it can be injected directly into tissues containing tumor cells; or it can be administered via a pump attached to a catheter, which may contain potential biosensors or homocysteine.

[0189] Therapeutic compositions containing modified CGL enzymes are typically administered intravenously, for example, by injecting a unit dose. When used in a therapeutic composition, the term "unit dose" refers to a physically separated unit as a single dose suitable for a subject, each unit containing a predetermined amount of active substance that can induce the desired therapeutic effect with respect to the desired diluent (i.e., carrier or medium).

[0190] This composition is administered in a manner appropriate to the dosage formulation and at a therapeutically effective amount. The amount administered depends on the subject being treated, the subject's systemic capacity to utilize the active ingredient, and the desired therapeutic effect. The precise amount of active ingredient to be administered depends on the physician's judgment and is specific to each individual. However, a dosage range suitable for systemic use is also disclosed herein, depending on the route of administration. Regimens suitable for initial administration and booster injections are also considered, typically involving a first administration followed by repeated administration every hour or more, and then injection or other routes of administration. Exemplary multiple administrations are described herein, with particular recommendation for maintaining high serum and tissue levels of the modified CGL enzyme, or conversely, maintaining low serum and tissue levels of homocysteine. Alternatively, continuous intravenous infusion may be considered to maintain blood concentrations within the therapeutically prescribed range in vivo. Notably, the weekly dose of the modified CGL enzyme for treating homocysteineuria and hyperhomocysteinemia is approximately one-quarter of the weekly dose required for cancer treatment. U.S. Patent No. 9,481,877.

[0191] V. Conjugate

[0192] The provided compositions and methods involve further modification of modified CGL enzymes for improvement, for example, by forming conjugates with heterologous peptide fragments or polymers (such as polyethylene glycol). The modified CGL enzyme can be linked to PEG to increase the enzyme's hydrodynamic radius, thereby increasing serum persistence. The disclosed peptides can be coupled to any targeting agent, such as ligands capable of specifically and stably binding to external receptors or binding sites on tumor cells (US Patent Publication 2009 / 0304666). The size of the PEG is between about 3,000 and 20,000 Daltons, with an exemplary size of 5,000 Daltons.

[0193] A. Fusion protein

[0194] In fusion proteins, the modified CGL enzyme can be linked to a heterologous domain at the N- or C-terminus. For example, fusions can also utilize leader sequences from other species to allow recombinant protein expression in a heterologous host. Another useful fusion involves the addition of protein affinity tags (such as serum albumin affinity tags or six-histidine residues) or immunologically active domains (such as antibody epitopes, preferably cleavable) to facilitate the purification of the fusion protein. Non-restrictive affinity tags include multihistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione S-transferase (GST).

[0195] Modified CGL enzymes may be linked to peptides that increase in vivo half-life, such as XTEN peptides (Schellenberger et al., 2009), IgG Fc domains, albumin, or albumin-binding peptides.

[0196] Methods for generating fusion proteins are well known to those skilled in the art. Such proteins can be generated, for example, through de novo synthesis of a fully fusion protein, or by ligation of a DNA sequence encoding a heterologous domain and expression of the fully fusion protein.

[0197] By linking a gene to a bridging DNA fragment encoding a peptide linker (which splices between tandemly linked polypeptides), the generation of fusion proteins that restore the functional activity of the parent protein can be facilitated. This linker should be of sufficient length to allow for proper folding of the resulting fusion protein.

[0198] B. Connector

[0199] Modified CGL enzymes can be chemically conjugated using bifunctional crosslinking agents or fused to peptide linkers at the protein level. These bifunctional crosslinking agents have been widely used for various applications, including the preparation of affinity matrices, modification and stabilization of various structures, identification of ligand and receptor binding sites, and structural studies. Suitable peptide linkers (such as Gly-Ser linkers) can also be used to ligate modified CGL enzymes.

[0200] Homobifunctional reagents carrying two identical functional groups can induce cross-linking between identical or different macromolecules or macromolecular subunits, and attach peptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By utilizing the different reactivity of the two functional groups, cross-linking can be selectively and sequentially controlled. Based on the specificity of the functional groups, bifunctional cross-linking reagents can be classified into amino-, mercapto-, guanidino-, indole-, and carboxyl-specific groups, etc. Among them, reagents directed by free amino groups are popular due to their commercial availability, ease of synthesis, and suitable mild reaction conditions.

[0201] Some heterobifunctional crosslinking agents contain a primary amine reactive group and a thiol reactive group. In another example, a heterobifunctional crosslinking agent and a method of using the crosslinking agent are described (US Patent No. 5,889,155, the entire contents of which are incorporated herein by reference). This crosslinking agent binds a nucleophilic hydrazide residue to an electrophilic maleimide residue, allowing, for example, the coupling of aldehydes with free thiols. The crosslinking agent can be modified to crosslink various functional groups.

[0202] In addition, any other linker / coupling agent and / or mechanism known to those skilled in the art can be used to bind modified CGL enzymes, such as antibody-antigen interactions, avidin-biotin bonds, amide bonds, ester bonds, thioester bonds, ether bonds, thioether bonds, phosphate ester bonds, phosphoramide bonds, acid anhydride bonds, disulfide bonds, ionic and hydrophobic interactions, bispecific antibodies and antibody fragments, or combinations thereof.

[0203] Crosslinking agents with reasonable blood stability are preferred. Many types of disulfide bond-containing linkers are known to be successfully used in conjugated targeting and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds are more stable in vivo. Therefore, these linkers constitute a group of connecting agents.

[0204] In addition to hindered crosslinking agents, non-hindered crosslinking agents may also be used as specified herein. Other useful crosslinking agents (not considered to contain or generate protected disulfide bonds) include SATA, SPDP, and 2-iminosulfide (Wawrzynczak and Thorpe, 1987). The use of such crosslinking agents is well understood in the art. Flexible joints may also be used.

[0205] Once chemically conjugated, peptides are typically purified to isolate the conjugate from non-conjugating agents and other contaminants. A wide range of purification techniques are available to provide conjugates of sufficient purity for clinical use.

[0206] Particle size-based purification methods (such as gel filtration, gel permeation, or high-performance liquid chromatography) are generally the most commonly used. Other chromatographic techniques (such as Blue-Sepharose separation) can also be used. Traditional methods for purifying fusion proteins from inclusion bodies can be useful, such as using mild detergents, like sodium lauryl sarcosinate (SLS).

[0207] C Polyethylene glycol

[0208] Methods and compositions relating to the polyethylene glycolation of modified CGL enzymes are disclosed. For example, the modified CGL enzyme can be polyethylene glycolated according to the methods disclosed herein.

[0209] PEGylation is the process of covalently linking a poly(ethylene glycol) polymer chain to another molecule, typically a drug or therapeutic protein. PEGylation is usually achieved by incubating a reactive derivative of PEG with the target macromolecule. The covalent linking of PEG to a drug or therapeutic protein can "mask" the agent against the host immune system (reduced immunogenicity and antigenicity), increase the drug's hydrodynamic size (size in solution), which can prolong its circulation time by reducing renal clearance. PEGylation can also impart water solubility to hydrophobic drugs and proteins.

[0210] The first step in polyethylene glycol (PEG) conversion is the appropriate functionalization of the PEG polymer at one or both ends. PEG with each end activated by the same reactive moiety is called "homobifunctional," while PEG derivatives with different functional groups are called "heterobifunctional" or "heterofunctional." Chemically active or activated derivatives of the PEG polymer are prepared to attach PEG to the desired molecule.

[0211] The selection of suitable functional groups for PEG derivatives is based on the type of reactive groups available on the molecule to be coupled to PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. N-terminal amino groups and C-terminal carboxylic acids can also be used.

[0212] The techniques used to form first-generation PEG derivatives typically involve reacting the PEG polymer with groups that can react with hydroxyl groups (usually acid anhydrides, acyl chlorides, chloroformates, and carbonates). In second-generation PEGylation chemistry, more efficient functional groups (such as aldehydes, esters, and amides) can be used for conjugation.

[0213] As polyethylene glycolation applications become increasingly sophisticated and complex, the demand for heterobifunctional PEGs for conjugation has grown. These heterobifunctional PEGs are well-suited for connecting two entities that require hydrophilic, flexible, and biocompatible spacers. Preferred end groups for heterobifunctional PEGs are maleic anhydride, vinyl sulfone, dithiopyridine, amine, carboxylic acid, and NHS esters.

[0214] The most common modifiers or linkers are based on methoxy PEG (mPEG) molecules. Their activity depends on adding protein-modifying groups to the alcohol terminus. In some cases, polyethylene glycol (PEG diol) is used as a precursor molecule. The diol is then modified at both ends to produce heterodimeric or homodimeric PEG linkers.

[0215] Proteins are typically PEGylated at nucleophilic sites, such as unprotonated thiols (cysteyl residues) or amino groups. Examples of cysteyl-specific modifiers include PEG-cis-imide, PEG-iodoacetate, PEG-thiols, and PEG-vinyl sulfones. All four exhibit strong cysteyl specificity under mild conditions and at neutral to slightly alkaline pH, but each has some drawbacks. The thioether formed by cis-imide can be slightly unstable under alkaline conditions, thus limiting the formulation options using this linker. The thiocarbamate bond formed by iodoPEG is more stable, but free iodine can modify tyrosine residues under certain conditions. PEG-thiols form disulfide bonds with protein thiols, but these bonds can also be unstable under alkaline conditions. PEG-vinyl sulfones are relatively slow to react compared to cis-imide and iodoPEG; however, the resulting thioether bonds are extremely stable. Their slower reaction rate also makes the PEG-vinyl sulfone reaction easier to control.

[0216] Site-specific PEGylation at native cysteine ​​residues is rarely performed because these residues are typically in the form of disulfide bonds or are required for biological activity. On the other hand, site-directed mutagenesis can be used to incorporate cysteine ​​PEGylation sites for thiol-specific linkers. Cysteine ​​mutations must be designed to make them accessible to the PEGylating agent and to retain biological activity after PEGylation.

[0217] Amine-specific modifiers include PEG NHS esters, PEG trifluoroethyl sulfonates, PEG aldehydes, and PEG isothiocyanates. They all react under mild conditions and are highly specific to amino groups. PEG NHS esters may be a more reactive agent; however, their high reactivity can make PEGylation reactions difficult to control on a large scale. PEG aldehydes form imines with amino groups, which are then reduced to secondary amines using sodium cyanoborohydride. Unlike sodium borohydride, sodium cyanoborohydride does not reduce disulfide bonds. However, this chemical is highly toxic and must be handled with care, especially at lower pH levels where it is volatile.

[0218] Site-specific PEGylation is challenging due to the multiple lysine residues on most proteins. Fortunately, because these reagents react with unprotonated amino groups, it is possible to direct PEGylation to lower pK amino groups by reacting at lower pH. Generally, the pK of the α-amino group is 1-2 pH units lower than that of the ε-amino group of the lysine residue. High selectivity for the N-terminus is usually obtained by PEGylating the molecule at pH 7 or lower. However, this is only feasible if the N-terminal portion of the protein is not required for biological activity. Furthermore, the beneficial pharmacokinetic effects of PEGylation generally outweigh the significant loss of in vitro biological activity, yielding products with greater in vivo biological activity, regardless of the PEGylation chemistry.

[0219] Developing PEGylation procedures requires consideration of several parameters. Fortunately, the number is typically no more than four or five. An "experimental design" approach to optimizing PEGylation conditions is very useful. For thiol-specific PEGylation reactions, parameters to consider include: protein concentration, PEG to protein ratio (in moles), temperature, pH, reaction time, and, in some cases, deoxygenation. (Oxygen can facilitate intermolecular disulfide formation through proteins, which reduces the yield of the PEGylation product.) The same factors (except oxygen) should be considered for amine-specific modifications, with pH potentially being even more critical, especially when targeting N-terminal amino groups.

[0220] Regarding amine and thiol-specific modifications, reaction conditions can affect protein stability. This can limit temperature, protein concentration, and pH. Additionally, the reactivity of the PEG linker should be known before initiating the PEGylation reaction. For example, if the PEGylation agent has only 70% activity, then the amount of PEG used should ensure that only the active PEG molecules are included in the stoichiometry of the protein-PEG reaction.

[0221] VI. Proteins and Peptides

[0222] Compositions comprising at least one protein or peptide (such as a modified CGL enzyme) are provided. These peptides may be contained in a fusion protein or conjugated to the reagents described above.

[0223] As used herein, protein or peptide generally refers to, but is not limited to, proteins of more than about 200 amino acids up to the full-length sequence translated from a gene; polypeptides of more than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein.

[0224] Therefore, the term "protein or peptide" encompasses at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unnatural amino acid.

[0225] Proteins or peptides can be prepared using any technique known to those skilled in the art, including expressing proteins, polypeptides, or peptides using standard molecular biotechnology, isolating proteins or peptides from natural sources, or chemically synthesizing proteins or peptides. Techniques disclosed herein or known to those skilled in the art can be used to amplify and / or express coding regions of known genes. Alternatively, various commercial formulations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0226] VII. Nucleic Acids and Vectors

[0227] Nucleic acid sequences encoding modified CGL enzymes or fusion proteins containing modified CGL enzymes are disclosed. Depending on the expression system used, the nucleic acid sequence can be selected based on conventional methods. For example, if the modified CGL enzyme is derived from human cystathionine and contains several codons rarely used in *E. coli*, it can interfere with expression. Therefore, free software (see Hoover & Lubkowski, 2002) can be used to design coding sequences free of rare codons to codon-optimize individual genes or their variants for *E. coli* expression. Various vectors can also be used to express proteins of interest, such as modified CGL enzymes. Exemplary vectors include, but are not limited to, plasmid vectors, viral vectors, transposon vectors, or liposome-based vectors.

[0228] VIII. Host Cell

[0229] The host cell can be any cell that can be transformed to allow the expression and secretion of the modified CGL enzyme and its conjugates. The host cell can be bacteria, mammalian cells, yeast, or filamentous fungi. Various bacteria include *Escherichia* and *Bacillus*. Yeasts belonging to the genera *Saccharomyces*, *Kluyveromyces*, *Hansenula*, or *Pichia* will be suitable as host cells. Various species of filamentous fungi can be used as expression hosts, including the following genera: Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus, and Pyricularia.

[0230] Examples of available host organisms include bacteria such as Escherichia coli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al., 1984), Staphylococcus aureus SAI123 (Lordanescu, 1975), or Streptococcus lividans (Hopwood et al., 1985); yeasts such as Saccharomyces cerevisiae AH 22 (Mellor et al., 1983) or Schizosaccharomyces pombe; and filamentous fungi such as Aspergillus nidulans, Aspergillus awamori (Ward, 1989), or Trichoderma reesei (Penttila et al., 1987; Harkki et al., 1989).

[0231] Examples of mammalian host cells include Chinese hamster ovary cells (CHO-K1; American Type Culture Collection (ATCC) No. CCL61), rat pituitary cells (GH1; ATCC No. CCL82), HeLa S3 cells (ATCC No. CCL2.2), rat liver cancer cells (H-4-II-E; ATCC No. CRL-1548), SV40-transformed monkey kidney cells (COS-1; ATCC No. CRL-1650), and mouse embryonic cells (NIH-3T3; ATCC No. CRL-1658). The foregoing is illustrative and not intended to limit the many possible host organisms known in the art. In principle, any host capable of secretion, whether prokaryotic or eukaryotic, can be used.

[0232] Mammalian host cells expressing modified CGL enzymes and / or their fusion proteins are cultured under conditions typically used for culturing parental cell lines. Generally, cells are cultured in standard media (e.g., standard RPMI, MEM, IMEM, or DMEM) containing physiological salts and nutrients, usually supplemented with 5%–10% serum (e.g., fetal bovine serum). Culture conditions are also standard, such as static or rotary culture at 37°C, until the desired protein levels are achieved.

[0233] IX. Protein Purification

[0234] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, the homogenization and crude separation of cells, tissues, or organs into peptide and non-peptide fractions. Unless otherwise specified, chromatographic and electrophoretic techniques may be used to further purify the protein or peptide of interest to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides include ion exchange chromatography, gel size exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography, and isoelectric point focusing. A particularly effective method for purifying peptides is rapid high-performance liquid chromatography (FPLC) or even high-performance liquid chromatography (HPLC).

[0235] Purified proteins or peptides refer to compositions that can be separated from other components, wherein the proteins or peptides are purified to any degree relative to their naturally occurring state. Therefore, isolated or purified proteins or peptides also refer to proteins or peptides isolated from their naturally occurring environment. Generally, "purified" will refer to a protein or peptide composition that has been fractionated to remove various other components, and that the composition substantially retains its expressed biological activity. When using the term "substantially purified," this name will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein in the composition.

[0236] Various techniques suitable for protein purification are well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies, etc., or by heat denaturation followed by centrifugation; chromatographic steps, such as ion exchange, gel filtration, reversed-phase chromatography, hydroxyapatite chromatography, and affinity chromatography; isoelectric point focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, the order of the various purification steps can be changed, or certain steps can be omitted, and these methods remain suitable for preparing substantially purified proteins or peptides.

[0237] Various methods for quantifying the degree of purification of proteins or peptides are known to those skilled in the art. These methods include, for example, determining the specific activity of an active fraction or assessing the amount of peptide within a fraction by SDS / PAGE analysis. A preferred method for analyzing the purity of a fraction is to calculate the specific activity of the fraction and compare it with the specific activity of the initial extract to calculate the degree of purity therein, which is assessed by a “purification fold”. Of course, the actual unit used to express the amount of activity depends on the specific detection technique selected after purification and whether the expressed protein or peptide exhibits detectable activity.

[0238] It is generally not required that proteins or peptides always be provided in their purest state. In fact, it can be expected that substantially impure products may be of practical use. Partial purification can be achieved by combining fewer purification steps or by utilizing different forms of the same general purification protocol. For example, it should be understood that cation exchange column chromatography using an HPLC apparatus typically yields a greater purification "fold" than the same technique using a low-pressure chromatography system. Methods with relatively lower purification levels may have advantages in terms of overall recovery of the protein product or in maintaining the activity of the expressed protein.

[0239] Proteins or peptides can be isolated or purified, such as modified CGL enzymes, fusion proteins containing modified CGL enzymes, or PEGylated modified CGL enzymes. For example, His tags or affinity epitopes can be included in such modified CGL enzymes to facilitate purification. Affinity chromatography is a chromatographic procedure that relies on the specific affinity between the substance to be separated and the molecules that it can specifically bind to. This is a receptor-ligand interaction type. Column materials are synthesized by covalently coupling a binding coupler to an insoluble matrix. The column material is then able to specifically adsorb substances from solution. Elution is performed by changing the conditions to those conditions that do not occur (e.g., changing pH, ionic strength, temperature, etc.). The matrix should be a substance that largely does not adsorb molecules and has broad chemical, physical, and thermal stability. The ligand should be coupled in a manner that does not affect its binding properties. The ligand should also provide a relatively tight binding. The substance should be eluted without destroying the sample or the ligand.

[0240] Size exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size or, in more technical terms, hydrodynamic volume. It is generally suitable for large molecules or large molecular complexes, such as proteins and industrial polymers. Typically, when an aqueous solution is used to deliver the sample through a column, this technique is called gel filtration chromatography, while when an organic solvent is used as the mobile phase, it is called gel permeation chromatography.

[0241] The basic principle of SEC (Separation of Size Limiting Columns) is that particles of different sizes elute (filter) through a stationary phase at different rates. This allows the particle solution to be separated based on size. If all particles are loaded simultaneously or nearly simultaneously, particles of the same size should elute together. Each size of size-limiting column has a separable molecular weight range. The size limit restricts the molecular weight to the upper end of this range and is the limitation that molecules are too large to be trapped in the stationary phase. The permeation limit restricts the molecular weight to the lower end of the separation range, where molecules of sufficiently small size can completely penetrate the pores of the stationary phase, and all molecules smaller than this molecular mass are too small to be eluted as a single band.

[0242] High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography commonly used in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column containing chromatographic packing material (stationary phase), a pump that moves the mobile phase through the column, and a detector that displays the retention times of molecules. Retention times vary depending on the interactions between the stationary phase, the analyte, and the solvent used.

[0243] X. Therapeutic Composition

[0244] The human cystathionine-γ-lyase gene contains several codons that are rarely used in *E. coli* and can interfere with expression. Therefore, to optimize protein expression in *E. coli*, codon-optimized oligonucleotides designed using DNA-Works software can be used to assemble the corresponding gene (Hoover et al., 2002). Each construct can contain an N-terminal NcoI restriction site, an in-frame N-terminal His6 tag, and a C-terminal EcoRI site to simplify cloning. After cloning into the pET28a vector (Novagen), *E. coli* (BL21) containing the appropriate cystathionine expression vector can be cultured at 37°C in TBTerrific Broth (TB) medium containing 50 μg / mL kanamycin at 250 rpm in shake flasks until OD is reached. 600= ~0.5-0.6. At this point, the culture can be transferred to a shaker at 25°C and induced with 0.5 mM IPTG for protein re-expression for 12 h. The cell pellet can then be collected by centrifugation and resuspended in IMAC buffer (10 mM NaPO4 / 10 mM imidazole / 300 mM NaCl, pH 8). After lysis using a French cell crusher, the lysate is centrifuged at 20,000 x g for 20 min at 4°C, and the resulting supernatant is applied to a nickel IMAC column. The column is washed extensively with IMAC buffer (90-100 column volumes) containing 0.1% TRITON™ 114, followed by 10-20 column volumes of IMAC buffer, and then eluted with IMAC elution buffer (50 mM NaPO4 / 250 mM imidazole / 300 mM NaCl, pH 8). The purified protein can be buffer-exchanged to 100 mM NaPO4 buffer pH 8.3 using a 10,000 MW CO (molecular weight cutoff) filter. The enzyme fraction can then be incubated at 25°C with 10 mM PLP for 1 h. 5000 MW of methoxyPEG succinimide carboxymethyl ester (JenKem Technology) can be added to CGL-8mut-1 at an 80:1 molar ratio, and the reaction can be allowed to proceed with constant stirring at 25°C for 1 h. The resulting mixture is extensively buffer-exchanged (PBS containing 10% glycerol) using a 100,000 MW CO filter (Amicon) and sterilized using a 0.2 μm syringe filter (VWR). Aliquots of the enzyme can be flash-frozen in liquid nitrogen and stored at -80°C. The homogeneity of CGL or CGL variants purified in this manner can be >95%, as assessed by SDS-PAGE and Coomassie staining. Under the conditions of a final buffer concentration of 6M guanidine hydrochloride, 20mM phosphate buffer, and pH 6.5, the extinction coefficient λ can be calculated. 280 =29870M -1 cm -1 Calculate the yield (Gill and von Hippel, 1989).

[0245] For example, the serum stability of polyethylene glycolated hCGL-8mut-1 was tested by incubating the enzyme in collected human serum at a final concentration of 10 μM at 37 °C. Aliquots were extracted and tested at different time points using a DTNB (Ellman's reagent; 5,5-dithio-bis-(2-nitrobenzoic acid)) detection method, as described in U.S. Patent Publication 2011 / 0200576, which is incorporated herein by reference in its entirety. The calculated half-life (T0) of polyethylene glycolated hCGL-8mut-1 was... 0.5 The time was 101 ± 4 hours.

[0246] This is not intended to limit the specific properties of therapeutic formulations. For example, such compositions may be provided in the formulation along with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic formulations may be administered to mammals for veterinary use, such as in livestock, and to humans for clinical use, in a manner similar to other therapeutic agents. Generally, the dosage required for therapeutic efficacy will vary depending on the type and mode of administration and the specific needs of the individual subject.

[0247] Such compositions are typically prepared as liquid solutions or suspensions for use in injection. Suitable diluents and excipients are, for example, water, saline, glucose, glycerol, and combinations thereof. Additionally, if desired, the composition may contain small amounts of auxiliary substances such as wetting agents or emulsifiers, stabilizers, or pH buffers.

[0248] When considering clinical applications, it may be necessary to prepare therapeutic compositions comprising proteins, antibodies, and drugs in a form suitable for the intended application. Generally, a pharmaceutical composition may contain an effective amount of one or more modified CGL variants or adjuncts dissolved or dispersed in a pharmaceutically acceptable carrier. The term “therapeutic or therapeutically acceptable” means that the molecular entity and composition, when administered to animals (e.g., humans) as appropriate, do not produce adverse, allergic, or other adverse reactions. The preparation of pharmaceutical compositions containing at least one modified CGL enzyme isolated by the methods disclosed herein, or other active ingredients, will be known to those skilled in the art, as exemplified in Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biostandards.

[0249] As used herein and known to those skilled in the art, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, gels, binders, excipients, similar materials and combinations thereof (see, for example, Remington Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions should be considered.

[0250] The composition may be administered subcutaneously, intravenously, intra-arterially, intraperitoneally, intramuscularly, by injection, infusion, continuous infusion, via catheter, in a lipid composition (e.g., liposomes), or by other methods as should be known to a person skilled in the art, or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference).

[0251] Modified polypeptides can be formulated into compositions in the form of free bases, neutral compounds, or salts. Therapeutically acceptable salts include acid addition salts, such as those formed by the free amino groups of the protein composition, or those formed by inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed by free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide); or organic bases (such as isopropylamine, trimethylamine, histidine, or procaine). In formulation, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The formulation can be administered in various dosage forms, such as formulations for parenteral administration, such as injectable solutions, or for delivery to the lungs as aerosols, or formulations for dietary administration, such as drug-release capsules, etc.

[0252] The disclosed compositions suitable for application may be provided in pharmaceutically acceptable carriers, with or without inert diluents. Use in an administerable composition for use in practice is appropriate unless any conventional medium, reagent, diluent, or carrier is harmful to the recipient or to the therapeutic efficacy of the composition contained therein. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, etc., or combinations thereof. The compositions may also contain various antioxidants to delay the oxidation of one or more components. Additionally, prevention of microbial action may be achieved by preservatives, such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0253] The disclosed composition and carrier can be combined in any convenient and practical manner, i.e., through solution, suspension, emulsion, blend, encapsulation, absorption, etc.

[0254] Use of therapeutic lipid mediator compositions comprising CGL enzymes, one or more lipids, and an aqueous solvent is provided. As used herein, the term "lipid" will be defined as any of a broad range of substances characterized by insolubility in water and extractability with organic solvents. This broad class of compounds is well known to those skilled in the art, and when the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., artificially designed or produced). However, lipids are generally biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, glycerol phosphates, steroids, terpenes, lysolipin, glycosphingolipids, glycolipids, thioesters, lipids having ether and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Of course, compositions and methods also encompass compounds that are understood by those skilled in the art to be lipids other than those specifically described herein.

[0255] Those skilled in the art will be familiar with the range of techniques that can be used to disperse compositions in lipid media. For example, modified CGL enzymes or their fusion proteins can be dispersed in solutions containing lipids, dissolved in lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently linked to lipids, contained in lipids as suspensions, contained in micelles or liposomes, or complexed with micelles or liposomes, or associated with lipids or lipid structures in any manner known to those skilled in the art. Dispersion may or may not result in the formation of liposomes.

[0256] The actual dosage of the composition administered to animal patients is determined by physical and physiological factors such as body weight, severity of illness, type of disease being treated, prior or concurrent treatment interventions, patient's spontaneous disease onset, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of times a therapeutically effective amount is administered may vary based on the subject's response. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject.

[0257] The therapeutic composition may contain, for example, at least about 0.1% of the active compound. The active compound may be contained, for example, between about 2% and about 75% by weight, or between about 25% and about 60% by weight, and any range from which it can be derived. Of course, the amount of active compound in each therapeutically available composition can be prepared in such a way as to obtain a suitable dose at any given unit dose of the compound. Those skilled in the art who prepare such pharmaceutical formulations will envision factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmaceutical considerations, thus multiple dosages and treatment regimens may be desirable.

[0258] Dosage may also include approximately 500 micrograms / kg body weight, approximately 1 microgram / kg body weight, approximately 5 micrograms / kg body weight, approximately 10 micrograms / kg body weight, approximately 50 micrograms / kg body weight, approximately 100 micrograms / kg body weight, approximately 750 mg / kg body weight or more per administration, and any range from which it can be derived. If administered weekly, the dose may be 5 mg / kg body weight, or, for example, a 70 kg subject requiring 350 mg of protein.

[0259] XI. Reagent Kit

[0260] Kits, such as therapeutic kits, may be available. For example, a kit may contain one or more therapeutic compositions described herein, along with optional instructions for their use. The kit may also contain one or more devices for administering such compositions. For example, a subject kit may contain a therapeutic composition and a catheter for administering the composition directly intravenously to a target tissue. The kit may contain pre-filled ampoules optionally formulated as a therapeutic composition or lyophilized modified CGL enzyme, used in conjunction with a delivery device.

[0261] The kit may contain labeled containers. Suitable containers include, for example, bottles, vials, and test tubes. Containers may be formed from a variety of materials, such as glass or plastic. Containers may contain compositions comprising modified CGL enzymes as described above, effective for therapeutic or non-therapeutic applications. Labels on the containers may indicate that the compositions described above are intended for specific therapeutic or non-therapeutic applications, and may also indicate instructions regarding in vivo or in vitro use. The kit of the present invention will generally contain the containers described above and one or more other containers containing materials necessary from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use.

[0262] XII. Example:

[0263] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention and can therefore be considered as preferred modes of practice of the invention. However, based on this disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtain the same or similar results.

[0264] Example 1 - A mouse model of hyperhomocysteinemia treated with a variant of human cystathionine-γ-lyase

[0265] Traditional homocystinuria is caused by a genetic defect in the cystathionine-β synthase gene. Disruption of the metabolic pathway leads to hyperhomocysteinemia, a condition characterized by severely elevated serum total homocysteine ​​(tHcy) levels. Homocystinuria is characterized by problems with the bones, eyes, blood vessels, and nervous system. These symptoms may result from the accumulation of homocysteine ​​in serum and intracellular cells.

[0266] The aim of this work was to develop a novel enzymatic therapeutic approach capable of degrading excess serum homocysteine, providing a pool of intracellular homocysteine, and creating a new method for treating homocystinuria. Human cystathionine-γ-lyase (CGL) was engineered to degrade homocysteine ​​and homocysteine. A library of CGL variants was generated using random and rational mutagenesis, and clones were identified through genetic selection based on their growth advantage over engineered strains of *E. coli* by degrading homocysteine ​​to α-butanone. Kinetic tests were performed on the CGL variants, and the results are shown in Table 1.

[0267] Hyperhomocysteinemia was induced in mice using a high-methionine diet (2.5% w / w methionine, instead of the 0.6% w / w methionine found in normal rodent diets) to establish a mouse model for evaluating the efficacy of the developed enzyme. Administration of a single dose of modified CGL at 50 mg / kg (intraperitoneal) to the high-methionine-diet mouse model significantly reduced tHcy from 380 ± 13 μM to 44.7 ± 4.8 μM within 24 hours. Figure 1 Note that the normal tHcy level in mice is approximately 4 ± 0.5 μM. Administration of 50 mg / kg (intraperitoneal) every 5 days for 30 days can induce a sustained decrease in tHcy (e.g., Figure 2 Furthermore, long-term, frequent administration did not lead to weight loss or any other signs of toxicity. In a diet-induced mouse model, administration of the enzyme successfully depleted tHcy without side effects. A high-methionine diet is a normal rodent diet but contains four times the amount of methionine.

[0268] To evaluate the therapeutic effect of modified CGL (hCGL-8mut-4; mutant 2; SEQ ID NO:6), cbs were used. - / - In mouse strains, this treatment can be fatal to newborns. Modified CGL and inactivated enzyme (20 mg / kg Ip, twice weekly) are administered from day 10 until day 50. A survival advantage is expected in treated mice. Both mothers and pups are fed betaine (a food additive believed to improve disease survival). Figure 3Treatment with the modified CGL (mutant 2) showed improved pup survival, suggesting that controlling Hcy levels is important for improving the condition.

[0269] Example 2 - Design and kinetic characteristics of highly specific homocysteine / homocysteine ​​degradation variants

[0270] As enzyme activity or selectivity for homocysteine ​​or homocysteine ​​increases, the gene encoding mutant 2 enzyme (SEQ ID NO: 6) was used as a starting point to generate more variants. Saturation mutagenesis was performed on active site residues hypothetically coordinating substrate binding, followed by the generation of corresponding libraries via overlap extension PCR (polymerase chain reaction). The assembled PCR products were digested with NcoI and EcoRI and ligated into the pET28a vector using T4 DNA ligase. The resulting ligations were directly converted to *E. coli* (Bl21) and subsequently screened on LB-kanamycin plates (as described in U.S. Patent No. 9,481,877, the entire contents of which are incorporated herein by reference). The number of colonies screened was twice the theoretical diversity of the library. Clones exhibiting significant activity were isolated and sequenced to identify mutants with improved activity. Three mutants were identified at the R119 site (mutant 3: R119D, SEQ ID NO: 37; mutant 4: R119H, SEQ ID NO: 38; mutant 5: R119G, SEQ ID NO: 39). These mutants were purified to a level of over 95% identity as determined by SDS-PAGE, and their degradation kinetics for methionine, cysteine, cystine, homocysteine, and homocysteine ​​were characterized kinetically in 100 mM PBS (phosphate-buffered saline) buffer at 37°C and pH 7.3 (Table 1) (as described in U.S. Patent No. 9,481,877, the entire contents of which are incorporated herein by reference).

[0271] Table 1. Kinetics of CGL mutants

[0272]

[0273] *ND = No data

[0274] Example 3 - Homocysteine ​​pharmacodynamics of mutant 3 in mice using a diet-induced mouse model of hyperhomocysteinemia

[0275] A high-methionine diet (2.5% w / w, instead of the normal 0.6% w / w described in Example 1) was used to induce hyperhomocysteinemia in mice. These mice were administered mutant 3 (SEQ ID NO:37) or the inactivating enzyme at a single dose of 50 mg / kg body weight (intraperitoneal), respectively. In the high-methionine diet mouse model, mutant 3 significantly reduced tHcy from 335 ± 37 μM to 42 ± 20 μM within 48 hours. Figure 4 ).

[0276] Example 4 - Treatment of a mouse model with hyperhomocysteinemia using mutant 3

[0277] To evaluate the efficacy of mutant 3 (R119D; SEQ ID NO:37) (which exhibits the highest substrate selectivity for homocysteine / homocysteine ​​compared to its methionine, cysteine, and cysteine ​​degradation activities), mutant 3 or mutant 3 enzymes that have been thermally deactivated by placing the sample in a boiling water bath for 10 minutes were used to treat cbs. - / - Young mice were treated. Injections were administered to the young mice starting on day 10 after birth at a dose of 25 mg / kg body weight (intraperitoneally, twice weekly) until the final treatment on day 50. Both the mother and young mice received a diet containing betaine (a food additive believed to improve disease survival rates). Figure 5 As shown, treatment with the active mutant 3 completely reduced CBS compared to animals treated with the same amount of inactivating enzyme. - / - Neonatal mortality rate in young mice.

[0278] **

[0279] In view of this disclosure, all methods disclosed and claimed herein can be performed and carried out without excessive experimentation. Although the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the methods described herein and the steps or the order of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein while obtaining the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to fall within the spirit, scope, and concept of the invention as defined by the appended claims.

[0280] References

[0281] The following references provide exemplary procedures or other details to some extent, which supplement those procedures or details described herein, and are expressly incorporated herein by reference.

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[0345] Yoshioka et al., Cancer Res., 58:2583-2587, 1998. sequence list <110> University of Texas System Board of Trustees <120> Human enzyme-mediated homocysteine ​​depletion used to treat patients with hyperhomocysteinemia and homocysteineuria. <130> UTFB.P1138WO <140> Not specified <141> 2018-05-11 <150> US 62 / 505,493 <151> 2017-05-12 <160> 66 <170> PatentIn version 3.5 <210> 1 <211> 405 <212> PRT <213> Homo sapiens <400> 1 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Glu Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Arg Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Glu Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 2 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 2 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 3 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 3 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 4 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 4 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 5 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 5 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 6 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 6 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80<00010​​​​​​100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro<上标 385 390 395 400 Ser Gly Ser His Ser 405 <210> 7 <211> 405 <212> PRT <213> Sumatran orangutan <400> 7 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Val Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Arg Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Glu Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Val Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 8 <211> 405 <212> PRT <213> Cynomolgus monkey <400> 8 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp<00011​​​​​Gln Ala Ala Pro Gly Gln His Ser Gly Phe Glu Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Arg Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Glu Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 9 <211> 405 <212> PRT <213> Chimpanzee <400> 9 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Glu Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Arg Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Glu Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 10 <211> 405 <212> PRT <213> Bonobo <400> 10 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Glu Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Arg Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Glu Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 11 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 11 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 [[ID=十四]]Ser Gly Ser His Ser 405 <210> 12 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 12 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly It should be noted that there seems to be an incorrect line break in the original Chinese translation for line 14. It should be "Ser Gly Ser His Ser" instead of "十四". This has been corrected in the translation above.65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 13 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 13 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 14 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 14 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 15 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 15 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 16 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 16 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 17 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 17 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 18 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 18 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 19 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 19 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 20 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 20 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 21 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 21 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 22 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 22 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 23 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 23 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 24 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 24 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45<e Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 25 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 25 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 26 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 26 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 27 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 27 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly [ 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Asp 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 28 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 28 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Asp 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 29 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 29 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Asp 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 30 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 30 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Asp 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 31 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 31 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Asp 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 32 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 32 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Ser Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Leu Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Lys His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Thr Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Glu 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ile Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 33 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 33 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Glu 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 34 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 34 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Leu Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Glu 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 35 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 35 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Glu 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 36 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 36 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Glu 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 37 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 37 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 38 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 38 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 6​​​​​​​​​​​​​​​​​​​130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 39 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 39 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Gly Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Gly Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Ser Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 40 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 40 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 41 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 41 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 42 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 42 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Gly Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 43 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 43 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 44 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 44 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 45 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 45 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Gly Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 46 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 46 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 47 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 47 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 48 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 48 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 49 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 49 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 50 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 50 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 51 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 51 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Ile Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Gly Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 52 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 52 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 53 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 53 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 54 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 54 Met Gln Glu Lys Glu Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Pro Ile Ser Pro Ser Val Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Ala Gly Thr Asn Gly Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Met Thr Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu Tyr Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu Gln Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Lys Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 55 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 55 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80​​​​​​​​​Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 56 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 56 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 57 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 57 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Glu Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Val Val Pro Leu Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Ala Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Val Leu Lys Met Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys Arg Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Cys 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Arg Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Leu Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Gly Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Ala Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Ile Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Val Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Pro Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Lys 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Asn 405 <210> 58 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 58 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 59 <211> 405 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 59 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly [[ID=²7]]50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu It should be noted that there seems to be a small error in line 27 where "²7" is present in the original. It's likely a typo and I've translated it as normal as possible while keeping the original structure.130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 60 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 60 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Arg Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Gly Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Met Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 61 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 61 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Asp Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 62 <211> 405 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 62 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn His Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 63 <211> 405 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 63 Met Gln Glu Lys Asp Ala Ser Ser Gln Gly Phe Leu Pro His Phe Gln 1 5 10 15 His Phe Ala Thr Gln Ala Ile His Val Gly Gln Asp Pro Glu Gln Trp 20 25 30 Thr Ser Lys Ala Leu Val Pro Pro Ile Ser Leu Ser Thr Thr Phe Lys 35 40 45 Gln Gly Ala Pro Gly Gln His Ser Gly Phe Asn Tyr Ser Arg Leu Gly 50 55 60 Asn Pro Thr Arg Asn Cys Leu Glu Lys Ala Val Ala Ala Leu Asp Gly 65 70 75 80 Ala Lys Tyr Cys Leu Ala Phe Ala Ser Gly Met Ala Ala Thr Val Thr 85 90 95 Ile Thr His Leu Leu Lys Ala Gly Asp Gln Ile Ile Cys Met Asp Asp 100 105 110 Val Tyr Gly Gly Thr Asn Ala Tyr Phe Arg Gln Val Ala Ser Glu Phe 115 120 125 Gly Leu Lys Ile Ser Phe Val Asp Cys Ser Lys Ile Lys Leu Leu Glu 130 135 140 Ala Ala Ile Thr Pro Glu Thr Lys Leu Val Trp Ile Glu Thr Pro Thr 145 150 155 160 Asn Pro Thr Gln Lys Val Ile Asp Ile Glu Ala Cys Ala His Ile Val 165 170 175 His Lys His Gly Asp Ile Ile Leu Val Val Asp Asn Thr Phe Met Ser 180 185 190 Pro Tyr Phe Gln Arg Pro Leu Ala Leu Gly Ala Asp Ile Cys Met Tyr 195 200 205 Ser Ala Thr Lys Tyr Met Asn Gly His Ser Asp Val Val Ile Gly Leu 210 215 220 Val Ser Val Asn Cys Glu Ser Leu His Asn Arg Leu Arg Phe Leu Gln 225 230 235 240 Asn Ser Leu Gly Ala Val Pro Ser Pro Ile Asp Cys Tyr Leu Cys Asn 245 250 255 Arg Gly Leu Lys Thr Leu His Val Arg Met Glu Arg His Phe Lys Asn 260 265 270 Gly Met Ala Val Ala Gln Phe Leu Glu Ser Asn Pro Trp Val Glu Lys 275 280 285 Val Ile Tyr Pro Gly Leu Pro Ser His Pro Gln His Glu Leu Val Lys 290 295 300 Arg Gln Cys Thr Gly Cys Gly Gly Met Val Thr Phe Tyr Ile Lys Gly 305 310 315 320 Thr Leu Gln His Ala Glu Ile Phe Leu Lys Asn Leu Lys Leu Phe Thr 325 330 335 Leu Ala Val Ser Leu Gly Gly Phe Glu Ser Leu Ala Glu Leu Pro Ala 340 345 350 Ser Met Thr His Ala Ser Val Leu Lys Asn Asp Arg Asp Val Leu Gly 355 360 365 Ile Ser Asp Thr Leu Ile Arg Leu Ser Val Gly Leu Glu Asp Glu Glu 370 375 380 Asp Leu Leu Glu Asp Leu Asp Gln Ala Leu Lys Ala Ala His Pro Pro 385 390 395 400 Ser Gly Ser His Ser 405 <210> 64 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 64 atgggaggcc atcaccacca tcatcatggc gggcaggaaa aggatgcgag ctcccagggc 60 tttcttccgc atttccagca ttttgcgacg caggcgatac acgtgggcca agacccggaa 120 cagtggacgt cgcgtgcggt tgtaccgccg attagcctga gcacgacctt taaacaaggt 180 gcgccgggcc agcatagcgg ttttatttat agccgtctgg gcaatcccac acggaattgc 240 ctggagaagg cggtggcggc tctggacggc gcgaagtatt gccttgcgtt tgcgagcgga 300 atggcggcca ccgtgaccat tacccacctg cttaaggctg gggaccagat tatttgcatg 360 gatgatgtgt atggtgggac caatgattat ttccgtcagg tggcgagcga gttcggcctg 420 aagatatcct ttgtcgactg ctcgaagatc aagctgttag aggcagcgat tacgccggaa 480 acaaaacttg tgtggataga aaccccgacg aacccgaccc agaaagtgat tgacattgaa 540 ggctgcgccc acattgtgca taaacacggc gatatcatcc tggtcgtgga taataccttc 600 atgagcccgt acttccagcg tccgctggcg cttggcgccg acattagcat gtattcggcg 660 accaagtata tgaacggcca tagcgacgtt gtcatgggcc tggtgagcgt gaattgcgag 720 agcctgcata atcgtctgcg ttttctgcaa aattcgcttg gagcggtgcc gagcccgatc 780 gattgctatc tgtgcaatcg tgggctgaag actctgcatg tgcggatgga gagacatttt 840 aagaatggca tggctgtggc gcagtttctg gaaagcaatc cgtgggtgga aaaagttatc 900 tacccgggac tgcccagcca cccgcagcat gaactggtca aacgtcagtg cacaggttgc 960 ggcggcatgg tgaccttcta tatcaagggc accctgcaac acgccgaaat ctttctgaaa 1020 aacctgaaac tgtttaccct ggcagtgagc ttgggcggct ttgaaagcct tgctgaactg 1080 ccggccagta tgactcatgc ctccgtgttg aaaaatgatc gtgatgttct gggcataagc 1140 gataccctga ttcgcctgtc cgtaggactg gaagatgaag aagatctgct ggaggatctg 1200 gatcaggcgc tgaaagcggc ccatccccca tcgggaagcc acagt 1245 <210> 65 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 65 atgggaggcc atcaccacca tcatcatggc gggcaggaaa aggatgcgag ctcccagggc 60 tttcttccgc atttccagca ttttgcgacg caggcgatac acgtgggcca agacccggaa 120 cagtggacgt cgcgtgcggt tgtaccgccg attagcctga gcacgacctt taaacaaggt 180 gcgccgggcc agcatagcgg ttttatttat agccgtctgg gcaatcccac acggaattgc 240 ctggagaagg cggtggcggc tctggacggc gcgaagtatt gccttgcgtt tgcgagcgga 300 atggcggcca ccgtgaccat tacccacctg cttaaggctg gggaccagat tatttgcatg 360 gatgatgtgt atggtgggac caatcattat ttccgtcagg tggcgagcga gttcggcctg 420 aagatatcct ttgtcgactg ctcgaagatc aagctgttag aggcagcgat tacgccggaa 480 acaaaacttg tgtggataga aaccccgacg aacccgaccc agaaagtgat tgacattgaa 540 ggctgcgccc acattgtgca taaacacggc gatatcatcc tggtcgtgga taataccttc 600 atgagcccgt acttccagcg tccgctggcg cttggcgccg acattagcat gtattcggcg 660 accaagtata tgaacggcca tagcgacgtt gtcatgggcc tggtgagcgt gaattgcgag 720 agcctgcata atcgtctgcg ttttctgcaa aattcgcttg gagcggtgcc gagcccgatc 780 gattgctatc tgtgcaatcg tgggctgaag actctgcatg tgcggatgga gagacatttt 840 aagaatggca tggctgtggc gcagtttctg gaaagcaatc cgtgggtgga aaaagttatc 900 tacccgggac tgcccagcca cccgcagcat gaactggtca aacgtcagtg cacaggttgc 960 ggcggcatgg tgaccttcta tatcaagggc accctgcaac acgccgaaat ctttctgaaa 1020 aacctgaaac tgtttaccct ggcagtgagc ttgggcggct ttgaaagcct tgctgaactg 1080 ccggccagta tgactcatgc ctccgtgttg aaaaatgatc gtgatgttct gggcataagc 1140 gataccctga ttcgcctgtc cgtaggactg gaagatgaag aagatctgct ggaggatctg 1200 gatcaggcgc tgaaagcggc ccatccccca tcgggaagcc acagt 1245 <210> 66 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 66 atgggaggcc atcaccacca tcatcatggc gggcaggaaa aggatgcgag ctcccagggc 60 tttcttccgc atttccagca ttttgcgacg caggcgatac acgtgggcca agacccggaa 120 cagtggacgt cgcgtgcggt tgtaccgccg attagcctga gcacgacctt taaacaaggt 180 gcgccgggcc agcatagcgg ttttatttat agccgtctgg gcaatcccac acggaattgc 240 ctggagaagg cggtggcggc tctggacggc gcgaagtatt gccttgcgtt tgcgagcgga 300 atggcggcca ccgtgaccat tacccacctg cttaaggctg gggaccagat tatttgcatg 360 gatgatgtgt atggtgggac caatggttat ttccgtcagg tggcgagcga gttcggcctg 420 aagatatcct ttgtcgactg ctcgaagatc aagctgttag aggcagcgat tacgccggaa 480 acaaaacttg tgtggataga aaccccgacg aacccgaccc agaaagtgat tgacattgaa 540 ggctgcgccc acattgtgca taaacacggc gatatcatcc tggtcgtgga taataccttc 600 atgagcccgt acttccagcg tccgctggcg cttggcgccg acattagcat gtattcggcg 660 accaagtata tgaacggcca tagcgacgtt gtcatgggcc tggtgagcgt gaattgcgag 720 agcctgcata atcgtctgcg ttttctgcaa aattcgcttg gagcggtgcc gagcccgatc 780 gattgctatc tgtgcaatcg tgggctgaag actctgcatg tgcggatgga gagacatttt 840 aagaatggca tggctgtggc gcagtttctg gaaagcaatc cgtgggtgga aaaagttatc 900 tacccgggac tgcccagcca cccgcagcat gaactggtca aacgtcagtg cacaggttgc 960 ggcggcatgg tgaccttcta tatcaagggc accctgcaac acgccgaaat ctttctgaaa 1020 aacctgaaac tgtttaccct ggcagtgagc ttgggcggct ttgaaagcct tgctgaactg 1080 ccggccagta tgactcatgc ctccgtgttg aaaaatgatc gtgatgttct gggcataagc 1140 gataccctga ttcgcctgtc cgtaggactg gaagatgaag aagatctgct ggaggatctg 1200 gatcaggcgc tgaaagcggc ccatccccca tcgggaagcc acagt 1245

Claims

1. An isolated and modified primate cystathionine-γ-lyase, wherein the amino acid sequence of the isolated and modified primate cystathionine-γ-lyase is shown in SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO:

39.

2. The isolated and modified primate cystathionine-γ-lyase according to claim 1, wherein the amino acid sequence of the isolated and modified primate cystathionine-γ-lyase is shown in SEQ ID NO:

37.

3. The isolated and modified primate cystathionine-γ-lyase according to claim 1, wherein the amino acid sequence of the isolated and modified primate cystathionine-γ-lyase is shown in SEQ ID NO:

38.

4. The isolated and modified primate cystathionine-γ-lyase according to claim 1, wherein the amino acid sequence of the isolated and modified primate cystathionine-γ-lyase is shown in SEQ ID NO:

39.

5. A nucleic acid encoding the isolated modified primate cystathionine-γ-lyase as described in any one of claims 1-4.

6. The nucleic acid of claim 5, wherein the nucleic acid is codon-optimized for expression in bacteria, fungi, insects, or mammals.

7. The nucleic acid of claim 6, wherein the bacteria is Escherichia coli.

8. An expression vector comprising the nucleic acid as described in any one of claims 5-7.

9. A host cell comprising the nucleic acid as described in any one of claims 5-7.

10. The host cell of claim 9, wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.

11. A therapeutic formulation comprising, in a pharmaceutically acceptable carrier, an isolated and modified primate cystathionine-γ-lyase as described in any one of claims 1-4 or a nucleic acid as described in any one of claims 5 or 6.

12. Use of the formulation of claim 11 in the preparation of a medicament for treating a subject suffering from homocystinuria or hyperhomocysteinemia or at risk of developing homocystinuria or hyperhomocysteinemia.

13. The use as described in claim 12, wherein the subject maintains a methionine-restricted diet.

14. The use as described in claim 12, wherein the subject maintains a normal diet.

15. The use as described in claim 12, wherein the subject is a human patient.

16. The use as described in claim 12, wherein the drug is prepared for administration via injection, infusion, continuous infusion, direct local perfusion of target cells, or via catheter into a vein, artery, peritoneum, lesion, joint, prostate, pleura, trachea, vitreous body, muscle, vesicle, or umbilical cord.

17. The use as claimed in claim 12, wherein the subject has previously undergone treatment for homocystinuria or hyperhomocysteinemia, and the drug is used to prevent recurrence of said homocystinuria or hyperhomocysteinemia.

18. The use as claimed in claim 12, wherein the medicament is prepared for combined administration to the subject with at least a second homocystinuria or hyperhomocysteinemia therapy.

19. The use as claimed in claim 18, wherein the treatment for second homocystinuria or hyperhomocysteinemia is high-dose vitamin B6 or betaine (N,N,N-trimethylglycine) therapy.

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